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ISBN 978-952-62-1103-9 (Paperback)ISBN 978-952-62-1104-6 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1343

ACTA

Marjut Sakko

OULU 2016

D 1343

Marjut Sakko

ANTIMICROBIAL ACTIVITY AND SUITABILITY OF2-HYDROXYISOCAPROIC ACID FOR THE TREATMENT OF ROOT CANAL INFECTIONS

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU, FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU;FINNISH DOCTORAL PROGRAM IN ORAL SCIENCES

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 1 3 4 3

MARJUT SAKKO

ANTIMICROBIAL ACTIVITY AND SUITABILITY OF 2-HYDROXYISOCAPROIC ACID FOR THE TREATMENT OF ROOT CANAL INFECTIONS

Academic dissertation to be presented with the assent ofthe Doctora l Tra in ing Committee of Health andBiosciences of the University of Oulu for public defence inLecture Hall 3, Biomedicum Helsinki 1 (Haartmaninkatu 8,Helsinki), on 26 February 2016, at 12 noon

UNIVERSITY OF OULU, OULU 2016

Copyright © 2016Acta Univ. Oul. D 1343, 2016

Supervised byProfessor Leo TjäderhaneDocent Riina Rautemaa-Richardson

Reviewed byProfessor Tuomas WaltimoDocent Pentti Kuusela

ISBN 978-952-62-1103-9 (Paperback)ISBN 978-952-62-1104-6 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2016

OpponentDocent Mataleena Parikka

Sakko, Marjut, Antimicrobial activity and suitability of 2-hydroxyisocaproic acidfor the treatment of root canal infections. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; MedicalResearch Center Oulu; Finnish Doctoral Program in Oral SciencesActa Univ. Oul. D 1343, 2016University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Microbial infection in dental root canal induces an inflammatory reaction called apicalperiodontitis. In post-treament disease, Enterococcus faecalis is the most commonly foundorganism, which may survive well in root canal despite calcium hydroxide paste medication. Inthese cases, effective irrigation or repeated chlorhexidine medication are recommended. Newmedications with long-lasting antimicrobial activity are needed for the treatment of persistent rootcanal infections. 2-hydroxyisocaproic acid (HICA) is a protein fermentation product oflactobacilli and few other bacterial or fungal species express enzymes required for its metabolism.However, mammalian cells can metabolise it and use it for protein production. It is known to bewell-tolerated by humans and have anti-inflammatory properties as well. Therefore, thehypothesis was that it affects microbe-specific metabolic pathways and have potential as a novelantimicrobial agent.

The aim of this study was to evaluate the antibacterial and antifungal spectrum of HICA usingin vitro microdilution methods for susceptibility testing and an ex vivo extracted tooth root canalinfection-model. The impact of dentine on the antimicrobial activity of HICA was also evaluated.The results showed that HICA has broad-spectrum bactericidal activity for gram-positive andgram-negative organisms. It is also fungicidal for several fungal species and it is only marginallyinactivated by clinically-relevant concentrations of dentine. It is at least as active against E.faecalis as presently-used interappointment medications in infected root canals after a seven-dayincubation ex vivo. Since HICA has a broad-spectrum and long-term antimicrobial activity as wellas an anti-inflammatory effect, it may be a useful new agent for clinical endodontology. However,controlled clinical studies are needed for evaluation of the efficacy of HICA in clinical conditions.

Keywords: 2-hydroxyisocaproic acid, antimicrobial, apical periodontitis, antisepticagent, calcium hydroxide, chlorhexidine, disinfectant, root canal medication

Sakko, Marjut, 2-hydroksi-isokapronihapon antimikrobiteho ja soveltuvuusjuurikanavainfektioiden hoitoon. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical ResearchCenter Oulu; Finnish Doctoral Program in Oral SciencesActa Univ. Oul. D 1343, 2016Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Hampaan juurikanavan mikrobi-infektio aiheuttaa apikaalisen parodontiitin eli tulehdusreaktionjuuren kärjen läheisyydessä oleviin kudoksiin. Silloin, kun infektio ei parane hoidon jälkeen,Enterococcus faecalis on yleisin löydös. Jos kalsiumhydroksidilääkitys ei ole tehokas, hoitoonsuositellaan huolellista desinfektiohuuhtelua ja uusittavaa lyhytkestoista klooriheksidiinilääki-tystä. Juurikanavainfektioiden hoitoon tarvitaan uusia antimikrobiaineita, joilla on pitkäaikainenvaikutus. Lactobacillus-bakteerit tuottavat proteiiniaineenvaihdunnassa 2-hydroksi-isokaproni-happoa eli HICAa, jonka metaboloimiseen tarvittavia entsyymejä tunnetaan vain muutamillamuilla bakteereilla. Ihmissolut metaboloivat ja käyttävät sitä proteiinituotantoon. Se on hyvinsiedetty aine ja se lieventää tulehdusreaktiota. Tutkimuksen hypoteesina oli, että HICA vaikut-taa mikrobien aineenvaihduntaan ja se voisi olla mahdollinen uusi antimikrobiaine.

Tässä tutkimuksessa HICAn antimikrobitehoa tutkittiin bakteereita ja sienilajeja vastaanpienerälaimennusmenetelmällä tehdyissä herkkyystesteissä. Sen soveltuvuutta hampaiden juuri-kanavainfektioiden hoitoon arvioitiin dentiinin läsnäollessa sekä potilailta poistetuissa ja kokeel-lisesti infektoiduissa hampaissa. Tulokset osoittavat, että HICA tappaa laajakirjoisesti gram-positiivisia ja gram-negatiivisia bakteereita sekä hiivoja ja muita sienilajeja. Dentiinin vaikutusHICAn antimikrobitehoon on vähäinen. Sen vaikutus E. faecalista vastaan poistettujen hampai-den juurikanavissa on viikon jälkeen yhtä hyvä tai parempi kuin nykyisten lääkeaineiden vaiku-tus. Laajakirjoisen, pitkäkestoisen antimikrobitehon ja anti-inflammatorisuuden vuoksi HICAvoisi olla uusi vaihtoehto juurikanavainfektioiden hoitoon. HICAn kliinisen tehokkuuden arvi-ointiin tarvitaan kontrolloituja kliinisiä tutkimuksia.

Asiasanat: 2-hydroksi-isokapronihappo, antimikrobiaine, apikaaliparodontiitti,antiseptinen aine, desinfiointiaine, juurikanavalääke, kalsiumhydroksidi,klooriheksidiini

7

Acknowledgements

This PhD thesis was a joint project between the University of Oulu and the

University of Helsinki, Finland, and the University of Manchester, United

Kingdom 2010–2015. I sincerely thank Professor Pertti Pirttiniemi, the head of the

Institute of Dentistry in the University of Oulu, for providing the research facilities

and the opportunity to conduct this study. I would like to thank also the

collaborative organisations: Department of Oral and Maxillofacial Diseases, the

Department of Bacteriology and Immunology in Haartman Institute at the

University of Helsinki, and the Mycology Reference Centre of the University of

Manchester.

Most of all I am grateful to my excellent supervisors Dr. Riina Richardson from

the University of Manchester and Prof. Leo Tjäderhane from the University of Oulu

and the University of Helsinki. It has been a great honour for me to work under the

encouraging and qualified guidance of these enthusiastic and inspiring scientists. I

wish to express my sincere appreciation to the reviewers of this thesis Prof. Tuomas

Waltimo and Dr. Pentti Kuusela for the constructive critique and valuable

commentary on the manuscript. I am also deeply thankful to the members the

doctoral study follow-up group Dr. Sakari Jokiranta and Dr. Teemu Kinnari for

sharing their expertise and providing support in the field of microbiological and

medical research.

It is a great pleasure for me to thank the collaborators Prof. Timo Sorsa, Prof.

Asko Järvinen, and Asst. Prof. Pentti Hietala from the University of Helsinki, and

Dr. Caroline Moore, Dr. Lily Novak-Frazer, Dr. Paul Bowyer and Vilma Rautemaa

from the University of Manchester. They all have provided a highly valuable

contribution to this research as co-authors. I sincerely thank Prof. Malcolm

Richardson for his expertised contribution to the language edition of all

manuscripts. I am thankful to Dr. Julie Morris for her expert opinions on statistical

methods. I kindly thank Dr. Jennifer Rowland for the language revision of this

thesis.

I wish to thank Heini Torkko and Kirsti Iivonen from the Clinical Microbiology

Reference Laboratory of the Helsinki University Hospital for the friendly co-

operation. I am thankful to Kirsti Kari for providing the excellent facilities at the

research laboratory. I warmly thank Saija Perovuo, Ritva Keva, Jukka Inkeri, Carin

Sahlberg, and Maarit Hakkarainen for their friendly co-operation, advice and

laboratory assistance as well as Dr. Leena Putro and Dr. Anne Kirilä, who carried

out their dental studies amid this project. I express my deepest thanks to Marjatta

8

Kivekäs due to her excellent technical guidance and skilful assistance in

histological works. I warmly thank Arja Haapasalo and Päivi Haataja for their kind

assistance in providing the dental technical facilities needed in this work. I wish to

thank Dr. Isabella Portenier from the University of Oslo, who provided the human

dentine powder as a kind gift for performing a part of the experiments of this study.

I am also thankful to the collegues and personnel of the Social and Health Services

of Lahti, who assisted in the study arrangements by collecting the extracted teeth

for the experiments.

I would like to thank for all hospitality of the personnel of the Laboratory of

the Mycology Reference Centre of the University of Manchester during the period

I was fortunate to work there. I wish to thank Dr. Mikko Nieminen, Dr. Lotta

Grönholm, Dr. Esa Färkkilä, Dr. Peter Rusanen and Dr. Sari Hotti from the Dr.

Riina Richardson’s group for the collegial discussions and co-operation. I am

thankful to Dr. Pirkko Pussinen, Dr. Taina Tervahartiala, and Dr. Kati Hyvärinen

for the expert advice and interesting conversations during this prosess. I would also

like to thank all collegues with clinical and research experience in Finland and

abroad, who have shown interest and pointed out valuable questions on this issue

in the congresses and meetings.

I express my warmest thanks to my beloved parents. This work is dedicated to

Ritva and Lauri with much gratitude for their encourangement, support, and endless

love, which they have given me, and also to our family. Their assistance in everyday

life has been priceless over the past years. I wish I could share this moment with

my father, too. I am more than grateful to our family. I express my deepest thanks

and appreciation to my beloved husband Sami for his understanding and loving

support by my side over this challenging, instructive, and enjoyable time. I owe my

greatest gratitude to our beloved children Matilda, Kristiina, and Samuli for giving

me so much happiness and joy, and for reminding me the meaning of life every day.

I am also deeply thankful to all my dear friends and dear relatives for the delightful

moments experienced together out of research in the meanwhile.

The Finnish Doctoral Program in Oral Sciences, Medical Research Center

Oulu, Finnish Dental Foundation, Finnish Dental Society Apollonia, Finnish

Association of Female Dentists, University of Helsinki and University Hospital of

Helsinki Research Funds (EVO), Academy of Finland, the UK National

Aspergillosis Centre are acknowledged for the financial support.

Tuusula, December 2015 Marjut Sakko

9

Abbreviations

AP apical periodontitis

ASM American Society of Microbiology

ATCC American Type Culture Collection

BHI brain heart infusion

BRU brucella agar

BSA bovine serum albumin

Ca(OH)2 calcium hydroxide

CCUG Culture Collection of the University of Gothenburg

CFU colony forming units

CHA chlorhexidine acetate

CHG chlorhexidine digluconate

CHX chlorhexidine

CLSI Clinical Laboratory Standards Institute

CMCP camphorated paramonochlorophenol

CP camphorated phenol

DL dextro and levo -enantiomers

DP dentine powder

EC Enzyme Commission of International Union of Biochemistry and

Molecular Biology

EDTA ethylenediaminetetraacetic acid

EFSA European Food Safety Authority

ESBL extended spectrum of beta-lactamase

EUCAST European Committee on Antimicrobial Susceptibility Testing

FAA fastidious anaerobe agar

FLU fluconazole

HA hydroxyapatite

HICA 2-hydroxyisocaproic acid

HicDH hydroxyisocaproic acid dehydrogenase

IC inhibitory concentration

IPI iodine potassium iodine

ITR itraconazole

KICA 2-ketoisocaproic acid

LTA lipoteichoic acid

LPS lipopolysaccharide

MIC minimum inhibitory concentration

10

MBC minimum bactericidal concentration

MRCM Mycology Reference Centre Manchester

MRSA methicillin-resistant Staphylococcus aureus

MSUD maple syrup urine disease

NaOCl sodium hypochlorite

NCTC National Collection of Type Cultures

NGS next-generation DNA sequencing

PCR polymerase chain reaction

PMN polymorphonuclear leucocytes

PW peptone water

RPMI Roswell Park Memorial Insitute media

SAB Sabouraud agar

SD standard deviation

SEM standard error of mean

TSA tryptic soya agar

UK NEQAS United Kingdom National External Quality Assessment Service

11

List of original publications

This thesis is based on the following publications, which are referred throughout

the text by Roman numerals:

I Sakko M, Tjäderhane L, Sorsa T, Hietala P, Järvinen A, Bowyer P, Rautemaa R (2012) 2-hydroxyisocaproic acid: a new potential topical antibacterial agent. International Journal of Antimicrobial Agents 39: 539–540.

II Sakko M, Moore C, Novak-Frazer L Rautemaa V, Sorsa T, Hietala P, Järvinen A, Bowyer P, Tjäderhane L, Rautemaa R (2014) 2-hydroxyisocaproic acid (HICA) is fungicidal for Candida and Aspergillus species. Mycoses 57: 214–221.

III Sakko M, Tjäderhane L, Sorsa T, Hietala P, Rautemaa R (2015) 2-hydroxyisocaproic acid (HICA) and chlorhexidine resist inactivation by dentine. International Endodontic Journal. doi: 10.1111/iej.12465.

IV Sakko M, Tjäderhane L, Sorsa T, Hietala P, Rautemaa R (2015) 2-hydroxyisocaproic acid (HICA) is bactericidal in human dental root canals ex vivo. Manuscript.

The original publications have been reprinted with permission of copyright holders.

In addition, this thesis contains unpublished data.

12

13

Contents

Abstract

Tiivistelmä

Acknowledgements 7 Abbreviations 9 List of original publications 11 Contents 13 1  Introduction 15 2  Review of the literature 19 

2.1  2-hydroxyisocaproic acid (HICA) ........................................................... 19 2.1.1  Distribution and biochemistry ...................................................... 19 2.1.2  Antimicrobial properties ............................................................... 21 2.1.3  Other uses and biocompatibility ................................................... 22 

2.2  Antimicrobials ......................................................................................... 23 2.2.1  Biocides ........................................................................................ 23 2.2.2  Antibiotics and antifungals ........................................................... 24 2.2.3  Non-susceptibility to biocides ...................................................... 24 

2.3  Endodontic disinfectants ......................................................................... 26 2.3.1  Alcohols ....................................................................................... 26 2.3.2  Biguanides .................................................................................... 27 2.3.3  Halogen-releasing agents .............................................................. 29 2.3.4  Hydroxyl ion-releasing agents ...................................................... 30 

2.4  Root canal infections ............................................................................... 31 2.4.1  Etiology and pathology................................................................. 31 2.4.2  Microbial species .......................................................................... 35 2.4.3  Treatment ...................................................................................... 37 

3  Aims of the study 43 4  Materials and methods 45 

4.1  Materials ................................................................................................. 47 4.1.1  Microbial isolates and strains ....................................................... 47 4.1.2  Culture conditions and dilutions ................................................... 47 

4.2  Methods ................................................................................................... 49 4.2.1  Susceptibility testing of bacteria and fungi against HICA ........... 49 4.2.2  Suitability testing of HICA for root canal medication .................. 51 

5  Results 57 5.1  Susceptibility of microbial isolates to HICA .......................................... 57 

14

5.1.1  Antibacterial activity (I) ............................................................... 57 5.1.2  Antifungal activity (II).................................................................. 59 

5.2  Suitability of HICA for root canal medication ........................................ 62 5.2.1  Antibacterial activity with potential inhibitors (III) ..................... 62 5.2.2  Antibacterial activity in dental root canals ex vivo (IV) ............... 66 

6  Discussion 71 6.1  Susceptibility testing studies ................................................................... 71 6.2  Methodological considerations ............................................................... 72 

6.2.1  Culture media ............................................................................... 73 6.2.2  Negative and positive controls ..................................................... 74 6.2.3  Test organisms .............................................................................. 76 6.2.4  Microbial cultures ......................................................................... 76 6.2.5  Potential inhibitors ........................................................................ 77 6.2.6  Human teeth .................................................................................. 80 6.2.7  Group size and statistical analysis ................................................ 80 

6.3  Antimicrobial activity ............................................................................. 81 6.3.1  HICA (I, II, III, IV) ....................................................................... 81 6.3.2  Calcium hydroxide (III, IV) .......................................................... 86 6.3.3  Chlorhexidine (III, IV) ................................................................. 90 

6.4  Future work ............................................................................................. 92 6.5  Potential of HICA for other uses (I, II) ................................................... 93 

7  Conclusion 95 References 97 Original publications 119 

15

1 Introduction

In general, microbial resistance to antimicrobials cause severe problems in the

treatment of infections. The excessive use of systemic antibiotics further increase

the emerging problem (World Health Organization 2014). Microbial colonisation

is generally seen as a prerequisite for a topical or systemic infection. Prevention of

colonisation in order to prevent infection process would be a useful target for

managing infection.

In the management of superficial infections, topical delivery of antimicrobial

agents may have several advantages in comparison to systemic use. At a site of

infection, high topical concentrations are easily achieved and the risk of systemic

toxicity is reduced. In addition, selection of species resistant to systemic

antimicrobials would decrease with the use of novel agents with different

antimicrobial mechanisms from the mode of action of current antimicrobials in use

and the use of antibiotics could be limited for management of severe deep infections.

In the development of new antimicrobials those agents unsuitable for systemic

treatments could be considered for topical use (McHugh et al. 2011).

Topical antimicrobials are the first line medications in the treatment of dental

root canal infections. Persisting root canal infection is the main reason for apical

periodontitis (AP) in post-treatment infections (Peciuliene et al. 2008, Siqueira &

Rôças 2014). Microbial biofilms, which are associated with AP, have an increased

ability to resist the effect of antimicrobials in comparison to free-floating cells (Ceri

et al. 1999, Lang et al. 2012).

The strategy of root canal treatment is based on microbial eradication from root

canals by both mechanical and chemical means, while interappointment medication

does not necessarily lead to significantly better treatment outcomes than single-

visit treatment (Weiger et al. 2000, Shuping et al. 2000, Kvist et al. 2004, Vivacqua-

Gomes et al. 2005, Paredes-Vieyra & Enriquez 2012, Vera et al. 2012).

Unreachable sites of complex root canal anatomy by mechanical instrumentation

or disinfection are potential niches for microbial persistence.

Highly alkaline calcium hydroxide (Ca(OH)2) is the most generally-used agent

in interappointment medication, but there are conflicting results regarding its

antimicrobial activity (Ørstavik et al. 1991, Reit et al. 1999, Peters et al. 2002, Vera

et al. 2012, Kim & Kim 2014, 2015). Ca(OH)2-resistant strains may cause difficult-

to-treat root canal infections. Chlorhexidine, sodium hypochlorite, and iodine

potassium iodine all have a different spectrum of activity in comparison to Ca(OH)2.

Despite their many positive properties in endodontic treatment, short-term activity,

16

potential of tissue irritation, allergic and toxic reaction caused by these agents

restrict their wider use as intracanal interappointment medication (Fedorowitz et al.

2012, Guleri et al. 2012, Pasternak & Williamson 2012).

Inorganic and organic content in root canals is reported to reduce antimicrobial

activity of disinfectants (Haapasalo et al. 2000, Portenier et al. 2001, 2002, 2006,

Oliveira de et al. 2010). In contrast, dentine extracellular matrix is shown to have

antimicrobial activity (Smith et al. 2012). New safer and more active antimicrobials

in comparison to presently-used agents are needed for achieving better treatment

outcomes in endodontology.

2-hydroxyisocaproic acid (HICA) could be a new and safe topical

antimicrobial agent, since it has antimicrobial properties (Hietala et al. 1979), it is

naturally found in nutrition (European Food Safety Authority 2012), and it is

metabolised by human cells (Hoffer et al. 1993, Dallmann et al. 2012). HICA is a

bacterial protein fermentation product, which is a by-product of the leucine-acetyl-

CoA pathway in the leucine degradation pathway. For HICA elimination

hydroxyisocaproic acid dehydrogenase (HicDH) enzyme is needed. Both are

mainly produced by lactobacilli (Hummel et al. 1985, Yamakazi & Maeda 1986,

Hummel & Kula 1989, Kallwass 1992, Bernard et al. 1994, Niefind et al. 1994,

1995, Dengler et al. 1997, Yvon & Rijnen 2001, Broadbent et al. 2004, Smit et al.

2004, 2005, Chambellon et al. 2009).

The present series of studies focuses on the antimicrobial spectrum of HICA.

Antibacterial activity of HICA was observed for a spectrum of human pathogenic

facultatively and obligately anaerobic gram-positive and gram-negative bacterial

species. The antibiotic-resistant strains of methicillin-resistant Staphylococcus

aureus (MRSA) and Escherichia coli with extended-spectrum beta-lactamase

(ESBL) were included. The antifungal spectrum of Candida and Aspergillus

species for HICA and azoles was tested. The impact of HICA on hyphal formation

of Candida albicans and filamentation of Aspergillus fumigatus, which are

associated with the pathogenesis and virulence, were evaluated.

A particular interest of this study was the potential of HICA for root canal

medication. Enterococcus faecalis has been the most common finding in persisting

root canal infections. The interactions between HICA and potential inhibitors such

as dentine, hydroxyapatite, and serum albumin were examined in 48 h incubation.

Longevity of antibacterial activity of HICA against E. faecalis was observed ex

vivo in the infected root canals of extracted human teeth for seven days. These test

conditions mimic the clinical conditions in an infected root canal system.

17

The hypothesis of this study was that HICA is a potential antimicrobial agent

for the treatment of topical infections. The purpose of this study was to evaluate the

antimicrobial spectrum and the suitability of HICA for the management of topical

infections in dental root canals.

18

19

2 Review of the literature

2.1 2-hydroxyisocaproic acid (HICA)

2.1.1 Distribution and biochemistry

HICA is also known as leucic acid, 2-hydroxy-4-methylvaleric acid, or 2-hydroxy-

4-methylpentanoic acid, with a molecular weight of 132.16 g/mol (Takayama 1933).

It is patented for antimicrobial and anti-inflammatory uses (Westermarck et al.

2008; EP 0871438 B1, “Use of alpha-hydroxy acids in the manufacture of a

medicament for the treatment of inflammation”). HICA is a by-product of the

leucine-acetyl-CoA pathway (Fig. 1). Together with isoleucine and valine, leucine

is one of the three essential branched-chain amino acids (BCAA) in humans, which

is primarily acquired from nutrition. HICA is produced in animal protein

fermentation of Lactobacillus plantarum (Hietala et al. 1979). The other organisms,

which are able to produce HICA are Clostridium difficile (Brooks et al. 1984), other

lactobacilli (Hummel et al. 1985, Bernard et al. 1994, Yvon & Rijnen 2001,

Chambellon et al. 2009), Peptostreptococcus anaerobicus (Hamid et al. 1997), and

Weissenella paramesenteroides (Ndagano et al. 2011). Some environmental

bacteria of Bacillus, Pseudomonas, Streptomyces species, and the Coryneform

group are also able to produce HICA by enantiospesific dehydrogenation of

different racemic aliphatic 2-hydroxyacids (Sawada et al. 1983).

HICA is formed by transamination of leucine to 2-ketoisocaproic acid (KICA),

which is followed by reduction of KICA to HICA (Fig. 1; Smit et al. 2004). Both

breakdown products are incorporated into normal biochemical pathways to energy

production in the citric acid cycle (Kanehisa et al. 2014). In yeast, these reactions

of the leucine metabolic pathway seem to be cytocolic reactions (Kohlaw 2003).

Aminotransferase enzymes (EC 2.6.1.42, EC 2.6.1.6) in conversion of leucine to

KICA have been described in a number of micro-organisms (Kanehisa et al. 2014).

HICA is thought to be the end product of KICA reduction (Hoffer et al. 1993, Yvon

& Rijnen 2001, Chambellon et al. 2009). Therefore, conversion by oxidation

reaction from HICA back to KICA is required for HICA elimination (Bossow &

Wandrey 2006). The reduction reaction is catalyzed by hydroxyisocaproic acid

dehydrogenase (Fig. 1; HicDH: EC 1.1.1.-), which is mainly detected in lactic acid

bacteria (Hummel et al. 1985, Yamakazi & Maeda 1986, Hummel & Kula 1989,

Kallwass 1992, Bernard et al. 1994, Niefind et al. 1994, Niefind et al. 1995,

20

Dengler et al. 1997, Yvon & Rijnen 2001, Broadbent et al. 2004, Smit et al. 2004,

2005, Chambellon et al. 2009).

Fig. 1. A schematic illustration of leucine degradation pathway.

Hydroxyisocaproic acid dehydrogenase

HicDH enzymes belong to a subgroup of lactate dehydrogenases (LDH) in the

family of 2-hydroxycarboxylate dehydrogenases (Dengler et al. 1997). They

catalyse NAD-dependent reversible stereospecific interconversion of D- or L-

isomers between 2-ketocarboxylic and 2-hydroxycarboxylic acids (Schütte et al.

1984, Smit et al. 2005). D-HicDH and D-LDH have a similar protein structure,

although their relationship is not clear fully (Yamakazi & Maeda 1986, Tamura et

al. 2002). Conversion of LDH to HicDH is also shown in lactobacilli (Tokuda et al.

2003). Another enzyme from the LDH subgroup is D-mandelate dehydrogenase,

which has been isolated from E. faecalis (Tamura et al. 2002). The enzymes in the

LDH subgroup are named after their main substrates, but crossreactions between

these enzymes and the substrates may occur (Yamakazi & Maeda 1986, Tamura et

al. 2002). The activity of HicDH enzyme differs between bacterial species and

isolates (Bachmann et al. 2009). The activity is regulated by panE gene.

Inactivation of this gene leads to reduction of HICA production in Lactococcus

lactis (Cadiñanos de et al. 2013). The D-HicDH coding gene is also detected in E.

21

coli (Lerch et al. 1989) and C. albicans (CaO19.2989 gene; Jones et al. 2004,

Kanehisa et al. 2014).

HICA in humans

The reactions of BCAAs and their derivatives take place mainly in muscles, liver,

brain, and kidneys (Hoffer et al. 1993, Suryawan et al. 1998, Holecek 2002). The

BCAA aminotranferases are both mitochondrial and cytosolic (Wu et al. 2004).

HICA is metabolised in an analogous manner in the leucine degradation pathway

in humans. It is isolated from human plasma (Dallmann et al. 2012), urine (Tanaka

et al. 1980), and amniotic fluid (Jacobs et al. 1984). Plasma concentration of HICA

(0.1–0.25 μmol/L) is normally 30–100 times lower than KICA, while it increases

during prolonged fasting and after exercise due to use of proteins as an energy

source. HicDH activity may also be bidirectional in humans. (Hoffer et al. 1993).

Defects in branched-chain keto acid dehydrogenase complex (EC 1.2.4.4) lead

to accumulation of several BCAA metabolites including HICA and odor of urine.

This is called maple syrup urine disease (MSUD). Unless diagnosed early in

newborns, it may cause extensive encephalopatic symptoms mainly because of

increased amounts of leucine and 2-keto acids. Oxidative stress is supposed to

participate in the neuropathology. (Mamer & Reimer 1992, Fontella et al. 2002,

Heldt et al. 2005, Frazier et al. 2014). Elevated HICA concentrations are also found

in diabetic ketoacidosis (Lieblich & Först 1984), in dihydrolipoyl dehydrogenase

deficiency (EC 1.8.1.4) (Haan et al. 1985), and in short bowel syndrome (Kuhara

et al. 1983). Deficiency of dihydrolipoyl dehydrogenase leads to atypical MSUD

and in short bowel syndrome an increased amount of lactobacilli in gut flora is also

seen (Haan et al. 1985). It is suggested that the D-enantiomer of HICA from

bacterial origin dominates in urine in short bowel syndrome and L-form from

human origin in MSUD (Spaapen et al. 1987).

2.1.2 Antimicrobial properties

Secondary metabolites of many lactic acid bacteria possess broad-spectrum

antimicrobial properties. Inter alia, Lactobacillus, Lactococcus, Streptococcus,

Leuconostoc, Pediococcus, Weissenella, and Enterococcus species belong to this

group of bacteria, which produce lactic acid as a major end product of carbohydrate

metabolism. Antimicrobial effects of microbial production of organic acids,

hydrogen peroxide, and bacteriocins have been widely examined (Olasupo 1996,

22

Ricke 2003, Todorov 2009). Antimicrobial compounds of phenyllactic acid, cyclic

dipeptides (Ström et al. 2002), peptides (Brogden et al. 2005, Hayes et al. 2006,

Gupta & Srivastava 2014), and 3-hydroxy fatty acids (Broberg et al. 2007) have

also been investigated. Hietala et al. (1979) also reported antimicrobial properties

of lactic acid and 2-hydroxypropionic acid. Mandelic acid or 2-hydroxy-2-

phenylacetic acid have been proposed for the treatment of urogenital infections

(Putten van 1979, Zaneveld et al. 2002). Antimicrobial peptides have recently also

been suggested for root canal medication (Lee et al. 2013, Freitas de Lima et al.

2015, Wang et al. 2015).

HICA has antibacterial activity against E. coli (Hietala et al. 1979), which is

the most general aerobic bacteria of human intestinal microflora and causes mainly

urogenital infections, enteric infections and enteritis. Antifungal activity of HICA

has been shown against Trichophyton (Hietala et al. 1979), Aspergillus and

Penicillium species (Broberg et al. 2007, Ndagano et al. 2011) and against C.

albicans biofilms (Nieminen et al. 2014a). C. albicans is the most common fungus

in human oral microflora (Ghannoum et al. 2010) and the most general

opportunistic pathogen in superficial and deep candidosis (Jones et al. 2004, Arana

et al. 2009). Aspergillus and Penicillium species are spore-forming environmental

moulds, which may cause spoilage of foods, allergic reactions in humans or even

invasive infections in immunocompromised patients (Lyratzopoulos et al. 2002,

Ndagano et al. 2011). In addition, A. fumigatus is the most common cause of

pulmonary aspergillosis. Trichophyton species are dermatophytic fungi (Nir-Paz et

al. 2003).

Interestingly, Hamid et al. (1997) showed that the amount of HICA production

by Peptostreptococcus anaerobicus is increased in the presence of metronidazole.

HICA is also one part of pleofungin molecules, which are new depsipeptide

antifungal antibiotics (Aoyagi et al. 2007). The amphipathic and hydrophobic

nature of the molecule may have a role in the antimicrobial mode of action of HICA

(Chen et al. 2007). The antimicrobial spectrum and potency of HICA for clinical

use as an antimicrobial agent has not been defined earlier.

2.1.3 Other uses and biocompatibility

Commensal bacteria, especially Lactobacillus species are widely studied and used

as probiotics for the treatment of unbalanced microflora in oral, intestinal,

genitourinal, and burn wound infections (Lombardo 2008, Barrons & Tassone 2008,

Peral et al. 2009, Hasslöf et al. 2010). Lactobacillus species are also frequently

23

utilized in the food industry as starter cultures for ripening foodstuffs by

fermentation and for prevention of spoiling. HICA is expressed in bovine whole

milk (Ehling & Reddy 2014), fermented dairy products, cheeses (Yvon & Rijnen

2001, Broadbent et al. 2004, Smit et al. 2005), and wines (Van Wyk et al. 1967).

In contrast, in Swiss cheese Propionibacterium freudenreichii did not show either

production of HICA or conversion of it to KICA (Thierry et al. 2002). The

overexpression of HicDH may lead to decrease in aroma or to delayed flavour

formation in cheese (Broadbent et al. 2004). As a non-nitrogen substitute in

nutrition, HICA increases weight and muscle mass in chicks and rats (Boebel &

Baker 1982) and it improves muscle recovery after immobilisation in rats (Lang et

al. 2013). In humans, HICA is used for speeding up muscle recovery after exercise

with a 1500 mg daily dose (Mero et al. 2010). In addition, HICA reduces C.

albicans biofilm-induced inflammatory responses in murine cells (Nieminen et al.

2014b).

The European Food Safety Authority (EFSA; 2012) has approved HICA as a

flavouring substance in nutrition. The maximimum survey-delivered daily intake

(MSDI) is 0.0012 μg/capita/day and theoretical maximum daily intake (mTAMDI)

is 3800 μg/capita/day. The dose of the threshold of concern is 1800 μg/capita/day.

Since HICA can be metabolised by human tissues and is found naturally in foods it

is well tolerated at least in the doses recommended by EFSA.

2.2 Antimicrobials

2.2.1 Biocides

Antiseptics, disinfectants, preservatives, and sterilants belong to the group of

topical antimicrobial agents called biocides. They have an important role in the

prevention of general out-patient infections and nosocomial infections. They are

also used for the treatment of superficial infections or for prevention of the growth

of foodborne pathogens. Topical antimicrobials are used superficially on living

tissues to prevent bacteremia and further generalised sepsis. In this context, they

are called antiseptics. On inanimate surfaces topical antimicrobials are used to

prevent infection and spreading of infection, whilst they are called disinfectants.

The general aim of topical antimicrobials is to prevent bacterial growth and kill

bacteria. In practice, subcidal concentrations or insufficient exposure time in

contact with target organisms may lead to inhibition of microbial growth to a lower

24

extent. Preservatives prevent deterioration of food and medical products. Sterilants

are used in medical devices for bacterial killing.

Antimicrobial mechanisms of different biocides are not well described in the

literature as the mode of action of different antibiotics and antifungals (Russell

2002, Sheldon 2005). Interaction between biocide and microorganism is needed for

antimicrobial activity. This may occur with one to three levels: in outer cellular

components, cytoplasmic membrane, or cytoplasmic constituents. (Maillard 2002).

Interaction with the cell surface allows invasion of chemical agents into

intracellular target components. Viability of the microbial cells may also be affected

by surface interaction alone. (McDonnell & Russell 1999). It is suggested that the

microbicidal effect and prevention of development of microbial resistance is based

on multiple targets of biocide on microbial cells at the same time (Sheldon 2005).

2.2.2 Antibiotics and antifungals

The main targets of systemic antimicrobials in bacterial cells are cell wall, protein,

folic acid, and nucleic acid biosynthesis. Penicillins, cephalosporins, and

glycopeptides belong to the group of bacterial cell-wall biosynthesis inhibitors.

Tetracyclines, aminoglycosides, and macrolides inhibit bacterial protein

biosynthesis. Sulfonamides and trimetoprime derivatives inhibit folic acid

synthesis. Peptide, polypeptide, and lipopeptide antibacterials inhibit cell-

membrane function or protein biosynthesis. Antifungal azoles belong to the group

of ergosterol-synthesis inhibitors and echinocandins to the fungal cell-wall

biosynthesis inhibitors. Polyenes inhibit cell-membrane function and nucleic acid

synthesis is inhibited by fluoropyrimidine. (Kanehisa et al. 2014). Resistance of

most common human pathogens to nearly every antibiotic and synthetic systemic

antimicrobials has been reported. Development of new treatment options for

infections will assist in tackling this worldwide problem. (World Health

Organization 2014).

2.2.3 Non-susceptibility to biocides

Permeability of microbial cell-wall structures and membranes is a principle

mechanism that dictates the sensitivity and resistance of microbial species to

antimicrobial agents and biocides, in general. Prokaryotic bacteria are simpler in

structure than eukaryotic organisms. Bacterial cell layers consist of phospholipids.

Gram-positive bacteria have a single cytoplasmic membrane and thick

25

peptidoglycan cell wall. Gram-negative bacteria possess a cytoplasmic membrane

and outer membrane with a thin peptidoglycan layer in the periplasmic space

(Maillard 2002). Bacteria also have fewer cell organelles than fungi and their

metabolism is simpler. The rigid cell wall structure of fungi includes glucan,

mannan, and chitin. The fungal cell membrane covers the nucleus of the cell and it

continues around the endoplasmic reticulum and cell organelles such as

mitochondria and ribosomes (Arana et al. 2009). The cell wall structure of

Aspergillus species is highly hydrophobic in comparison to that of bacteria and

yeasts. The ability of microbes to adhere, colonize, and invade into tissues; and to

produce capsules, endotoxins, and exotoxins, may increase virulence (Peterson

1996). Hyphal formation and filamentation of dimorphic fungal species is

associated with virulence and biofilm formation (Shapiro et al. 2011).

According to the current literature, non-susceptibility to biocides can be

divided into intrinsic and acquired mechanisms. An intrinsic mechanism is

regulated by the microbial innate genome. It may be expressed by impermeability

of membranes, biofilm formation, efflux systems, and enzymatic degradation.

Gram-positive bacteria are more susceptible to biocides than gram-negative

bacteria, mycobacteria, or spores of spore-forming organisms, due to a more

permeable cell wall. Hydrophilic porin channels in the outer membrane of gram-

negative bacteria prevent and regulate the transport of hydrophilic substances

(Denyer & Maillard 2002). Similarly, lipopolysaccharide (LPS) of the bacterial

outer membrane prevents permeability (McDonnell & Russell 1999). In some cases,

the same efflux pump systems may be responsible for antibiotic resistance and non-

susceptibility to biocides (Moken et al. 1997, McMurry et al. 1998, Chuanchuen et

al. 2001). In bacterial and fungal biofilms, non-susceptibility to biocides may

depend on altered growth rate, adherence of the biocide to biofilms, and

inactivation of enzymatic degradation of biocide (Gilbert & McBain 2003, Sheldon

2005, Soto 2013).

Acquired non-susceptibility may be a consequence of mutation or

overexpression of the target, plasmid-regulated efflux, or inactivation by enzymes

(McDonnell & Russell 1999, Soto 2013). Surface hydrophobicity, dictated by the

fatty acid and protein structure of the outer membrane, has an impact on acquired

non-susceptibility (Maillard 2002). In staphylococci, plasmid-mediated non-

susceptibility for cationic biocides, chlorhexidine digluconate, and quaternary

ammonium compounds, was observed (Russell 2000, Poole 2002). In addition,

multi-drug efflux-pump coding genes have been found in multidrug-resistant

plasmids of S. aureus isolates (Costa et al. 2013). The intrinsic and acquired

26

mechanisms of antifungal resistance are well described, but the fungal non-

susceptibility to biocides is not known precisely (Vandeputte et al. 2012).

Resistance of fungal biofilms has been reported for nearly all presently-used

antifungals, except for echinocandins and lipid formulations of amphotericin B

(Chandra et al. 2005, Perlin et al. 2015). Therefore, biocides are one primary part

in the control of fungal infections. It is well recognised that the same strategies of

adaptation to toxicity of antibiotics and biocides are used by microorganisms

(Sheldon 2005). Bacteria expressing the genes encoding antibiotic resistance

mechanisms have been detected also in the oral environment from saliva, supra-

and subgingival plaque, tongue, and root canals (Moraes et al. 2015), but it does

not nesessarily mean non-susceptibility of these isolates to topical antiseptics or

disinfectants.

Disinfection is a function of the concentration of an agent and time of exposure

in given conditions. The presence of organic load, interactions with other chemicals,

formulation methods, temperature, dilution, and test method may have an impact

on their antimicrobial activity. Increasing contact time and different substitutes may

enhance the activity of biocides. (McDonnell & Russell 1999). The role of

antimicrobial agents in root canal treatment is to improve mechanical eradication

by reducing or killing of microbial cells as irrigants or interappointment

medications. Topical antibiotics have provided no benefit over Ca(OH)2 intracanal

treatment (Longman et al. 2000). Potential resistance and narrower spectrum of

antibiotics makes them unwarranted instead of biocides with broad-spectrum

activity (Zehnder 2006). The most generally used antimicrobials, agents with

clinical antimicrobial relevance for disinfection and interappointment medication

in root canal treatments, and agents chemically in close relation to them are

discussed below.

2.3 Endodontic disinfectants

2.3.1 Alcohols

The most widely-used antimicrobial alcohols are ethyl alcohol, isopropyl alcohol,

and n-propanol. They have broad-spectrum activity against bacteria and fungi, but

they are not sporicidal. Ethanol is mainly used for antisepsis, and isopropanol for

disinfection and preservation. (Block 1991) Optimal antimicrobial concentration is

60–90%. Isopropyl alcohol is more lipophilic than ethyl alcohol. Membrane

27

damage and protein denaturation are their suspected mode of actions that,

especially in water solutions, lead to interferences in metabolism and to lysis of

microbial cells. Denaturation of dehydrogenases of E. coli is reported to support

that suggestion. (McDonnell & Russell 1999). Alcohols are used as a diluent in

commercial disinfectant products in endodontics.

2.3.2 Biguanides

Biguanides are a large group of chemical agents consisting of biguanide molecule

(C2H7N5). Some have hypoglycemic, antimicrobial (Weinberg 1968), or even

antineoplastic activity (Pollak 2012). Chlorhexidine is a well-known member of

this group. It is a positively-charged synthetic bisbiguanide, in which two biguanide

groups and 4-chlorophenyl rings are attached with a central hexamethylene chain

(Greenstein et al. 1986). This strong base is soluble and stable in water as a

gluconate, acetate, or hydrochloride salt. Their activity is prevented by anionic and

non-ionic surface-active agents and reduced by phospholipids and organic matter.

(Russell & Day 1993).

In general, a low concentration of chlorhexidine increases the cell-membrane

permeability of microbes and may cause leakage of agents with low molecular

weight out of the cell, especially potassium and phosphorous. Higher concentration

of chlorhexidine leads to cell death by precipitation of the cytoplasmic contents.

(Hugo & Longworth 1964). Chlorhexidine has rapid broad-spectrum activity

against gram-positive and gram-negative bacteria (Mohammadi & Abbott 2009,

Haapasalo et al. 2010, Gomes et al. 2013, Kulik et al. 2015). It is also highly active

against Candida spp. (Salim et al. 2013). As a lipophilic and hydrophobic agent,

chlorhexidine targets microbial cell-membrane phospholipids and

lipopolysaccharides (Athannassiadis et al. 2007, Cheung et al. 2012). Microbial

cell-membrane phosphate groups are negatively charged and the chlorhexidine

molecule interacts with them (Hugo & Longworth 1964). Rapid permabilisation of

the cell membrane is followed by its disruption (Athanassiadis et al. 2007). The

inner membrane of gram-negative bacteria is the main target for further interactions

in cytosol (Russell & Furr 1987, McDonnell & Russell 1999). Fast intake into the

cell is reported in E. coli (Hugo & Longworth 1966, Fitzgerald et al. 1989, Cheung

et al. 2012), S. aureus (Hugo & Longworth 1966), and Pseudomonas aerunginosa

cells (Fitzgerald et al. 1989). Hiom et al. (1992) showed it also enters yeast cells

of C. albicans, C. glabrata, and Saccharomyces cerevisiae. Cheung and collegues

(2012) showed that potential sites of chlorhexidine activity were in different parts

28

on the cell wall of gram-positive and gram-negative bacteria. They also found up-

regulation and down-regulation of several proteins of Bacillus subtilis and E. coli,

but these proteins were totally different between these species. Chlorhexidine also

attenuates inflammatory responses caused by lipoteichoic acid (LTA) of E. faecalis

in murine macrophages, in which tumor necrosis factor-α receptor activity and toll-

like receptor-2 stimulation are reduced (Lee et al. 2009). Development of microbial

resistance to chlorhexidine during long-time use might be more evident than in

short-time use (Kulik et al. 2015)

In endodontics, chlorhexidine is used mainly as a root canal irrigant, but it is

recommended in the treatment of Ca(OH)2-resistant strains as an interappointment

medication. The activity of chlorhexidine is pH-dependent and can be decreased

with organic matter (Russell & Day 1993, Haapasalo et al. 2000, Oliveira de et al.

2010). Chlorhexidine forms a precipitate with NaOCl or EDTA, which may have a

harmful influence on the activity of antimicrobials and on the dissolving capacity

of organic and inorganic tissues of the two latter ones (Rossi-Fedele et al. 2012).

Chlorhexidine has no tissue-dissolution capacity (Naenni et al. 2004, Okino et al.

2004). The substantivity of chlorhexidine to dentine may be one useful property of

chlorhexidine in comparison to present root canal medicaments. It can be adsorbed

into and released from hydroxyapatite of dentine. (Carrilho et al. 2010). This may

lengthen the antimicrobial activity (Souza de et al. 2011) and reduce bacterial

colonisation in dentine tubules (Basrani et al. 2003).

In clinical medicine, allergic reactions such as contact dermatitis, immediate

type of hypersensitivity, and even anaphylaxis, caused by chlorhexidine have been

diagnosed (Calogiuri et al. 2013, Nakonechna et al. 2014). Increased risk for

contact dermatitis may be a dose-dependent consequence of chlorhexidine use in

hand disinfectants (Nagendran et al. 2009). The risk for anaphylaxis could be

higher with mucosal application (Okano et al. 1989). Allergic reactions may be

related to the exposure of chlorhexidine in former surgery operations or in other

invasive procedures (Garvey et al. 2001, Aalto-Korte & Mäkinen-Kiljunen 2006).

Even though, the probability of strict contact of disinfectants to living tissues and

induction of new allergic responses in root canal treatment is low; patients with a

history of mild reactions to chlorhexidine should be recognized to prevent severe

reactions later.

29

2.3.3 Halogen-releasing agents

Sodium hypochloride (NaOCl) is a bactericidal and fungicidal agent. NaOCl

dissolves to hypochlorite ions and hypochlorous acid in water. Temperature and pH

have an impact on its antimicrobial activity. Undissolved hypochlorous acid is more

active than dissolved hypochlorite. Hypochlorous acid dominates nearby neutral

and slightly acidic conditions. (McKenna & Davies 1988, Barrette et al. 1989). The

main mode of action of NaOCl is protein denaturation. Chlorine-releasing agents

may cause chlorinated derivatives of nucleotide bases in bacterial DNA (Dennis et

al. 1979). Disruption in membrane-associated activity by hypochlorous acid is

reported, especially in oxidative phosphorylation (Barrette et al. 1989). DNA

synthesis is disrupted more than protein synthesis or bacterial membrane

(McKenna & Davies 1988).

Because of its antimicrobial activity, tissue-dissolution capacity, and activity

against bacterial biofilms it is considered to be the most important agent in root

canal irrigation (Gutiérrez et al. 1990, Haapasalo et al. 2010, 2014). NaOCl rapidly

loses its activity due to its reactivity. Therefore, thorough application is needed and

it cannot be used as an interappointment medication. Inactivation of NaOCl has

also been shown by dentine (Haapasalo et al. 2000) and by serum albumin, which

may be present in inflammatory exudate (Pappen et al. 2010). High concentrations

of NaOCl with long application time may weaken the strength of dentine (Sim et

al. 2001, Marending et al. 2007). It is noteworthy that tissue irritation and cell

toxicity of NaOCl is a major caution, which should be paid attention to in clinical

use (Spångberg et al. 1973, Hülsmann & Hahn 2000).

Iodine is less reactive than chlorine, but its broad-spectrum antimicrobial

action occurs rapidly, too. After invasion through cell membrane, iodine attacks

proteins such as cysteine and methionine, as well as nucleotides and fatty acids

resulting in the death of microbial cells. Iodine molecule (I2) plays the main role in

antimicrobial activity in aqueous solutions (Gottardi 1991). Iodine potassium

iodine (IPI) has no capacity to dissolve tissues. In a case of persistent root canal

infection, a final rinsing with IPI may extend the antimicrobial effect of

conventional irrigation regimen (Molander et al. 1999, Kvist et al. 2004, Haapasalo

et al. 2010). In addition, there are some promising results of its ability to disinfect

dentine tubules (Safavi et al. 1990), which may also increase the activity of

Ca(OH)2 as an intracanal dressing (Cwikla et al. 2005). Toxic effects caused by

overdose oral administration of iodine compounds and reactions to iodinated

contrast agents in imaging techniques are better described in the literature than

30

potential allergies and staining effects of iodine compounds in topical use (Backer

& Hollowell 2000, Pasternak & Williamson 2012).

2.3.4 Hydroxyl ion-releasing agents

Hydrogen peroxide is a renowned biocide with a rapid release of hydroxyl ions,

which is in wide use in antisepsis and disinfection. It is a fast-acting, broad-

spectrum, antimicrobial agent for bacteria and yeasts, which denatures proteins,

lipids, and DNA. Free hydroxyl radicals oxidize thiol groups of proteins and

enzymes. DNA strands are broken by hydrogen peroxide. (McDonnell & Russell

1999). Hydrogen peroxide decreases the microhardness of dentine (Lewinstein et

al. 1994). The antimicrobial activity of hydrogen peroxide has not been very good

in comparison to more commonly used irrigants in endodontics (Viljaykumar et al.

2010).

Ca(OH)2 paste is well-known due to its slow release of hydroxyl ions. This

chemical property is utilized in dentistry and environmental chemistry. The uses in

dentistry are related to its antimicrobial activity and its ability to stimulate

mineralisation (Foreman & Barnes 1990). Ca(OH)2 is slightly soluble in water. The

physical constant of hydrolysed ions in aqueous saturated solution of Ca(OH)2 is

1.65 mg/mL at 20ºC and at body temperature this is a little lower (1.45 mg/mL;

Dean 1973). There is also a marginal decrease in pH, if the temperature is increased

(Bates et al. 1956). The antimicrobial activity of Ca(OH)2 is attributed to high

alkalinity (pH 12.5) of saturated solution. Undersaturation of solution leads to

reduction of hydroxyl-ion concentration and pH. The targets of Ca(OH)2 are in the

bacterial cytoplasmic membrane, protein denaturation, and possibly in DNA

(Halliwell 1987, Imlay & Linn 1988, Siqueira & Lopes 1999, Estrela & Holland

2003). It has activity on the outer membrane antigen, bacterial lipopolysaccharide

(LPS) of gram-negative bacteria (Buck et al. 2001, Silva et al. 2002), and on E.

faecalis lipoteichoic acid (LTA). Deacylation of LTA leads to attenuation of

inflammatory reaction in apical periodontitis. (Baik et al. 2011).

Byström et al. (1985) showed that Ca(OH)2 was more active in situ than

camphorated phenol (CP) or camphorated paramonochlorophenol (CMCP) during

a month-long incubation. Sjögren et al. (1991) showed its antimicrobial effect

during seven-days of medication in vivo. Nowadays, Ca(OH)2 paste is the most

widely used interappointment medication in endodontics (Law & Messer 2004).

Ca(OH)2 has the ability to dissolve tissues (Hasselgren et al. 1988), which enhances

tissue dissolution effect of NaOCl after appointment (Türkün & Cengiz 1997).

31

However, there is conflicting evidence of its antimicrobial activity in root canal

treatments both in vitro and in vivo (Ørstavik et al. 1991, Reit et al. 1999, Peters et

al. 2002, Vera et al. 2012, Kim & Kim 2014, 2015). Especially, its antimicrobial

activity is weaker against E. faecalis and C. albicans than other microorganisms

tested (Waltimo et al. 1999, Portenier et al. 2003, Delgado et al. 2010, 2013,

Siqueira & Rôças 2014). Alkaline-resistant strains are found mostly in post-

treatment infections, but also sometimes in primary infections (Lew et al. 2015).

According to present knowledge, antibacterial activity of Ca(OH)2 against E.

faecalis does not increase by mixing it with chlorhexidine (Saatchi et al. 2014).

Ca(OH)2 may cause tissue irritation. In a safety study, Barbosa et al. (2009)

demonstrated the toxicity of Ca(OH)2 (500 mg/mL) on mouse fibroblasts. Despite

its weaknesses, Ca(OH)2 remains a first line choice for interappointment intracanal

dressing, if single-visit root canal treatment is not indicated (Law & Messer 2004,

Sathorn et al. 2007).

2.4 Root canal infections

Apical periodontitis (AP) seems to be more general in prevalence than severe forms

of periodontitis (Pilot & Miyazaki 1991, Suominen-Taipale et al. 2004). In Nordic

countries, the prevalence of AP in adults is over 40% (Kirkevang et al. 2012,

Haikola et al. 2013, Huumonen & Ørstavik 2013). In the Finnish adult population,

root canal treatment was carried out in 7% of all teeth and it was more general in

women than in men (Huumonen et al. 2012). In a ten-year follow-up study of root-

filled teeth, 13% were extracted, 12% were revised, and 42% had AP (Kirkevang

et al. 2014). The infection after dental trauma is a common reason for the need of

root canal treatment in children (Humphrey et al. 2003, Bakland 2012). General

changes in population age-distribution may lead to increase in prevalence of

periapical lesions of elderly dentate people (Ainamo et al. 1994, Haikola et al.

2013). Immunocompromised patients have a higher risk for generalized infections

also from dental origin. Two thirds of odontogenic maxillofacial abscesses

requiring hospital care are from pulpal origin. (Grönholm et al. 2013).

2.4.1 Etiology and pathology

AP is a root canal microbial infection-induced inflammatory reaction in the

periapical tissues (Kakehashi et al. 1965, Sundqvist 1976). Already in 1894, Miller

showed the presence of bacteria in necrotic pulps. However, much later Kakehashi

32

et al. (1965) demonstrated that microbial organisms are causative factors for AP.

Other studies have later confirmed that necrotic pulp without microbial infection

does not induce periapical inflammation (Sundqvist 1976, Möller et al. 1981).

According to present knowledge, microorganisms are commonly arranged as

biofilms in human root canals during the disease (Nair 2004, Svensäter &

Bergenholz 2004, Ricucci & Siqueira 2010, Siqueira & Rôças 2014). AP is an

inflammation reaction induced by intraradicular infection, but occasionally the

infection may spread to extraradicular tissues. Sunde and collegues (2003) showed

bacteria in over 50% of periapical lesions of asymptomatic root-filled teeth that

required apical surgery by fluoresence in situ hybridization. Dental caries, fractures,

and leakages in coronal or root fillings are the most common routes of infection,

from which any oral organism may potentially contaminate pulpal tissues.

Additional root canals, malformations in dental and dentine structures, active root

resorption and occlusal trauma increase the risk for pulpal infection. Microbial

colonization from root surface via cemento-deficient areas and hematogenous

routes have also been suggested (Stashenko et al. 1998, Nair et al. 2004).

Microbes

Bacteria are considered to be the main causative organisms of AP, even though

viruses, archaea, and fungi are also found (Siqueira & Rôças 2014). About one third

of 500 bacterial species detected in endodontic infections are culturable. One

quarter of them have been detected with both conventional culture methods and

with molecular biology methods such as polymerase chain reaction (PCR),

checkerboard, broad-range PCR, cloning, sequencing, terminal-restriction

fragment length polymorphism, reverse-capture checkerboard, real-time PCR, or

microarray analyses. (Siqueira & Rôças 2009). Next-generation DNA sequencing

(NGS) techniques, such as pyrosequencing, have already shown that the divergence

of microbial species in endodontic infections is far wider than was previously

thought and it will broaden our knowledge further in the future (Siqueira & Rôças

2014). Both culture and molecular methods are needed for comprehensive analyses.

The organisms distinguished by culture methods are living organisms, and with

molecular methods living organisms, and lifeless genomes can be detected.

Growth conditions, especially availability of nutrients, oxygen, and pH in root

canal affect the selection of the colonising microbial species and their position in

root canal. Therefore, obligately anaerobic species dominate in primary infection

and the flora is modified to facultatively aerobic species in secondary infection or

33

post-treatment disease. (Sundqvist & Fidgor 2003, Peciuliene et al. 2008).

Nutritional environment, structure of dentine and bacterial adhesion may influence

bacterial invasion to dentinal tubules. Gram-positive streptococcal, enterococcal,

and Actinomyces species are able to invade into human root dentinal tubules, while

gram-negative species are less frequently recovered (Love & Jenkinson 2002).

Dentine tubules invasion of C. albicans is mainly superficial. Some single cells

were reported throughout the dentine specimens with approximately 2 mm of

thickness ex vivo (Waltimo et al. 2000). In caries lesions, this hardly exists in vivo

(Maijala et al. 2007).

Host response

Periapical inflammation induced by microbial biofilms in the root canal resembles

the immunological reactions in periodontal infection. Initial infiltration of

immunocompetent cells and alveolar bone destruction may begin before pulpal

necrosis (Kawashima et al. 1996). Dental pulp is prepared for antigenic challenges

via dentine tubules. Innate immune reactions mediate the initial antigenic

challenges, which include inflammatory-cell infiltration (neutrophils, macrophages,

and dendritic cells), and synthesis of inflammatory mediators. Thereafter, mainly

T- and B-lymphocyte-mediated adaptive immune reactions are initiated for more

specific responses to certain antigens. Inflammatory reactions with restricted blood

circulation and decrease in environmental compliance lead to irreversible

destruction of pulp tissue. (Kawashima & Suda 2008). Increased levels of cytokines

have been detected from inflammatory exudate in periapical lesions (Matsuo et al.

1994) and from fibroblasts in inflammed pulp (Barkhordar et al. 2002, Silva et al.

2009). Bacterial lipopolysaccharide (LPS) induces inflammatory responses, which

lead to function of polymorphonuclear (PMN) cells (Hou et al. 2000, Ubagai et al.

2015). In primary infections, the microflora is potentially composed of anaerobic

gram-negative rods and LPS-induced reactions may be expressed. Interestingly,

Smith et al. (2012) showed that extracellular matrix extracts of dentine and pulp

have antimicrobial properties.

Remodelling of bone is a balance stage between resorption caused by

osteoclasts and formation by osteoblasts. In pathologic states, bone resorption is

increased in comparison to bone formation. Regulation mechanisms of this

phenomen are multidimensional. Briefly, bone destruction is mediated and caused

by osteoclasts, which are macrophage-derived multinuclear cells. Osteoblastic

cytokines, such as macrophage-colony stimulating factor and receptor activator of

34

NF-κB ligand (RANKL) increase osteoclast formation from osteoclast precursor

cells. T- and B-lymphocytes also regulate osteoclast formation by expressing

RANKL. Osteoclasts are able to secrete hydrochloric acid and proteases, such as

cathepsin K, to dissolve matrix and mineral constituents of bone, which occurs

beneath a ruffled part of the osteoclastic basal membrane. (Teitelbaum 2000). Root

cementum and dentine may be resorbed by odontoclasts, which have histochemical

and structural similarities with osteoclasts (Sahara et al. 1994).

Biofilms

Microbes in biofilms are far more resistant to antimicrobials and host response

reactions in comparison to planktonic cells (Ceri et al. 1999, Hajo et al. 2009, Lang

et al. 2012, Wang et al. 2012). Microbial adherence to a surface is a precondition

for biofilm formation. In a mature biofilm, several layers of microbial cells are

surrounded by extracellular polymeric substance (EPS), which contains mainly

extracellular polysaccharides and proteins. It takes part in aggregation and adhesion

of the cells. EPS plays a role in microbial biofilm formation, in protection of

microbial cells from environmental impulses, and it prevents diffusion of

antimicrobials. (Vu et al. 2009). In dental biofilms, growth-dependent killing may

be also reduced, because of slower microbial growth in them. In addition, drug-

inactivating enzymes may increase microbial resistance in biofilms. (Hajo et al.

2009). Resistance mechanisms in biofilms may also be attributed to nutritional

conditions and adaptation to resistant phenotype (Kishen et al. 2010).

Stojicic et al. (2013) showed that resistance of microbial cells to disinfectants

increases in experimental multispecies biofilms on collagen-coated hydroxyapatite

disks with time. Intraradicular biofilm formation was reported in 74–80% of

untreated and treated teeth with AP (Ricucci & Siqueira 2010). Chávez de Paz

(2007) showed that species of bacteria usually living in acidic environments such

as Streptococcus spp., Lactobacillus spp., and F. nucleatum were able to adapt and

stay viable in up to pH 10.5 biofilms for 4 h. The biofilm community is well

protected from the defence system of the host (Lang et al. 2012). Especially,

biofilms may be detected on root canal walls, whereas some microbial cells may

disperse into dentinal tubules.

35

2.4.2 Microbial species

In primary endodontic infections

In primary infections, commonly isolated organisms are anaerobic gram-negative

rods (Fusobacterium nucleatum, Cambylobacter rectus, Tannerella forsythia,

Prevotella and Porphyromonas spp.), and spirochetes (Treponema spp.), anaerobic

and facultative gram-positive cocci and rods (Peptostreptococcus spp.,

Eubacterium spp., Propionibacterium spp., Actinomyces spp., streptococci,

lactobacilli) (Sundqvist 1994, Sousa de et al. 2003, Gomes et al. 2004, Rôças &

Siqueira 2008, Sakamoto et al. 2009, Siqueira & Rôças 2014). Bacterial species

altogether from nine different phyla (Actinobacteria, Bacteroidetes, Firmicutes,

Fusobacteria, Proteobacteria, Spirochetes, Synergistetes, SR1, and TM7) have

been detected by culture or molecular methods. Bacterial species from nine other

phyla have been detected by pyrosequencing techniques, but they may be rare

species. There is clearly more divergence in the microflora of symptomatic versus

asymptomatic infections, which may be related to higher virulence and

pathogenicity (Santos et al. 2011, Siqueira & Rôças 2014).

In post-treatment endodontic infections

Facultatively anaerobic gram-positive cocci and rods dominate the microflora in

post-treatment disease such as Streptococcus, Enterococcus, Peptostreptococcus,

and Actinomyces species. The proportion of Lactobacillus species and anaerobic

gram-negative rods is lower than in primary infection. In addition, enteric rods have

often been isolated from root canals in post-treatment AP. (Molander et al. 1998,

Peciuliene et al. 2001, 2008, Chávez de Paz et al. 2003, 2004, Pinheiro et al. 2003,

Rôças & Siqueira 2008, Siqueira & Rôças 2014). Fungal species have also been

relatively often (in 5–20% of cases) isolated from AP, especially in persisting

infections. The most commonly isolated species is the dimorphic fungus C.

albicans. C. glabrata, C. guilliermondii, C. tropicalis, and C. krusei are isolated to

a lesser extent (Waltimo et al. 1997, 2001, 2003, Sinha et al. 2013, Kumar et al.

2015).

36

Enterococcus faecalis

E. faecalis is widely used as a test organism in endodontic studies during the last

two decades. A PubMed search (October 15th, 2015) yielded 535 studies with the

terms “E. faecalis and root canal”. The extent of research into this topic is based on

the fact that E. faecalis has been the most-often detected organism from post-

treatment infections (Portenier et al. 2003, Stuart et al. 2006, Zehnder &

Guggenheim 2009, Haapasalo et al. 2011, Lew et al. 2015, Zhang et al. 2015).

Some molecular method studies support the major prevalence of E. faecalis in

persisting AP (Rôças et al. 2004, Sedgley et al. 2006), while some studies do not

(Rolph et al. 2001).

E. faecalis is a gram-positive facultatively aerobic cocci. It is a saprophytic

commensal organism (Toledo-Arana et al. 2001), which belongs to the normal

intestinal flora and may also cause endocarditis, urinary tract, and skin wound

infections. E. faecalis is rarely found in the oral cavity but may originate from food

items (Razavi et al. 2007). It has intrinsic resistance to antibiotics such as β-lactams

and clindamycin: and may have acquired resistance to several antibiotics such as

tetracyclines, macrolides, glycopeptides, chloramphelicol, and aminoglycosides

(Portenier et al. 2003, Moraes et al. 2015). E. faecalis is able to invade into human

dentinal tubules (Safavi et al. 1990, Love & Jenkinson 2002) and is highly resistant

to alkaline stress (Flauhaut et al. 1997, Weckwerth et al. 2013). The ability of E.

faecalis to adhere to dentine is reported to improve the resistance to Ca(OH)2

(Brändle et al. 2008). Proton pump function is necessary for the survival of E.

faecalis in high alkalinity (Evans et al. 2002).

Especially in post-treatment disease, LTA of E. faecalis may increase

inflammatory responses (Lee et al. 2009, Baik et al. 2011). Differentiation of

osteoclasts may be reduced by E. faecalis, while macrophages sustain their capacity

to phagocytose and to induce secretion of proinflammatory cytokine and

chemokine (Park et al. 2015). However, E. faecalis may be present in the infected

root canals also without visual AP (Kaufman et al. 2005). In the experimental

conditions, Ozdemir et al. (2010) showed biofilm formation of E. faecalis in the

root canals of human teeth after 24 h.

Microbial species in extraradicular infections

Bacterial Actinomyces species and Propionibacterium propionicum are causative

organisms of independent extraradicular actinomycosis, but its role in endodontic

37

failures is controversial (Nair 2004, Siqueira & Roças 2014). Fungal Aspergillus

species are common colonisers of the upper and lower airways and they belong to

group of the most important pathogens in fungal infections together with yeasts.

They are spore forming and dimorphic moulds. Filamentation of A. fumigatus is

associated with pathogenesis. Systemic infections typically arise from colonisation.

Most Aspergillus infections are related to immunodeficiency. (Kousha et al. 2011).

The role of dental root-canal fillings and the over-fillings have been discussed in

connection with Aspergillus sinusitis (Legent et al. 1989). Surgical treatment is

needed in extraradicular dental infections.

2.4.3 Treatment

Root canal treatment is the treatment of choice in irreversible pulpitis, pulpal

necrosis, or AP. The overall aim of root canal treatment is to eradicate root canal

infection and to prevent or to heal AP. Three different parts of the treatment

protocol affects the prognosis of the disease: mechanical preparation, chemical

disinfection, and prevention of infection reactivation. This work is focused on the

second one. Destruction of biofilms by mechanical preparation is a cornerstone of

the treatment of biofilm infections including endodontic infections (Byström &

Sundqvist 1981, Dalton et al. 1998). Mechanical preparation includes hand and

rotary instrumentation and hydrodynamic effects during irrigation. Peters et al.

(2001) showed that even 35% of root canal system may be left uninstrumented after

preparation with hand or rotary instrument. Therefore, chemical agents are needed

for improvement of antimicrobial effect by irrigation and interappointment

medication.

The aim of root canal medication is to kill bacteria that either grow

planktonically or are arranged in biofilms. Subcidal concentration of an

antimicrobial agent in contact with microbes or insufficient exposure time can lead

to bacteriostatic effect, which may assist in infection control in favourable

conditions, too. NaOCl is the most active root canal irrigant against microbial

biofilms: the higher the concentration or the longer the contact time, the better

result is achieved (Zehnder 2006, Haapasalo & Shen 2012). Ever since Byström

and collegues (1985) showed better activity of Ca(OH)2 in comparison to CP or

CMCP, and Safavi et al. (1985) and Sjögren et al. (1991) showed its antimicrobial

activity in vivo Ca(OH)2 has become the most generally used interappointment

medication in endodontics (Law & Messer 2004). One explanation for the superior

activity of Ca(OH)2 may be that in the group of Ca(OH)2-treated teeth, Byström et

38

al. (1985) used 5% NaOCl in irrigation of nearly half of the subjects (n=15 of 35).

On the contrary, all subjects of the group of CMCP or CP-treated teeth were

irrigated only with 0.5% of NaOCl. Sjögren et al. (1991) used 0.5% NaOCl for all

subjects and they confirmed these results by 18 subjects with Ca(OH)2 medication

for seven days. Nine of them were bacteria free after the irrigation before

interappointment medication and all of them were disinfected after the medication.

It is noteworthy that the results of the clinical microbiological studies show the

combined effect of disinfection and interappointment medication, although these

are not clearly reported separately. Safavi et al. (1985) observed the effect of

interappointment medication after disinfection. All initial samples, which were

taken after irrigation from root canals were free of bacterial growth, but after seven

days of medication with Ca(OH)2, 77% of the subjects were bacterial free. The

same was evident for 66% of teeth with IPI intracanal medication. Despite

conflicting evidence for antimicrobial activity of Ca(OH)2 in root canal treatments

both in vitro (Ørstavik et al. 1991, Reit et al. 1999, Kim & Kim 2014) and in vivo

(Table 1), new approaches have not displaced it. The results of the clinical

outcomes analysed by molecular methods (Souza de et al. 2005, Siqueira et al.

2007, Rôças et al. 2011) and histobacteriological methods (Vera et al. 2012) are in

line with studies analysed by conventional culture methods shown in the underlying

comparison.

E. faecalis, which was described in the previous section of this thesis, and C.

albicans are especially non-susceptible to Ca(OH)2 (Waltimo et al. 1999, Portenier

et al. 2003, Delgado et al. 2013, Siqueira & Rôças 2014). Alkaline-resistant strains

are found mostly in post-treatment infections, but also sometimes in primary

infections (Lew et al. 2015). In addition, interactions with dentine, necrotic soft

tissue, or serum albumin may lead to inactivation of root canal disinfectants

(Haapasalo et al. 2000, 2007, Portenier et al. 2001, 2002, 2006, Oliveira de et al.

2010). More active irrigation and disinfection with chlorhexidine, NaOCl, and IPI,

or short chlorhexidine interappointment medication with broader spectrum activity

against Ca(OH)2-tolerant species are recommended in the treatment of these

infections (Waltimo et al. 1999, Haapasalo & Shen 2012). However, their potential

tissue toxicity or allergic reactions cannot be disgarded (Aalto-Korte & Mäkinen-

Kiljunen 2006, Pasternak & Williamson 2012, Guleri et al. 2012, Gomes et al.

2013), and the evidence of their clinical efficacy is lacking.

39

Table 1. Antimicrobial activity of disinfective irrigation and Ca(OH)2 medication detected

after 7 days up to 2 months in clinical conditions. A specific detection time was not

described in one study.

Year Author Analyses N N/group 7d 14d 1–2mon ND

1985 Byström et al. Culture 65 30–35 ++

1985 Safavi et al. Culture 1030 173–517 +

1991 Sjögren et al. Culture 30 12–18 ++

1997 Barbosa et al. Culture 120 36–45 +

1999 Molander et al. Culture 50 22 +

2000 Shuping et al. Culture 47 5–26 + (7–200d)

2002 Peters et al. Culture 43 21 +

2004 Tang et al. Molecular 31 6–25 -

2005 Zerella et al. Culture 40 20 + (7–10d)

2005 Souza de et al. Molecular 12 12 +

2006 Chu et al. Culture 88 12–19 +

2007 Manzur et al. Culture 33 11 +

2007 Vianna et al. Culture 24 8 -

2007 Siqueira et al. Molecular 11 11 +

2011 Rôças et al. Molecular 24 12 +

2012 Vera et al. Histological 13 6–7 +

2013 Sinha et al. Culture 90 30 +

++: good activity (90% of the subjects free of bacterial growth), +: limited activity (50–90%), -: no activity

(<50%), ND: not described,

Finally, at the end of and after root canal treatment, reactivation of infection is

mechanically prevented by tight root canal filling and coronal filling. The pathways

between the root canal and periapical tissues, and to the oral cavity should be sealed.

Nutrition of potentially present microbial cells has to be prevented.

Conventionally-used gutta-percha filling material lacks antimicrobial activity.

Some epoxy resin-based, polymethacrylate resin-based and calcium silicate-based

sealers may have antimicrobial properties in some experimental conditions (Ustun

et al. 2013), but the results are controversial (Heyder et al. 2013). In addition to

good sealing ability, mineral trioxide aggregate (MTA), which includes calcium,

silica, and bismuth, has antimicrobial activity (Estrela et al. 2000, Parirokh &

Torabinejad 2010).

40

Single-visit treatment

The strategy of one-visit treatment is based on the fact that mechanical preparation

and disinfection during irrigation at the appointment is sufficient and further

operation would not improve the quality of root canal treatment. In non-infected

diseases, such as in pulpitis or sterile pulpal necrosis, this is accurate (Trope &

Bergenholtz 2002), but in root canal infections the evidence is lacking. Although,

it has been shown, that the elimination of microbes from the root canal system in

multiple-visit treatment with Ca(OH)2 is not significantly better than single-visit

treatment ex vivo (Vivacqua-Gomes et al. 2005) or in vivo (Weiger et al. 2000,

Shuping et al. 2000, Kvist et al. 2004, Molander et al. 2007, Penesis et al. 2008,

Paredes-Vieyra & Enriquez 2012, Vera et al. 2012). However, Byström &

Sundqvist (1983) showed before the first clinical studies of Ca(OH)2 that with 0.5%

NaOCl irrigation, and with saline as an interappointment root-canal dressing,

bacterial growth was still detected in 50% of the root canals at second appointment.

Sjögren and collegues (1987) also confirmed this after chemo-mechanical

preparation. Vera et al. (2012) showed lower amounts of bacteria in different parts

of root canal system in histological analyses, including dentine tubules after

interappointment medication versus single-visit treatment. Therefore,

interappointment medication appears to be useful for the reduction of bacterial

growth in infected root canal system.

Prognosis of the disease

Population studies have shown successful healing in 60–74% of the treatment of

AP and the studies performed in universities have revealed around 90%. (Ray &

Trope 1995, Trope & Bergenholtz 2002). Surrounding deep periodontal pockets

(Ørstavik et al. 2004) and large lesion sizes (Chugal et al. 2001) are preconditions

that may lead to poorer clinical outcomes of root canal treatment. In a long run, the

best treatment outcomes in radiological analyses are associated with good quality

of both root canal filling and coronal restoration (Gillen et al. 2011, Song et al.

2014, Kirkevang et al. 2014). According to the literature, major associations with

failure in root canal treatment is because of microbial factors (Molander et al. 1998,

Waltimo et al. 2005, Peciuliene et al. 2008, Haapasalo et al. 2011, Siqueira & Rôças

2014), whereas other factors, such as true cysts and cholesterol crystals may cause

the disease far more rarely (Nair 2004). Insufficient microbial elimination from the

root canal system may lead to difficult-to-treat persisting infections, in which

41

enterococci: mainly E. faecalis, and enterobacteriae and C. albicans are often found

(Sen et al. 1995, Peciuliene et al. 2001, 2008, Waltimo et al. 2003, Portenier et al.

2003, Kumar et al. 2015). Despite the promising results of the activity of

chlorhexidine and sodium hypochlorite in vitro the ability to eliminate E. faecalis

in vivo has only been moderate (Estrela et al. 2008).

In clinical follow-up studies, the mean outcome of healed cases in the treatment

of primary AP has been 79% (range 46–97%) and in post-treatment disease 76%

(range 28–98%) (Friedman 2008). Clinical outcomes can still be improved. Better

chemomechanical methods and more active antimicrobials in comparison to

currently used ones would assist to resolve this problem. In general, microbial

susceptibility to antimicrobials can be determined in minimum inhibitory

concentration (MIC) assays. Microbial resistance leads to increases in the

minimum inhibitory concentration (MIC) of systemic antimicrobials, which is

associated with poorer clinical outcomes and infection complications during

treatment and prophylaxis (Pfaller 2012). The concentration as a function of time

is the main question for the disinfectant activity (Russell & McDonnell 2000). The

higher antimicrobial activity of an agent is without doubt a benefit in the complex

microanatomy of root canal system including dentine tubules. Safe antimicrobial

agents with new mode of actions are needed for the management of topical

infections in root canals.

42

43

3 Aims of the study

The aims of the present study were:

1. To evaluate the susceptibility of facultative and strictly anaerobic gram-

positive and gram-negative bacterial species to HICA for the evaluation of its

antibacterial spectrum.

2. To determine the susceptibility of yeasts and moulds to HICA to evaluate its

antifungal spectrum and further observe the influence of HICA on hyphal

formation of C. albicans and filamentation of A. fumigatus cells.

3. To compare the antibacterial activity of HICA to currently-used root canal

medicaments and the impact of potential inhibitors, such as dentine,

hydroxyapatite and serum albumin, on their activity.

4. To examine the antibacterial activity of HICA in the experimentally-infected

root canals of extracted human teeth and to compare the activity to presently-

used intracanal interappointment medications in the environment, which

mimic clinical conditions.

44

45

4 Materials and methods

The main objectives of this project were the antimicrobial properties of DL-HICA

in general and its suitability for the treatment of root canal infections. The project

consisted of two study entities. In the first two studies, the antimicrobial spectrum

of HICA was evaluated, in vitro (Study I and II). In the other two studies the

suitability of HICA for the treatment of root canal infections was evaluated, in vitro

(Study III) and ex vivo (Study IV). (Table 2).

Table 2. Summary of the studies (I–IV) and experiments (a–d).

Experiment Description of intervention

Ia Susceptibility testing of aerobically cultured bacteria

Ib Susceptibility testing of anaerobically cultured bacteria

IIa Susceptibility testing of Candida species

IIb Susceptibility testing of Aspergillus species

IIc Susceptibility testing of fungi for azoles

IId Hyphal formation in C. albicans and A. fumigatus isolates

IIIa Antibacterial activity with inhibitors in nutrient-deficient conditions

IIIb Antibacterial activity with inhibitors in nutrient-rich conditions

IV Antibacterial activity in the human dental root canals, ex vivo

The susceptibility of the isolates of human pathogenic gram-positive and gram-

negative bacterial species (n=18) was examined to HICA. Methicillin-resistant S.

aureus (MRSA) isolate and E. coli isolate with extended spectrum beta-lactamase

(ESBL) enzyme were included. The susceptibility of Candida isolates (n=10) and

Aspergillus isolates (n=9) to HICA and to azole antifungal agents was determined.

(Table 3). The modifications of the standard microdilution methods for

susceptibility testing were used (Study Ia, Ib, IIa, IIb, and IIc). The impact of HICA

on hyphal formation, filamentation, and cell-wall structures of C. albicans and A.

fumigatus isolates was evaluated in germination studies (Study IId).

The antibacterial activity against E. faecalis and suitability of HICA for root

canal medication was evaluated with potential inhibitors (Study IIIa and IIIb) and

in the infected human dental root canals (n=73; Study IV). The Ethics Committee

of Northern Ostrobothnia Hospital District approved the use of human teeth

(decision number 19/2006). The antimicrobial-inhibitor interactions were tested in

nutrient-deficient environment by the dentine powder method (Haapasalo et al.

2000) and nutrient-rich environment by microdilution method (Jorgensen et al.

46

2007). Bacterial invasion into dentine tubules was observed in the ex vivo

experiment (Study IV).

Table 3. Bacterial and fungal strains, designations, and sources used in the studies (I–

IV) and experiments (a–d).

Organism and phenotype Designation Source Experiment

Staphylococcus aureus1 ATCC 25923 NA Ia

Staphylococcus aureus T-54136 Blood Ia

Staphylococcus aureus, MRSA UKNEQAS 3/2011 NA Ia

Enterococcus faecalis1 ATCC 29212 Urine Ia, IIIb

Enterococcus faecalis T-75359 Root canal Ia, IIIa,b, IV

Streptococcus salivarius1 ATCC 13419 Saliva Ia

Streptococcus gordonii T-75354 Root canal Ia

Actinomyces israelii2 ATCC 12102 Brain abscess Ib

Actinomyces israelii T-90969 Gingival sulcus Ib

Lactobacillus rhamnosus2 ATCC 53103 Feces Ia

Lactobacillus spp. T-75020 Root canal Ia

Escherichia coli3 ATCC 25922 NA Ia

Escherichia coli T-54107 Blood Ia

Escherichia coli, ESBL NCTC 13353 NA Ia

Pseudomonas aeruginosa3 ATCC 27853 Blood Ia

Pseudomonas aeruginosa T-50866 Blood Ia

Fusobacterium nucleatum3 ATCC 25586 Cervico-facial lesion Ib

Fusobacterium nucleatum PD 788 Gingival sulcus Ib

Candida albicans4 ATCC 10231 Bronchomycosis IIa,c,d

Candida albicans D 17 Root canal IIa,c,d

Candida dubliniensis T-98 QC 2000 NA IIa,c

Candida dubliniensis EO 323 Oral IIa,c

Candida tropicalis ATCC 750 Bronchomycosis IIa,c

Candida tropicalis D 213 Oral IIa,c

Candida glabrata CCUG 32725 Blood IIa,c

Candida glabrata G 212 Oral IIa,c

Candida krusei ATCC 6258 Sputum IIa,c

Candida krusei D 206 B Oral IIa,c

Aspergillus fumigatus5 MRCM 24290 Sputum IIb,c,d

Aspergillus fumigatus MRCM 32208 Sputum IIb,c

Aspergillus fumigatus MRCM 32221 Sputum IIb,c,d

Aspergillus fumigatus MRCM 32552 Sputum IIb,c

Aspergillus flavus ATCC 204304 Sputum IIb,c

Aspergillus flavus MRCM 23135 Sputum IIb,c

Aspergillus flavus MRCM 24832 Sputum IIb,c

Aspergillus terreus MRCM 25730 Sputum IIb,c

Aspergillus terreus MRCM 26158 Bronchial wash IIb,c 1Gram-positive cocci (n=7); 2Gram-positive bacilli (n=4); 3Gram-negative bacilli (n=7); 4Yeasts (n=10); 5Moulds (n=9); NA: not applicable

47

4.1 Materials

4.1.1 Microbial isolates and strains

Clinical isolates were obtained from the Clinical Microbiology Laboratory Helsinki

University Central Hospital HUSLAB (T and PD isolates), the Institute of Dentistry,

University of Helsinki (D and G isolates), and the Mycology Reference Centre

Manchester (MRCM; UK). The reference strains were obtained from the American

Type Culture Collection (ATCC; USA), the Culture Collection of the University of

Gothenburg (CCUG; DK), National Collection of Type Cultures (NCTC; UK), and

United Kingdom National External Quality Assessment Service (UK NEQAS).

Bacterial isolates had been speciated by colony morphology, microscopy,

biochemical methods, or by 16s sequencing. Fungal isolates had been identified by

morphological and biochemical conventional methods, and by ITS1 and ITS2

rRNA sequencing (White et al. 2011). They were stored in milk-glycerine at -70ºC

and cultured twice on agar before the experiments for 48 h, except in anaerobic

conditions for seven days. E. faecalis isolates (n=2) were chosen for the suitability

testings of HICA for endodontic use.

4.1.2 Culture conditions and dilutions

The reagents, agars, culture broths, and their pHs, and potential inhibitors used in

the experiments are listed in Table 4. All incubations were performed at 37ºC: in 5%

CO2 for facultatively anaerobic bacteria, in anaerobiosis for Fusobacterium

nucleatum and Actinomyces israelii, and in aerobic conditions for fungi. For

bacterial susceptibility testing, HICA was diluted from 200 mg/mL aqueous

solution into broth (Study Ia, Ib, IIa, IId and IIIb). For susceptibility testing of

Aspergillus species for HICA and fungi for azoles, the test concentrations of

antimicrobials were dissolved directly into the broth (Study IIb and IIc). In the

dentine powder and root canal infection models antimicrobials were mixed with

sterile deionised water (Study IIIa and IV). In the susceptibility testings,

commercial chlorhexidine digluconate (CHG) of 2 mg/mL (Corsodyl®, Study Ia

and IIb; Curasept®, Study IIa, IIb, IIc and IIIb) were used as positive controls to

avoid precipitation and to fulfill the optical requirements of the spectrophotometric

analyses. The culture broth was used as a negative control to determine the

maximum microbial growth, except in the nutrient-deficient environments where

48

1.7 mg/mL of peptone water (Study IIIa) or sterile saline (Study IV) served as the

negative controls.

Table 4. Final test concentrations (mg/mL) of the reagents used in studies (I–IV) and

experiments (a–d) including antimicrobials, comparators, culture media, and potential

inhibitors and their pHs.

Reagents Manufacturer pH mg/mL I II III IV

DL-HICA TCI Europe, BE 5.2 0.6–36 a - - -

18–72 - a,b,d - -

33.3 - - a -

36–108 - - b -

400 - - - +

5.8 4.5–36 b - - -

DL-leucine TCI Europe, BE 5.2 4.5–36 a,b - - -

18–72 - a - -

DL-KICA TCI Europe, BE 5.2 18–72 a a,d - -

Fluconazole (mg/L) Pfizer, UK 0.125–64 - c - -

Itraconazole (mg/L) Sigma-Aldrich, UK 0.015–8 - c - -

Calcium hydroxide Sigma-Aldrich, UK 12.5 0.55 - - a -

100 - - - +

Chlorhexidine digluconate GlaxoSmithKline, GE 6.0 1.8 a,b - - -

Chlorhexidine digluconate Curaprox, UK 6.0 0.9 - - b -

1.8 - a,b,c,d - -

Chlorhexidine acetate Univ. Pharmacy, FI 6.0 0.17 - - a -

Chlorhexidine digluconate Univ. Pharmacy, FI 6.0 0.17 - - a -

20 - - - +

Thiglycollate Oxoid, UK 5.2 a a,b b -

5.8 b - - -

RPMI-1640 Sigma-Aldrich, UK 7.0 - c - -

Horse serum Oxoid, UK 5.2 - d - -

Peptone water Difco, USA 7.0 1.7 - - a -

Sterile saline 7.0 - - - +

Brain heart infusion BD, FR 7.0 - - - +

Brucella agar BD, UK 6.7 a - - -

Fastidious anaerobe agar LAB-M, UK 7.2 b - - -

Tryptic soy agar Difco, USA 7.3 - - a +

Blood agar Oxoid, UK 7.4 - - b -

Lysed blood agar BD, UK 7.0 - - b +

Sabouraud agar Oxoid, UK 5.6 - a,b,c,d - -

Dentine powder Univ. Oslo, NO 1–100 - - b -

1.87–187 - - a -

Hydroxyapatite Bio-Rad, DE 1–100 - - b -

187 - - a -

Bovine serum albumin Sigma-Aldrich, UK 1–100 - - b -

187 - - a -

49

Autoclaved human dentine powder (DP) prepared according to the previously

published model (Haapasalo et al. 2000, Portenier et al. 2001) was kindly provided

by Dr. I Portenier (University of Oslo). Briefly, extracted human third molars were

kept in 5 mg/mL NaOCl to remove soft tissue and prevent bacterial growth, rinsed

and autoclaved (121°C, 15 min) in distilled water to remove sodium hypochlorite.

The crowns were removed with a diamond saw (Accutom, Struers, Denmark). The

roots were crushed with a mixer mill (Retsch, Haan, Germany) to obtain powder

with 0.2–20 µm particle size. DP was stored dry at room temperature to avoid

contamination until use. Hydroxyapatite (HA) and bovine serum albumin (BSA)

were tested as comparators for DP.

4.2 Methods

4.2.1 Susceptibility testing of bacteria and fungi against HICA

The standard methods, modifications, density of microbial inoculums, incubation

volume, and breakpoints for detection of the results are listed in Table 5. Broth

medium, pH, DL-HICA concentrations, and microbial inoculum were selected

based on pilot studies. Cell densities of bacteria and yeasts in suspensions were

estimated with McFarland standard and confirmed by quantitative plate counts. The

density of Aspergillus spores was confirmed by cell cytometry. Solutions of 20, 40,

and 80 mg/mL of HICA were prepared in broth, all at pH 5.2. Controls free of

antimicrobial agents were included in all experiments. Microbial suspensions (20

µL) were added to broth (180 µL) in 96-well cell-culture microtiter plates (F96

MicroWell Plates, NUNC, ThermoFisher Scientific, Leicestershire, UK).

Microbial growth was determined by a spectrophotometer Multiscan RC

analyser (ThermoFisher Scientific) at 490 nm. The cultures in microtiter plates

were scanned at 0, 18, 24, 36, and 48 h, and the cultures in anaerobiosis at 0, 48,

and 72 h. The inhibitory concentration of HICA that prevented 50% and 90% of the

bacterial or candidal growth (IC50 and IC90) was determined by changes in optical

density. The growth of Aspergillus isolates was determined by 100% inhibitory

concentration (IC100) of the growth by visual inspection. The tests were done with

three to five replicate wells and repeated: 2-ketoisocaproic acid (KICA) and leucine

were used once as comparators. The results of spectrophotometrical analyses are

presented as means and standard error of the mean (SEM) of two experiments.

50

The microbicidal activity of HICA and comparators was observed by culture

on agar after final scanning for Candida species and after visual inspection for

Aspergillus species. Twenty µL of test suspension from representative wells were

cultured on agar for 48 h and four days for anaerobic bacteria. Microbial growth

was recorded as present or absent. The results of the microbicidal activity are

shown as minimum bactericidal and fungicidal concentration (MBC/MFC). The

breakpoints for detection are listed in Table 5. In the susceptibility tests, the

microbicidal value was set to >99.9% killing. In the suitability tests, bactericidal

value was set to 100% killing.

Susceptibility of Candida isolates was tested to fluconazole and Aspergillus

isolates to itraconazole (Study IIc). The growth of Candida species was determined

spectrophotometrically (OD490) and Aspergillus species was detected by visual

inspection. The minimum inhibitory concentration (MIC) was the lowest

fluconazole concentration, which reduced the optical density by 50% (IC50) in

comparison to the drug-free control at 48 h. The MIC for itraconazole was the

concentration, which prevented the growth of Aspergillus species visually (IC100)

at 48 h. The results of the impact of azole on fungal growth are presented as ICs.

In the germination study (Study IId), the impact of increasing concentrations

of HICA on hyphal formation of C. albicans and A. fumigatus in horse serum and

on their cellular morphology was visualised by Blankophor staining (100 nM;

Blankophor, Leverkusen, Germany) in a similar manner. In a pilot study, the impact

of KICA on C. albicans hyphal formation was tested in the same manner and only

Gram staining was done. Test-solution samples (5 µL) were used. The morphology

of the fungal cells was observed under bright field and fluoresence microscopy (490

nm excitation and 510 nm emission Nikon Eclipse 80i, Nikon, Japan).

51

Table 5. Summary of the reference methods, the densities of the microbial inoculums,

incubation volumes, and breakpoints for detection of inhibitory concentrations (IC),

minimum bactericidal or fungicidal concentrations (MBC/MFC).

Reagent Organism Method Inoculum

(cfu/mL; range)

Volume

(μL)

Breakpoints

(IC/MBC or

MFC)

DL-HICA Aerobic bacteria (Ia) ASM manual1

104

(2.5x103–5.9x104)

200 24h / 48h

Anaerobic bacteria (Ib) CLSI M11–A71 106

(2.0x106–1.0x107)

200 48h / 72h

Candida (IIa) CLSI M27–A31 103

(1.1x103–1.1x104)

200 24h / 48h

Aspergillus (IIb) EUCAST1 3x104

(cell cytometry)

200 48h / 48h

C. albicans (IId) Germ tube testing

106

(4.8x106–2.0x107)

200 24h / 48h

A. fumigatus (IId) Germ tube testing

106

(cell cytometry)

200 48h / 48h

FLU Candida (IIc) CLSI M27–A3 103

(1.1x103–1.1x104)

200 24h / 48h

ITR Aspergillus (IIc) EUCAST 3x104

(cell cytometry)

200 48h / 48h

ASM manual: Susceptibility test methods: dilution and disk diffusion methods. In book: Manual of Clinical

Microbiology, American Society of Microbiology: Jorgensen et al. 2007; CLSI M11–A7: Methods for

antimicrobial susceptibility testing of anaerobic bacteria, Clinical Laboratory Standards Institute, 2007;

CLSI M27–A3: Reference method for broth dilution antifungal susceptibility testing, 2008; EUCAST

Technical note on the method for the determination of broth dilution minimum inhibitory concentrations of

antifungal agents for conidiaforming moulds. Subcommittee on Antifungal Susceptibility Testing of the

ESCMID European Committee for Antimicrobial Susceptibility Testing, 2008; Germ tube testing: Johnson

et al. 1982; 1modifications based on pilot studies, cfu: colony forming units

4.2.2 Suitability testing of HICA for root canal medication

The reference methods, microbial inoculums, incubation volumes, and breakpoints

for the detection of bactericidal effect, which were used in the suitability testing of

HICA for root canal medication, are listed in Table 6. Some modifications were

performed in comparison to reference methods. In Study IIIa, 48 h detection was

added to the dentine powder method. In Study IIIb, potential inhibitors were added

to the antimicrobial solutions (Study Ia). In Study IV microbial growth was

52

detected in the root blocks departing the reference method of experimental root

canal infection model.

Table 6. Summary of the reference methods, the final densities of the microbial

inoculums, test volumes, and breakpoints used in the suitability testings of HICA.

Nutritional

conditions

Organism Method Inoculum

(cfu/mL;range)

Volume

(μL)

Breakpoint

Deficient (IIIa)

E.faecalis Dentine powder model1 3x108

(2.8–6.3x109)

150 48h

Rich (IIIb) ASM Manual1 1x106

(1.0–2.5x106)

200 48h

Deficient (IV) Root canal infection

model1

1.5x108

(not cultured)

3.8 7 days

Dentine powder model: Haapasalo et al. 2000; ASM manual: Susceptibility test methods: dilution and

disk diffusion methods. In book: Manual of Clinical Microbiology, American Society of Microbiology:

Jorgensen et al. 2007; Root canal infection model: Basrani et al. 2002; 1modifications based on pilot

studies

Antibacterial activity in the presence of potential inhibitors (IIIa, IIIb)

In the nutrient-deficient conditions (Study IIIa), the impact of three potential

inhibitors present in human dental root canals on the antibacterial activity of HICA

solution: DP suspension, HA suspension, and BSA solution was tested. The

antibacterial activity of DP without antimicrobials was observed also for control.

All antimicrobials and inhibitors were prepared in sterile deionised water. Each

antimicrobial agent (50 μL) was pre-incubated with each potential inhibitor (50 µL)

and controls in sealed Eppendorf tubes (2 mL) for 1 h. E. faecalis suspension (50

μL) was added to antimicrobial-inhibitor solutions and suspensions. They were

vortexed thoroughly and incubated for 48 h. All antimicrobials and controls could

be mixed well with DP and HA forming homogenous suspensions. BSA was mainly

in solution, but with Ca(OH)2 slurry it formed an insoluble precipitation, which

could not be analysed.

The samples were taken by a standard loop (10 μL) into peptone water (990

μL) after careful mixing at 0, 1, 24, and 48 h. They were further diluted in ten-fold

serial dilution to 10-4 (detection limit) for viability testing. 20 μL of each dilution

was cultured on TSA and incubated for 24 h. Colonies were counted and the purity

of the cultures was checked under stereomicroscope. The results were expressed as

53

relative viable counts (cfu%) with standard deviations. The tests were performed

once in three analogue experiments.

In the nutrient-rich conditions (Study IIIb), the influence of potential inhibitors

on increasing concentrations of HICA was tested by a modification of the broth

microdilution method for bacterial susceptibility testing used in Study I. The

potential inhibitors were added and one dilution higher concentrations of HICA

were used to test a hundred times higher density of microbial suspensions (Table

6). Two strains of E. faecalis were used in this experiment. The final test

concentrations of antimicrobials and potential inhibitors are summarised earlier in

Table 4. The positive control was commercial broth-soluble CHG 2 mg/mL diluted

1:1 in the broth. The amounts of 1.11 mg, 11.1 mg and 111.1 mg of potential

inhibitors were added into 1 mL of antimicrobial-broth solutions. Minimum

bactericidal concentration (MBC) for HICA was determined by culture (20 µL) on

blood agar at 1, 24, and 48 h. The purity of the cultures was checked. The tests were

performed once with three replicate wells.

Antibacterial activity in human dental root canals (IV)

Altogether 310 human vital teeth were collected at the Social and Health Services

of the City of Lahti during 2010–2012, after receiving patient’s informed consent.

The majority of the teeth were third molars and extracted mainly for malposition

reasons. Only teeth with a clinical diagnosis of vital pulp tissue were accepted and

the teeth with infected roots were excluded. This was rechecked thorougly during

the preparation of root blocks. The experimental infection model is summarised in

Fig. 2. After extraction, teeth were cleaned with tap water and stored frozen until

used. Root cementum was removed with a Carborundum drill, and one of the roots

of multi-rooted teeth was separated with a diamond drill. Standard root blocks with

root canals in 0.9 mm diameter were prepared with Gates-Glidden (GG) drill #3.

All drilling was done under water-cooling. Altogether 73 root blocks with no

apparent isthmus or lateral canals were accepted for the final testing. After smear-

layer removal and autoclaving of the root blocks they were stabilised in six-well

microtiter plates (Costar, Corning Incorporated, Corning, NY, USA) with dental

wax (Astynax, Associated Dental Products, Wiltshire, UK) in random order. Root

canals were dried with paper points (Orbis, Pontault Combault, France) before

filling them with E. faecalis suspension in BHI.

During 21 days of infection, a fresh bacterial suspension was delivered every

second day and BHI broth in the days between. The roots were incubated in 5%

54

CO2 at 37°C. Five roots were randomly chosen for histology. After 48 h

preservation in 4% paraformaldehyde, the root blocks were dehydrated in ethanol-

xylene dilution series and demineralised for 90 days in 17% EDTA. The root blocks

were rehydrated in xylene-ethanol dilution series and were stabilised into paraffin.

Then they were cut up into 3 μm sections with a mechanical microtome and stained

by the Brown and Brenn method (Brown & Brenn 1931, American Registry of

Pathology 1968). The remaining root blocks were randomised in four test groups

(n=17/group). Root canals were dried with paper points after irrigation with 10 mL

of sterile saline. Antimicrobials were prepared in deionised water. CHG was used

as a positive control. Sterile saline was an inactive negative control. Test reagents

were delivered once into root canals, which were closed tightly with Relyx Unicem

(3M Espe, Dental Products, Seefeld, Germany). They were incubated further in the

same conditions for seven days.

Sterile instruments and trays were used in all phases of the method for

performing strict asepsis, and ocular loops were used. Roots were not disinfected

with any additional chemicals. The fillings were removed before sampling. Root

canals were irrigated with 10 mL of sterile saline and they were dried with paper

points. After detachment from dental wax, the root blocks were attached tightly

with sterile locking instruments for each sampling. The inner and deeper root canal

dentine samples (first 0.1 mm, second 0.1 mm) were collected separately with

GG#4 (diameter 1.1 mm) and GG#5 (diameter 1.3 mm) drills into 2 mL of BHI

after irrigation and drying of the canals. Each dentine sample was collected using

a separate sterile drill from root canal walls by pushing the drill gently down and

up and using low frequency (<1000 r/min). The surface of the drill was checked

visibly to be clear after the dentine waist was delivered and mixed into BHI (2 mL)

in Eppendorf tubes. Dentine in broth was vortexed carefully. The residual roots

were also cultured in BHI. After 24 h incubation, 200 μL of each sample was

delivered to 96-well microtiter plates for the spectrophotometric analyses (OD490).

Bacterial growth was observed as changes in turbidity. In addition, 20 μL of all test

solutions were cultured on lysed blood agar for viability testing and checking of

the culture purity. Bactericidal activity was evaluated in the inner and deeper

dentine and in the residual roots. The results of spectrophotometric analyses were

depicted as bacterial growth inhibition (%) in comparison to negative control.

Bactericidal activity was reported as no growth percentage of samples in the broth

and in the culture on the agar.

The data did not follow a normal distribution (Study IV). Therefore, non-

parametric methods were used for statistical analysis. The analysis of the OD values

55

between the groups of different sampling depths was done with Kruskal-Wallis test,

and with Friedman and Wilcoxon Signed-rank tests between the different sampling

depths within each group. The analysis of bacterial growth between the groups in

different sampling depths was performed with Pearson Chi-Square and Mann-

Whitney test. Between the different sampling depths the analyses were performed

with Cochran and McNemar tests within each group.

Fig. 2. Main stages of the antibacterial activity testing of 400 mg/mL of DL-HICA, 400

mg/mL of Ca(OH)2, 20 mg/mL CHG and 9 mg/mL of sterile saline in experimentally-

infected human dental root canals ex vivo. (IV, reprinted with permission John Wiley &

Sons.)

56

57

5 Results

5.1 Susceptibility of microbial isolates to HICA

5.1.1 Antibacterial activity (I)

HICA inhibited the growth of facultatively aerobic bacteria in a dose-dependent

fashion, which was not observed in anaerobically cultured isolates (Fig. 3 and 4).

In addition, it showed a time-dependent effect on bacterial growth of most isolates.

The antibacterial activity of HICA against the gram-positive bacteria in subcidal

concentrations was reduced with time. For P. aeruginosa, the antibacterial activity

increased with time at all concentrations. In the incubations of E. coli or the

anaerobically cultured bacteria, no time-dependent effect by HICA was seen. IC50

and IC90 comparisons are shown in Fig. 3 and 4. The isolates of S. aureus were

most susceptible to HICA (IC90 ≤1.1 mg/mL) at 24 h, including MRSA. IC90 was

seen at four-fold lower concentrations compared to the other gram-positive bacteria

(1.1 mg/mL vs. 4.5 mg/mL). The ESBL positive E. coli isolate was also sensitive

for HICA (IC90 ≤4.5 mg/mL). HICA was less active against the other E. coli isolates

(IC90 ≤18 mg/mL for both). Over 90% of the growth of the reference isolate and

79% of the clinical isolate of P. aeruginosa was reduced by 4.5 mg/mL of HICA.

IC90 was achieved for the clinical isolate at 36 h. HICA inhibited in 9 mg/mL

concentration 90% of the growth of E. faecalis and Lactobacillus isolates.

MBC comparisons at 48 h are shown in Table 7. P. aeruginosa and F.

nucleatum isolates were most susceptible for HICA (MBC ≤4.5 mg/mL). MBC for

all E. coli isolates was 18 mg/mL. Both of E. faecalis and Lactobacillus isolates

were susceptible at the highest concentrations to HICA (MBC ≤36 mg/mL), in

which HICA had an equal bactericidal effect as the positive control, CHG. Leucine

did not have any influence on aerobically cultured bacterial isolates. In contrast,

the reference strain of Actinomyces israelii and the clinical isolate of F. nucleatum

were killed in 36 mg/mL of leucine.

58

Fig. 3. In aerobic conditions, the growth inhibition of a representative panel of clinical

isolates of bacteria in the presence of HICA and chlorhexidine (CHX; control)), including

antibiotic-resistant strains of MRSA and ESBL. IC50 and IC90 of HICA for bacterial strains

are shown at 18, 24, 36 and 48 h. Data is presented as means (±SEM). The reference

strains and the other clinical strains showed essentially the same with two exceptions:

IC90 of HICA for two other isolates of E. coli was higher (18 mg/mL); and IC90 of HICA for

the reference strain of P. aerunginosa in all test concentrations was achieved six hour

before (data not shown).

a. Staphylococcus aureus MRSA

0 0.6 1.1 2.3 4.5 CHX

10

50

90

mg/mL

Inhi

bitio

n % 18h

24h

48h

36h

c. Streptococcus gordonii

0 4.5 9 18 36 CHX

10

50

90

mg/mL

Inhi

bitio

n %

e. Escherichia coli ESBL

0 4.5 9 18 36 CHX

10

50

90

mg/mL

Inhi

bitio

n %

b. Enterococcus faecalis

0 4.5 9 18 36 CHX

10

50

90

mg/mL

Inhi

bitio

n %

d. Lactobacillus spp.

0 4.5 9 18 36 CHX

10

50

90

mg/mLIn

hibi

tion

%f. Pseudomonas aerunginosa

0 4.5 9 18 36 CHX

10

50

90

mg/mL

Inhi

bitio

n %

59

Fig. 4. In anaerobiosis, growth inhibition of two clinical isolates of bacteria in the

presence of HICA and chlorhexidine (CHX; control). IC50 and IC90 of HICA for bacterial

strains is shown at 48 and 72 h. Data is presented as means (±SEM). The reference

strains showed essentially the same (data not shown).

Table 7. Summary of minimum bactericidal concentrations (MBC) for HICA and leucine

(LEU) at 48 h.

MBC (HICA) MBC (LEU)

≥4.5 mg/mL ≥9 mg/mL ≥18 mg/mL ≥36 mg/mL ≥36 mg/mL

P. aerunginosa S. aureus Streptococci L. rhamnosus F. nucleatum

F. nucleatum (incl. MRSA) A. israelii Lactobacillus spp. A. israelii

E. coli

(incl. ESBL)

E. faecalis

5.1.2 Antifungal activity (II)

Dose-dependent inhibition induced by HICA was observed in the growth of all

candidal strains (Fig. 5). C. albicans and C. dubliniensis were more susceptible to

HICA than non-C. albicans Candida yeasts (C. glabrata, C. krusei and C.

tropicalis). In the subcidal concentrations, antifungal activity of HICA decreased

with time for most isolates. However, the activity increased for clinical isolate of

C. tropicalis up to 48 h.

In IC90 comparisons, C. albicans, C. dubliniensis, and C. tropicalis were most

susceptible to HICA (IC90 ≤18 mg/mL) at 24 h (Table 8). One dilution higher

concentration of HICA inhibited (IC90 ≤36 mg/mL) the growth of C. glabrata and

C. krusei. All isolates of Candida species were sensitive for fluconazole. A.

fumigatus and A. terreus isolates were the most susceptible species of the moulds

tested for HICA (Table 9). The IC100 was 36 mg/mL for all (n=6) and 18 mg/mL for

a. Fusobacterium nucleatum

0 4.5 9 18 36 CHX

10

50

90

mg/mL

Inhi

bitio

n % 48h

72h

b. Actinomyces israelii

0 4.5 9 18 36 CHX

10

50

90

mg/mL

Inhi

bitio

n %

60

five of them. HICA was not active against two isolates of A. flavus. All A. flavus

isolates were sensitive for itraconazole. Three of four isolates of A. fumigatus were

resistant to itraconazole, but one isolate (MRCM 32221) was sensitive even in 1

mg/mL concentration.

Fig. 5. Antifungal activity of HICA and chlorhexidine (CHX; control) for a representative

panel of clinical Candida isolates. IC50 and IC90 are shown at 18, 24, 36, and 48 h. Data

is presented as means (±SEM). The influence of HICA was essentially the same for the

reference strains with one exception: C. tropicalis inhibition was not reduced in

subcidal concentrations (data not shown).

a. Candida albicans

0 18 36 72 CHX

10

50

90

mg/mL

Inhi

bitio

n %

c. Candida tropicalis

0 18 36 72 CHX

10

50

90

mg/mL

Inhi

bitio

n %

e. Candida krusei

0 18 36 72 CHX

10

50

90

mg/mL

Inhi

bitio

n %

36h

24h

18h

48h

b. Candida dubliniensis

0 18 36 72 CHX

10

50

90

mg/mL

Inhi

bitio

n %

d. Candida glabrata

0 18 36 72 CHX

10

50

90

mg/mL

Inhi

bitio

n %

61

In MFC comparisons, 72 mg/mL of HICA was fungicidal for all Candida isolates

at 48 h (Table 8). The reference strain of C. dubliniensis was killed in 36 mg/L of

HICA. Fungicidal activity was similar with HICA or in 1.8 mg/mL CHG. KICA

and leucine were not active against Candida species. The MFC for the A. fumigatus

and A. terreus isolates was ≤36 mg/mL of HICA (Table 9), but A. flavus isolates

were not killed. Leucine was inactive for Aspergillus species up to 18 mg/mL,

which was the highest test concentration.

Table 8. Antifungal activity of fluconazole (FLU), 2-hydroxyisocaproic acid (HICA), 2-

ketoisocaproic acid (KICA) and leucine (LEU) are shown. Minimum inhibitory

concentration (MIC/24 h) for fluconazole was detected by spectrophotometric analyses

and minimum fungicidal concentration (MFC/48 h) for HICA, KICA, and LEU on agar.

Reagent Observation C. albicans C.dubliniensis C.tropicalis C. glabrata C. krusei

FLU MIC (mg/L) ≤0.5 ≤0.25 ≤1 ≤16 ≤32

HICA MFC (mg/mL) 72 ≤72 72 72 72

KICA MFC (mg/mL) >72 >72 ND >72 >72

LEU MFC (mg/mL) >72 >72 >72 >72 >72

ND: not determined

Table 9. Antifungal activity of itraconazole (ITR), 2-hydroxyisocaproic acid (HICA), and

leucine (LEU) are shown. At 48 h, fungal growth reduction of 100% (IC100) was

determined by visual inspection of the culture broth. Minimum fungicidal concentration

(MFC) was detected by culture on agar.

Reagent Observation A.fumigatus A.terreus A.flavus

ITR IC100 (mg/L) >8 nt 0.5

HICA IC100 (mg/mL) ≤36 18 >72

MFC (mg/mL) ≤36 ≤36 >72

LEU MFC (mg/mL) >181 >181 >181

1The highest test concentration

C. albicans germ tube formation was inhibited in 18 mg/mL of HICA and was

prevented in 36 mg/mL as observed in Gram staining (Fig. 6). In a pilot study, the

effect of 72 mg/mL of KICA was similar to 18 mg/mL of HICA. The morphology

of C. albicans cells incubated in 72 mg/mL of HICA or in the positive control CHG

was similar. The intensity of Blankophor cell wall staining was also decreased. The

size of the Candida daughter cells was diminished at 36 mg/mL of HICA. The

filamentation and sporulation of A. fumigatus was inhibited in 36 mg/mL and

prevented at 72 mg/mL.

62

Fig. 6. The impact of HICA on Candida albicans (D17) hyphal formation and expression

of the cells by Gram staining in the negative control of horse serum (a), 72 mg/mL of

KICA (b), 36 mg/mL of HICA (c), and 72 mg/mL of HICA (d), and in 1.8 mg/mL of CHG, at

24 h (a–c) and at 48 h (d, e). The influence of 72 mg/mL of KICA on germ tube formation

was similar to 18 mg/mL of HICA.

5.2 Suitability of HICA for root canal medication

5.2.1 Antibacterial activity with potential inhibitors (III)

Growth conditions and antibacterial activity without inhibitors

Nutrient-deficient conditions had an impact on bacterial growth in Study IIIa.

Bacterial counts decreased to hundred-fold (107 cfu/mL) immediately after mixing

at 0 h, which was verified by culture control on agar. Thereafter, in the negative

control of peptone water, bacterial viability reduced to 28% at 24 h and to 23% at

63

48 h from the initial counts, which demonstrated the nutrient-deficient test

conditions (Fig. 7). In comparison to maximum bacterial growth in the negative

control, HICA showed limited antibacterial effect with 73% of reduction at 1 h, but

it completely prevented bacterial growth as well as chlorhexidine acetate (CHA) or

CHG at 24 and 48 h. In contrast, Ca(OH)2 slurry showed good antibacterial activity

(93.5–98.5%) at 1 and 24 h, but the activity was totally lost in 48 h incubation (Fig.

7). Ca(OH)2 solution showed moderate reduction of bacterial viability (74%) at 1

h, but it was inactive after that.

Fig. 7. Antibacterial activity of antimicrobials at 1, 24, and 48 h. Means of relative viable

counts are demonstrated with standard deviations. Control: 1.7 mg/mL of peptone water,

Ca(OH)so: 0.55 mg/mL of calcium hydroxide solution, Ca(OH)sl: 100 mg/mL of calcium

hydroxide slurry, HICA: 33.3 mg/mL of 2-hydroxyisocaproic acid, CHA: 0.17 mg/mL of

chlorhexidine acetate, CHG: 0.17 mg/mL of chlorhexidine digluconate. (10 mg/mL = 1%)

Impact of dentine on antibacterial activity

Human DP reduced antimicrobial activity of HICA in a time- and dose-dependent

manner. The highest concentration of DP (187 mg/mL) prevented the bactericidal

effect of HICA against E. faecalis. In contrast, 1.87–18.7 mg/mL of DP inhibited

0–3% of its antibacterial activity in comparison to the maximum bacterial growth

Control Ca(OH)so Ca(OH)sl HICA CHA CHG 0.01

0.1

1

10

100

Rel

ativ

e vi

able

cou

nts

(%)

48

241

64

in peptone water control at 48 h. (Fig. 8a). Ca(OH)2 slurry demonstrated better

antibacterial activity with DP than without it, especially in 18.7–187 mg/mL

concentrations. The positive effect was lost with the lowest DP concentration at 48

h. (Fig. 8b). The activity of Ca(OH)2 solution was 91.5% with 187 mg/mL DP at

48 h, but with lower concentrations of DP it was ineffective (data not shown). DP

did not notably inhibit CHA (<1%, data not shown) or CHG (0%) (Fig. 8c).

Interestingly, the highest concentration (187 mg/mL) of DP without antimicrobials

decreased bacterial growth ≥86% in comparison to viable bacterial cells in peptone

water control after 24 h incubation (Fig. 8d).

Fig. 8. Impact of dentine powder (DP 0.0–187 mg/mL) on antibacterial activity of HICA

solution (a), Ca(OH)2 slurry (b), CHG solution (c) and with no antimicrobials (d) against

E. faecalis (T-75359) at 1, 24 and 48 h. The negative control was 1.7 mg/mL of peptone

water. Mean relative viable counts are presented with standard deviations (SD).

Detection limit: 0.01%.

Control 187 18.7 1.87 0.00.1

1

10

100

Rel

ativ

e vi

able

cou

nts

(%)

a.

12448

Control 187 18.7 1.87 0.00.1

1

10

100

Rel

ativ

e vi

able

cou

nts

(%)

c.

12448

Control 187 18.7 1.87 0.00.1

1

10

100

Rel

ativ

e vi

able

cou

nts

(%)

b.

Control 187 18.7 1.870.1

1

10

100

Rel

ativ

e vi

able

cou

nts

(%)

d.

65

Impact of hydroxyapatite or bovine serum albumin on antibacterial activity

At the end of incubation, HICA reduced >95% of bacterial growth in comparison

to negative control in spite of the presence of 187 mg/mL of HA (Fig. 9a). An error

in the diagram of the original article describing the interactions between HICA and

HA is corrected in Fig. 9a. In the presence of BSA, the activity of HICA was

eliminated and the activity of CHG was inhibited less than 11% after 24 h (Fig. 9b).

CHG showed ≥99% reduction of bacterial viability with HA at all time points. The

activity of CHA was inhibited by HA and BSA at first, but after 24 h incubation the

activity was normal. Ca(OH)2 solution and slurry were inactive with both of these

reagents. (Fig. 9a, 9b), and slurry precipitated with BSA.

Fig. 9. In nutrient-deficient conditions, the impact of 187 mg/mL hydroxyapatite (a) or

bovine serum albumin (b) on the antibacterial activity of antimicrobials is presented.

Mean relative viable counts (%) and standard deviations (SD) are depicted. Control: 1.7

mg/mL of peptone water, Ca(OH)so: 0.55 mg/mL of calcium hydroxide solution,

Ca(OH)sl: 100 mg/mL of calcium hydroxide slurry, HICA: 33.3 mg/mL of 2-

hydroxyisocaproic acid, CHA: 0.17 mg/mL of chlorhexidine acetate, CHG: 0.17 mg/mL

of chlorhexidine digluconate. Detection limit: 0.01%.

Impact of inhibitors on antibacterial activity in nutrient-rich conditions

In nutrient-rich conditions, thiglycollate broth was used as bacterial growth media

in all test solutions and therefore it was the negative control of Study IIIb. HA and

BSA reduced HICA’s antibacterial activity in a dose-dependent manner at 48 h (Fig.

10). The most active inhibitor was HA and the least active was BSA. MBC of HICA

increased to 72 mg/mL with 1 mg/mL of HA, 10 mg/mL of DP or BSA.

Antibacterial effect of HICA was prevented by 100 mg/mL of DP or 10 mg/mL of

HA.

Control Ca(OH)soCa(OH)sl HICA CHA CHG 0.1

1

10

100

Rel

ativ

e v

iab

le c

ou

nts

(%)

a.

12448

Control Ca(OH)so HICA CHA CHG

0.1

1

10

100

Re

lativ

e vi

abl

e c

ount

s (%

)

b.

66

Fig. 10. In nutrient-rich conditions, minimum bactericidal concentration (MBC) of 36–

108 mg/mL of HICA against E. faecalis with potential inhibitors (1–100 mg/mL): dentine

powder (DP), hydroxyapatite (HA) or bovine serum albumin (BSA), and without them

(Control) at 48 h.

5.2.2 Antibacterial activity in dental root canals ex vivo (IV)

One root block was excluded from histological analyses because of incomplete

demineralisation, which affected the cutting, attachment, and staining of the

sections. Invasion of E. faecalis into dentine tubules over the depth corresponding

to the sampling depth of 0.2 mm from pulpal wall surface was observed in all roots

(Fig. 11). Penetration of bacterial cells was discontinuous in the tubules. On

histological analysis bacteria were seen to invade beyond 1.0 mm from the pulpal

wall in all roots examined.

Control 1 10 1000

36

72

108

Concentration (mg/mL)

MB

C fo

r H

ICA

(4

8 h

)DP

BSA

HA

67

Fig. 11. E. faecalis penetration into dentine tubules is shown up to 0.4 mm depth from

pulpal wall before medication under light microscope. Gram-positive cocci stain blue

by Brown and Brenn method. Magnifications: 20x. (IV, reprinted with permission from

John Wiley & Sons.)

Bacterial growth inhibition

In comparison to sterile saline treatment, HICA inhibited 43% of E. faecalis growth

in the inner dentine (first 0.1 mm), 91% in deeper dentine (second 0.1 mm), and

21% still in the residual roots after 7 days incubation (Fig. 12). Respectively, CHG

inhibited 56% of the growth in the inner dentine, 43% in the deeper dentine and

was fully inactive in the residual roots. Ca(OH)2 did not show any antimicrobial

activity in comparison to sterile saline (negative control). In addition, HICA and

CHG inhibited the bacterial growth in the inner and deeper dentine significantly

better than in the residual roots (p<0.01; Wilcoxon Signed Rank test).

68

Fig. 12. In spectrophotometric analyses, mean bacterial growth inhibition (%) with

standard error of mean (SEM) in comparison to sterile saline treatment are shown in the

inner dentine (≤0.1 mm from root canal surface), deeper dentine (0.1–0.2 mm from root

canal surface) and in the residual roots after medication with 400 mg/mL of HICA paste,

400 mg/mL of Ca(OH)2 paste or 20 mg/mL of CHG solution (n=17/group). (IV, reprinted

with permission from John Wiley & Sons.)

Bactericidal activity

In the inner dentine, HICA or CHG medication resulted in a bactericidal effect and

roots free of bacterial growth in 82% or 88% of the samples (Fig. 13). In 59% of

Ca(OH)2 and 65% of sterile saline samples, no growth was detected. In the deeper

dentine, bactericidal effect was found in 100%, 88%, 76%, and 71% of HICA, CHG,

Ca(OH)2, or sterile saline samples, respectively. The differences between the groups

in the inner or deeper dentine were not statistically significant. In the residual roots,

29% of HICA and 18% of CHG samples showed bactericidal effect, whilst no

sample with Ca(OH)2 and sterile saline treatment were free of bacterial growth. The

difference between HICA and Ca(OH)2 or sterile saline groups were significant

(p=0.008; Pearson’s Chi-Square test with Mann-Whitney pairwise comparison),

Ca(OH)2 CHG HICA-50

0

50

100

Inhi

bitio

n (%

)Inner dentine

Deeper dentine

Residual root

69

but not between CHG and Ca(OH)2 or saline groups. (Fig. 13). The results of

viability testing on agar showed essentially the same, in which 35% of residual

roots with HICA medication were successfully disinfected and no growth was

observed. The results of viability testings are in line with the results of

spectrophotometric analyses in the present study.

Fig. 13. In spectrophotometric analyses, relative amount of samples with no bacterial

growth after medication with 400 mg/mL of HICA paste, 400 mg/mL of Ca(OH)2 paste, 20

mg/mL of CHG solution and 9 mg/mL of sterile saline (n=17/group) is shown in the inner

dentine (≤0.1 mm from root canal surface), deeper dentine (0.1–0.2 mm from root canal

surface), and residual roots in broth. (IV, reprinted with permission from John Wiley &

Sons.)

70

71

6 Discussion

This series of studies were performed in order to gain insight regarding the

antimicrobial spectrum of HICA and its potential for root canal medication. The

hypothesis of the study that HICA is a potential antimicrobial agent for the

treatment of topical infections was tested and found to be correct within the

limitations of in vitro and ex vivo methodology. The results indicate that HICA has

broad-spectrum antimicrobial activity. It is bactericidal against several species of

gram-positive and gram-negative cocci or rods in aerobic and anaerobic test

conditions. It is fungicidal against yeasts and some species of spore forming moulds.

HICA inhibits the hyphal formation and filamentation capacity of dimorphic fungi,

which are considered to be important virulence factors for these species. The

interactions between HICA and dentine that affect the antibacterial effect are dose-

dependent and moderate. In comparison to presently-used root canal medicaments,

HICA has better long-term antibacterial activity in infected human dental root

canals. The principle findings of the study are discussed with the methodological

considerations, and the results are compared to the earlier literature.

6.1 Susceptibility testing studies

Repetition of the studies is important for the assessment of the susceptibility of

microbial species and the activity of antimicrobial agents. Standardization and

conformity of the methods assists in the repetition and comparison of the results of

different studies. E. faecalis has been widely studied since it has shown to be the

most prevalent organism in persisting infections, especially over the past two

decades. A PubMed search with keywords of “calcium hydroxide and Enterococcus

faecalis” resulted in 223 articles from the year 1983 to date, 17 of which were

clinical trials. In contrast, “calcium hydroxide and Candida” resulted in 88 studies,

including one clinical study. A search with keywords of “chlorhexidine and

Enterococcus faecalis” found 333 papers from the year 1969 to date, 20 of them

being clinical trials. Ca(OH)2 studies were endodontic studies and in chlorhexidine

studies urinary infections were widely represented. In general, biocides are less

frequently studied in comparison to antibiotics. Hundreds of different clinical trials

on fungal species and azoles were found. The present study series is the first to

assess the susceptibility of E. faecalis to HICA, which was performed in three

different environments: in nutrient-rich (Study I, II and IIIb) and nutrient-deficient

conditions in vitro (Study IIIa) and in human root canals ex vivo (Study IV). In

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addition, interactions of HICA with potential inhibitors were observed for the first

time in two different nutritional conditions in vitro (Study IIIa and IIIb).

6.2 Methodological considerations

The susceptibility studies of microbes to HICA were performed in nutritionally rich

environments with broth control. Bacterial species in Study Ia and Ib, and candidal

species in Study IIa were analysed according to standard susceptibility

microdilution testing methods by spectrophotometer (Jorgensen et al. 2007,

Clinical and Laboratory Standards Institute 2001, 2008). These analyses were

supported by viability testing on agar. Susceptibility of spore-forming moulds in

Study IIb were analysed by visual inspection (Subcommittee on Antifungal

Susceptibility Testing of the ESCMID European Committee for Antimicrobial

Susceptibility Testing 2008). Histological analyses were performed in germination

study of dimorphic fungal species (Study IIb) to show the impact of HICA on

hyphal formation. (Fig. 14).

The suitability studies of HICA for root canal medication were performed

following a modified direct exposure method in vitro (Study IIIa; Haapasalo et al.

2000) by another modification of direct exposure method based on standard

microdilution methods for comparison in vitro (Study IIIb; Jorgensen et al. 2007)

and by experimental infection model ex vivo (Study IV; Basrani et al. 2002). Direct

exposure methods were modified by adding potential inhibitors in Study IIIa and

IIIb, in which the results were analysed by viability testing on agar. No

spectrophotometric analyses could be performed in these, because of the non-

soluble consistence of Ca(OH)2 paste and potential inhibitors: DP and HA. Agar

diffusion test was not accepted for these studies, because it is an unsuitable method

for comparing agents with different chemical structures and with heterogenous

ability to diffuse into medium (Estrela et al. 2001). Histological analyses were

performed in root canal infection models ex vivo (Study IV) to confirm the

penetration of bacterial cells into dentine tubules. (Fig. 14).

Simulation of clinical conditions in the root canals is very difficult (Siqueira

2008). Despite the promising results of the present study, they do not necessarily

correlate to the treatment outcomes in vivo and any conclusions of clinical efficacy

of HICA cannot be driven so far. However, this study is the largest study of the

antibacterial and antifungal spectrum of HICA and the first study about its potential

for root canal medication.

73

Fig. 14. Experimental methods (Study I–IV, experiments a–d), exposure time of

antimicrobials with microbial species, time of experimental infection (Study IV),

microbial species, nutritional conditions and analyses performed are shown. ND:

nutrient-deficient, NR: nutrient-rich.

6.2.1 Culture media

Culture media is an essential prerequisite for microbial growth. In general, pH,

temperature, and availability of oxygen have an impact on the microbial growth,

too. Thioglycollate broth was accepted for the experiments of Study I and II as a

culture medium, which is suitable for both aerobic and anaerobic organisms.

74

Importantly, thioglycollate allowed the growth of anaerobic bacteria, which were

the most demanding organisms to culture included in these studies (Clinical and

Laboratory Standards Institute 2001). Mueller-Hinton broth is recommended for

susceptibility testing of aerobic and facultative bacteria (Clinical and Laboratory

Standards Institute 2012), and RPMI-1640 for yeasts and spore-forming moulds

(Clinical and Laboratory Standards Institutes 2008, Subcommittee on Antifungal

Susceptibility Testing of the ESCMID European Committee for Antimicrobial

Susceptibility Testing 2008). Therefore, RPMI-1640 was used in susceptibility

testing of fungal species to azoles (Study IIc). In Study IId, horse serum was used

for induction of hyphal formation (Johnson et al. 1982). Thioglycollate broth was

used also in Study IIIb to evaluate the impact of nutritional conditions on

interactions of HICA and potential inhibitors. The use of same culture media in

different experiments makes the results comparable to each other (Study Ia, Ib, IIa,

IIb and IIIb). The results of the present studies are also comparable to other similar

studies in microbiology and in development of new antimicrobial agents.

In Study IIIa, the use of peptone water for microbial suspension was adapted

from the previous studies (Haapasalo et al. 2000, Portenier et al. 2001). The further

dilutions (2:1) resulted in low final concentration of peptone water, which provided

a nutrient-deficient test environment for all incubations in Study IIIa. Deficiency

of nutrition was supported by a finding in a pilot study, in which the same

concentration of peptone water reduced the E. faecalis viability by 77%, at 48 h

incubation. In the root canal infection model (Study IV), three weeks incubation of

root canals with bacterial suspension in brain heart infusion was also adapted from

previous studies (Basrani et al. 2002, 2003). This provides bacterial penetration

into dentine tubules before medication (Basrani et al. 2003, Zapata et al. 2008, Du

et al. 2015). No additional culture media were used in Study IV during medication.

The cultural difference between antimicrobial susceptibility testing methods in

microbiology and endodontology is based on nutrient-deficient environment in root

canals during root canal treatment (Sundqvist & Fidgor 2003) and nutrient-rich

conditions usually in other human tissues.

6.2.2 Negative and positive controls

Negative control together with positive control forms the frame of the observation

in antimicrobial activity studies. Positive control was used for microbicidal control

and negative control was used for control of microbial growth in media, which

allows microbes to grow freely in nutrient-rich conditions (Study I, II ad IIIb) or to

75

cause passive reduction in microbial viability in nutrient-deficient conditions

(Study IIIa and IV). These terms are also used in an opposite manner in the

literature to reflect bacterial growth or killing (Han et al. 2001, Shokraneh et al.

2014). Thioglycollate broth allowed maximum growth of the organisms in the

experiments and served as a negative control in Study I, II, and IIIb. In Study IIIa,

a maximum amount of residual bacteria was detected in peptone water control and

in sterile saline control in Study IV, and they were used as negative controls. The

main positive control of the present study series, CHG, killed all the organisms at

0.17–1.8 mg/mL concentration in a 48 h incubation in Studies I–III. In contrast,

ten-fold higher concentration of 20 mg/mL of CHG failed to achieve full

antibacterial effect in root canals in Study IV. This difference could be explained

with the polymorphous biological environment of the intradental system.

Since saline is widely used in endodontic studies as a negative control, it is

noteworthy that it fails to increase bacterial growth in long incubations in vitro

(Ballantyne 1929, Valu 1965), in dental root canals ex vivo (Maekawa et al. 2013)

or in vivo (Byström & Sundqvist 1983). This may be explained by normal bacterial

growth curves with exponential growth, stationary state, and decline phase (Roszak

& Colwell 1987). The passive effect of sterile saline on bacterial viability was also

confirmed in a pilot study, in which viability of E. faecalis cells was reduced by

99% over a six-day incubation. Saline and 5% NaOCl solution have been used

earlier as root canal irrigants. For toxicity reasons from high concentration of

NaOCl, Byström & Sundqvist (1983) observed and showed the better antimicrobial

activity of 0.5% of NaOCl solution in comparison to saline solution in root canals.

In their study, after five appointments with two to four days interval and with saline

medication for all subjects no bacterial growth was observed in twelve of fifteen

roots (80%) with NaOCl irrigation and in eight of fifteen roots (53%) with saline

irrigation. Nowadays, saline is still widely used in the treatment and prevention of

wound infections (Weiss et al. 2013). In experimental infection studies of root

blocks, saline is the most generally used alternative medication and negative

control (Table 11; see page 89). The use of control groups without infection,

without medication (Han et al. 2001, Zehnder et al. 2006), and with microbial broth

could assist in the assesstment of antimicrobial activities of different agents, too. In

particular, the trend of reduction in bacterial viability in saline refers to long-term

incubations of interappointment medication studies. This would not necessarily be

seen in short-term incubations of irrigation agent studies.

76

6.2.3 Test organisms

For evaluating the microbial spectrum of HICA in Study I and II, representatives

of seventeen different general human pathogenic organisms were chosen from

facultatively aerobic and anaerobic gram-positive and gram-negative bacterial

species (n=9), and from yeasts (n=5) and spore-forming moulds (n=3). At least two

isolates of each microbial species were included: clinical isolate and reference

strain of each bacterial and candidal species were used for control. Aspergillus

species were mainly clinical isolates. Although the number of isolates from single

species was restricted, the spectrum of tested organisms was a good selection of

most general human pathogens. The spectrum of test organisms is a representative

panel of microbial species commonly found in different phases of root canal

infection and covers well the most common findings in persistent root canal

infection such as E. faecalis and C. albicans. All test organisms have general

importance as human pathogens. Antibiotic-resistant strains were tested because of

their particular importance in the development of new antimicrobials.

E. faecalis was chosen as a test organism to evaluate the suitability of HICA

for root canal medication in Study III and IV, because it is the most general finding

in persistant root canal infections (Portenier et al. 2003, Stuart et al. 2006, Zehnder

& Guggenheim 2009, Haapasalo et al. 2011, Lew et al. 2015, Zhang et al. 2015).

The ability of E. faecalis to penetrate deep into the dentine tubules and to escape

immediate contact with root canal medicament results in problems in eradication

by presently-used chemo-mechanical methods. In Study I, E. faecalis was the most

tolerant bacterial species for HICA, together with Lactobacillus species. Only

yeasts and A. flavus were less susceptible to HICA.

6.2.4 Microbial cultures

Antimicrobials were exposed to planktonic organisms in Studies I, II, and III. The

incubation time was mainly 48 h, but longer incubation time was only needed for

anaerobic cultures to allow sufficient growth (Study Ib; 72 h). ICs and MBCs were

detected following the breakpoints of standard methods. In Study IV, the

medication time was one week after three weeks experimental infection. This time

should have been sufficient for the bacteria to form biofilm on dentine prior to

exposing them to antimicrobials as in previous studies. Bacterial biofilm formation

on root canal wall has been shown to take place already at 24 h incubation (Ozdemir

et al. 2010). E. faecalis penetration into dentine tubules and biofilm maturation has

77

been shown by confocal laser scanning microscopy after three weeks of

experimental infection (Zapata et al. 2008, Du et al. 2015). However, this could not

be detected in the study model used in the present study. In the present study the

penetration of E. faecalis cells up to 1.0 mm from root canal surface in dentine

tubules in Study IV are in line with previous reports. Bacterial invasion has been

reported from 0.1 mm to 1.5 mm depth in human teeth in histological analyses, by

scanning electron microscopy or by confocal laser scanning microscopy (Basrani

et al. 2003, George et al. 2005, Zapata et al. 2008, Harrison et al. 2010).

The depth of bacterial invasion into dentine tubules depends on the microbial

adherence, availability of nutrition and oxygen (George et al. 2005) and this may

not be continuous (Love 2002, George et al. 2005). In addition, dentine tubulus

wall may provide a barrier for dyes in Gram staining, which may lead to a lesser

extent of visibly stained bacteria than is present in reality. Only blue-coloured round

spots should be accepted for detection of coccal bacteria. Black or brownish

findings may contribute to artefacts caused by air bubbles. Bacterial penetration

and dentine infection was shown to be successful before medication in Study IV.

Non-susceptibility of microbial cultures in dentine tubules to disinfectants could be

a consequence of the lack of direct contact between antimicrobials and target

organisms or due to microbial resistance in biofilms.

6.2.5 Potential inhibitors

Hydroxyapatite

According to previous studies, HA would be the main component of dentine, which

is responsible for the inactivation of antimicrobial effect of Ca(OH)2 in the root

canal (Portenier et al. 2001, Haapasalo et al. 2007). Increasing concentrations of

HA increased the MBCs of HICA in Study IIIb. In the nutrient-rich conditions

(Study IIIb), the impact of HA on the antimicrobial activity of HICA was stronger

than in nutrient-deficient conditions at 48 h (Study IIIa). High concentration of HA

reduced the activity of HICA only by 4.3% in comparison to bacterial growth in

peptone water control; but in nutrient-rich conditions, twenty-fold concentration of

HA was sufficient to prevent the activity of HICA at 48 h. This may be explained

by the higher amount of proteins present in thiglycollate broth (Study IIIb), in

which protein binding and interactions of HA may be more probable than in low

concentration of peptone water in Study IIIa. This is in line with the finding that

78

BSA reduced the antimicrobial activity of HICA in a dose-dependent manner in a

nutrient-rich environment (Study IIIb). In nutrient-deficient conditions, the

inactivation of HICA by HA was substantially less than that of Ca(OH)2. In line

with previous results, the antimicrobial activity of Ca(OH)2 was completely

eliminated by 187 mg/mL of HA in Study IIIa (Portenier et al. 2001).

Serum albumin

The inflammatory exudate may be the main source for albumin and other serum

proteins in the infected root canals, while the levels of them are not known

(Haapasalo et al. 2007). In the present study, BSA was used instead of human serum

albumin or other serum proteins as in similar experiments earlier (Portenier et al.

2001, Sassone et al. 2003, Pappen et al. 2010). Therefore, the results may not

precisely reflect the conditions in the root canal in clinical infection, although BSA

and human serum albumin are highly uniform in structure. Earlier results were

supported by a finding that Ca(OH)2 solution lacked any antimicrobial effect in the

presence of BSA in Study IIIa (Portenier et al. 2001). In contrast, BSA formed an

insoluble precipitation with Ca(OH)2 slurry and therefore they could not be

analysed in Study IIIa. Although, the antimicrobial activity of HICA was prevented

by 187 mg/mL of BSA in Study IIIa, 10–100 mg/mL of BSA increased the MBC

of HICA only by one dilution from 36 mg/mL to 72 mg/mL in Study IIIb. This

finding indicates that in spite of a possible flow of inflammatory exudate into the

root canal, HICA would potentially be able to sustain its antimicrobial activity.

Dentine powder

Dentine remnants in root canal are probably mixed with soft tissue remnants at least

at the first appointment. In addition, interactions between antimicrobials and DP

may be enhanced by notably larger surface area in powder in comparison to dentine

on the unbroken root canal wall. The highest test concentration of 187 mg/mL of

DP would not reflect clinical conditions in the well-prepared and irrigated root

canals. Earlier results were supported by a finding that Ca(OH)2 solution lacked

any antimicrobial effect in the presence of DP in Study IIIa (Portenier et al. 2001).

In contrast, the antibacterial activity of Ca(OH)2 slurry was enhanced with the

highest test concentration of DP in Study IIIa. This kind of enhancement in

antimicrobial activity of bioactive glass and MTA is also shown with bioactive

glass and DP (Zehnder et al. 2006, Zhang et al. 2009). However, DP failed to

79

prevent the antibacterial activity of HICA at clinically relevant concentrations

(≤18.7 mg/mL) in Study IIIa and IIIb, unlike it did in 1.87 mg/mL concentration

for the activity of Ca(OH)2 slurry in Study IIIa.

Impact of preincubation time

Haapasalo and collegues (2000) also showed that increasing preincubation time (0–

24 h) between antimicrobials and potential inhibitors before adding microbial

suspensions to the test solution increases the interaction effect. However, this

merits further research. It is noteworthy that the results of the present study

similarly indicate that one hour of preincubation may enhance the interaction

effects in comparison to drug-free controls. In addition, the order of test component

delivery is opposite to clinical conditions, in which microbial species are present in

root canals at first. Secondly, the amount of DP could be increased after preparation.

Finally, antimicrobial agents are delivered into root canal during root canal

treatment, especially interappointment medication is the last component used

during the appointment. The interactions may happen between interappointment

medication and dentine in root canal before next appointment. This order could be

the same in a case of reinfection. In this context, one hour preincubation is far too

short. However, preincubation of antimicrobials with potential inhibitors may aid

in the assessment of differences in vitro.

Comparisons and clinical relevance

In nutrient-deficient conditions, the impact of DP on antibacterial activity of HICA

was dose-dependent (Study IIIa). The effect of all potential inhibitors (DP, HA, and

BSA) on the antibacterial activity of HICA was similarly dose-dependent in

nutrient-rich conditions (Study IIIb). Only one concentration of HA and BSA were

tested in Study IIIa as technical comparators in nutrient-deficient environment.

Interactions between the tested reagents may also be bidirectional and antimicrobial

activity may be reduced or enhanced. In addition, the interactions may also change

the direction over the incubation time as was shown in Study IIIa between Ca(OH)2

slurry and 1.87 mg/mL of DP.

While keeping the clinical relevance of the results in mind, the albumin

concentration in human serum is 35–55 mg/mL, which is the major source of

albumin in inflammatory exudate. Relative amount of HA in human dentine is 70%.

Therefore, the comparisons between dentine and HA should be done in that context.

80

The test concentrations of ≤100 mg/mL of BSA and ≤10 mg/mL of HA in the

present study may have some clinical relevance. In this context, the presence of HA

would lead more probably to inhibition of HICA than the presence of serum

albumin. Therefore, the results of the present in vitro study show some basic

evidence of potential interactions between present root canal medicaments or HICA

and dentine, causing improved or worse antimicrobial action.

6.2.6 Human teeth

Study IV was performed in the root canals of extracted human teeth. Six mm long

root blocks of the canals were prepared uniformly to minimize the natural

morphological discrepancy between the roots. A total of 73 roots of initial were

carefully selected from 310 teeth, excluding the roots with apparent isthmus or

lateral canals. The test volume in root canals was made analogous (3.8 μL) in all

incubations with as minimum variation as possible. No additional antimicrobials

were used during the collection, preservation and preparation of the blocks. The

method was practiced in pilot studies. At the commencement of experiments,

sterility of the root blocks after autoclaving was confirmed by incubation in broth.

During the relatively long infection and medication time, contamination of the

blocks was the major concern for the success of the whole experiment. However,

this was succesfully prevented with strict asepsis, which was confirmed with the

purity of the culture controls on agar at the end of the study. The results of the

present study can be adapted well to human root canal treatments. However,

controlled clinical trials are needed to show the relevance of the results in clinical

conditions.

6.2.7 Group size and statistical analysis

For appropriate statistical analyses, the amount of replicates and repeats should

have been larger in Studies I–III. It is inappropriate to analyze small amounts

individual isolates statistically, especially when they represent technical replicates

instead of true biological replicates. The used experimental setting set-up could be

improved by higher number of microbial species. Definition of MIC90 for a new

antimicrobial agent means that the agent kills at least 90% of different isolates

(Hoogkamp-Korstanje 1997, Schwarz et al. 2010). Therefore, the use of IC90 was

prefered to describe over 90% reduction of bacterial growth of single species in the

present study. However, the spectrum of tested organisms in the susceptibility

81

testings was a representative selection of most general human pathogenic

organisms (Study I and II). Studies with a large amount of single species of bacteria

would be especially difficult to perform in the endodontic field, because the root

canal findings consist of numberless variation of species and microbial sampling is

not a routine operation during clinical root canal treatment (Molander et al. 1998,

Chávez de Paz et al. 2003, 2004, Peciuliene et al. 2001, 2008, Rôças & Siqueira

2008, Siqueira & Rocas 2014).

In general, the availability of human teeth, especially of single-rooted teeth,

restricts the amount and the size of the test and control groups in reality in

endodontic studies. In Study IV, there were a larger amount of replicates included

per group (n=17) and one repeat could be performed. Single isolate of E. faecalis

was used as a technical replicate in all incubations and different human teeth

represented true biological replicates in each incubation. Statistical analyses were

performed as appropriate in the present study. Significant differences in

antibacterial activity were seen between the group of HICA and Ca(OH)2 in residual

roots in >0.2 mm depth of dentine. The present differences in the inner dentine and

deeper dentine group would have been statistically significant in comparison to

sterile saline treatment, if the size of test groups would have been extended by eight

root blocks. However, this study series provides basic and moderate evidence of

antimicrobial potential of HICA for planning of clinical investigations in the future.

6.3 Antimicrobial activity

6.3.1 HICA (I, II, III, IV)

Susceptibility of bacteria and fungi (I, II)

Antimicrobial HICA has broad-spectrum antibacterial and antifungal activity.

Microbicidal activity of HICA arises slowly, but it is long lasting in 36 mg/mL

concentration for gram-positive and gram-negative bacterial strains (Study Ia and

Ib) and in 72 mg/mL concentration for Candida and Aspergillus species (Study IIa

and IIb). S. aureus isolates, including MRSA were the most sensitive species for

HICA with an IC90 of 1.1 mg/mL and P. aerunginosa with a MBC of 4.5 mg/mL.

The most tolerant bacterial isolates belonged to Lactobacillus and Enterococcus

species. The growth of Lactobacillus species and E. faecalis was reduced over 90%

by 9 mg/mL of HICA. In subcidal concentrations, HICA was bacteriostatic for all

82

bacterial isolates, except clinical and reference strains of E. coli and F. nucleatum

in Study Ia and Ib. Since MBC was 18 mg/mL for all E. coli, including ESBL isolate,

the present results were in line with the findings of Hietala and collegues (1979),

in which the MBC for E. coli was 4 mg/mL. It is also noteworthy that IC90 was 4.5

mg/mL for ESBL isolate of E. coli in the present study.

One interesting finding of a pilot study in anaerobic test conditions in pH 7.0

was that ≤36 mg/mL of HICA was bactericidal also for both isolates of obligately

anaerobic Porphyromonas gingivalis detected from human gingival sulcus (T-

75048 and ATCC 33277) at 48 h. MBC for F. nucleatum isolates increased to 36

mg/mL of concentration in neutral pH. For A. israelii isolates, MBC in 36 mg/mL

of HICA was not achieved until 96 h incubation. P. gingivalis isolates were

excluded from the present investigation, because they did not survive in the test

condition of Study Ib (pH 5.8) in pure thioglycollate broth. These findings support

the previous knowledge of the antimicrobial activity of HICA, which is at its best

near pH 5.2 (Hietala et al. 1979). The effect is also seen in neutral pH in

anaerobiosis, which was not observed in aerobic conditions for the other bacterial

species.

A fungistatic effect of HICA was clearly seen for C. albicans, C. dubliniensis,

and C. tropicalis in 18 mg/mL concentration and at least in 36 mg/mL concentration

for C. glabrata and C. krusei, whilst MBC was 72 mg/mL for all yeast isolates in

Study IIa. IC50 is the detection criteria for MICs for fluconazole (Clinical and

Laboratory Standards Institute 2008). When the same criterion was used, IC50

would be 18 mg/mL of HICA for all Candida species. Earlier, fungicidal activity

has been demonstrated in 4–10 mg/mL concentration of HICA against

dermatophytic Trichophyton sp. (Hietala et al. 1979) and against spore forming

moulds: A. fumigatus, A. niger, and A. tubingensis, Penicillium anomala, P.

roqueforti, and P. crustosum (Broberg et al. 2007, Ndagano et al. 2011). The results

of Study IIb supported previous findings of the susceptibility of A. fumigatus being

≤36 mg /mL. In conclusion, MBCs and MFCs were marginally higher in the present

study than in formerly-published studies. However, ten-fold differences in

microbicidal concentrations between different studies can be explained by normal

variation or minor changes in test conditions, for example in the density of original

microbial inoculum.

83

Antimicrobial mechanisms

In Study I and II, antimicrobial activity was associated to HICA molecule and not

its precursors: leucine or KICA. This is line with the findings of Ward and collegues

(1999), who showed that E. faecalis growth is increased in the presence of KICA,

because of its ability to use KICA as an energy source. For HICA elimination,

oxidation of HICA to KICA is needed. In anaerobic conditions, spontaneous

oxidation reactions are naturally restricted. Gram-positive bacteria seem to react

similarly with bacteriostatic inhibition in subcidal concentrations regardless of the

presence or absence of oxygen. In the present study, the bacteriostatic effect of

HICA was also seen under bactericidal concentrations in anaerobiosis for A. israelii.

Strict bactericidal effect of HICA was seen against all other isolates of gram-

negative bacterial species, except for the ESBL isolate of E. coli, for which

bacteriostatic inhibition in subcidal concentrations was also seen.

The highest MBCs of HICA were seen for those species, which are known to

express activity of enzymes from HicDH family. The presence of these enzymes

may strengthen the tolerance of gram-positive bacteria to HICA. It may be

explained by the regulation of oxidation in the presence of HicDH in Lactobacillus

species and mandelate-DH in E. faecalis isolates, because of potential cross-

reactions with substrates of the relative enzymes (Yamakazi & Maeda 1986,

Tamura et al. 2002, Chambellon et al. 2009). Extended production of betalactamase

enzymes might increase the sensitivity of gram-negative bacterial strains of E. coli.

In contrast, E.coli may have potential for increased tolerance, also because of the

D-HicDH coding gene in its genome (Lerch et al. 1989). Low oxidoreductase

enzyme activity or absent microbial capacity to regulate HICA metabolism may

explain the higher sensitivity of microbial species, especially in S. aureus (Study

Ia).

Since a fungistatic effect was also seen for yeasts in subcidal concentration in

Study IIa, potential impact of oxidoreductase enzymes on the antifungal effect of

HICA cannot be excluded. This is supported by earlier findings of a regulator gene

of HicDH in C. albicans (Jones et al. 2004, Kanehisa et al. 2014). In addition,

HICA may have an impact on the virulence of C. albicans and A. fumigatus by

inhibiting their hyphal formation (Study IIc), which is in line with the findings of

Nieminen and collegues (2014a), who showed weakening of hyphal structures in

terms of C. albicans biofilm formation in the presence of HICA.

In Study IIb, HICA was fungicidal for the majority of Aspergillus isolates. All

A. fumigatus and A. terreus isolates were sensitive for HICA, whilst all A. flavus

84

isolates were resistant (Study IIb). Interestingly, the azole-resistant isolates were

sensitive for HICA, and vice versa. This finding may contribute to both the

antifungal mechanism of HICA and to the resistance mechanism of A. flavus for

HICA. The main antifungal mode of action of azoles is inhibition of ergosterol

synthesis of fungal cell membrane. Modification of this target is one possible

resistance mechanism for azoles. The other ones are active efflux or changes in

cytochrome P-450 enzyme (Ghannoum & Rice 1999). Therefore, these

mechanisms may be involved in the tolerance of Aspergillus isolates for HICA, too.

Bactericidal and fungicidal activity of HICA may contribute to its amphipatic,

hydrophilic, and hydrophobic properties, which allow the potential ability to reach

the targets and to be active in different environments. A hydroxyl group replaces

the amino group of leucine in HICA. As an amino acid derivative, HICA probably

affects protein metabolism and protein structures of the cells. Connection via an

amino group is needed for formation of peptide and protein chains. If HICA is

attached to a protein chain instead of leucine or other amino acids, the connection

of a protein chain by an amino group could be prevented. This may be a mechanism

needed for protein chain reproduction in living cells, but could be destructive to a

larger extent. Earlier mentioned genes and enzymes may participate in the

regulation of HICA-derived reactions.

Suitability for root canal medication (III, IV)

The physical property of 400 mg/mL of HICA paste is suitable for the delivery and

long retention in root canals. The activity of HICA against E. faecalis and Candida

species is favourable to endodontic use. At least in theory, other more sensitive

species may not be able to colonize root canal in the presence of active

concentrations of HICA. The antibacterial effect of HICA emerges slowly, but it is

long lasting. No considerable activity was seen after the first hour of incubation. It

is in line with the earlier results, in which HICA had no effect against Candida

biofilms after 12 h, but was highly active at 24 h incubation (Nieminen et al. 2014a).

A single potentially-vulnerable concentration of HICA for interactions was

chosen to observe the impact of biological factors such as dentine, hydroxyapatite,

and serum albumin on its antibacterial activity (Study IIIa). This concentration was

equal to MBC of HICA for E. faecalis (Study Ia). The concentrations of

comparators and controls: Ca(OH)2, CHA, and CHG solutions were adapted from

previous studies (Haapasalo et al. 2000, Portenier et al. 2001). In Study IIIb, dose

dependence of the impact of biological factors on three increasing concentrations

85

of HICA was evaluated. The results indicated that increasing concentration of

HICA was shown to provide more tolerance to interactions with DP, HA, and BSA.

Therefore, a ten-fold higher concentration of HICA was chosen for the observations

in the experimental infection study (Study IV). This dose resulted in 1.5 mg of

HICA in each root canal, which is below the daily dose of threshold of concern by

oral administration (1.8 mg/capita/day), according to EFSA. In Study IV, CHG was

tested in a hundred-fold higher concentration and Ca(OH)2 slurry was tested in

four-fold higher concentration, which was an equal concentation to HICA paste.

This concentration of CHG was comparable to that used in clinical conditions (5–

20 mg/mL). Concentrations of Ca(OH)2 slurry are generally not informed, whilst

the antimicrobially active concentration of saturated solution is a temperature-

dependent physical constant, being 1.45 mg/mL at 37ºC (Dean 1973). This is

almost ten-fold higher than the concentration of chlorhexidine solutions in Study

IIIa and over ten-fold lower than the CHG concentration in Study IV.

In the interaction study (Study IIIa and IIIb), HICA maintained its antibacterial

activity in the presence of clinically relevant low concentrations of dentine (≤18.7

mg/mL) despite the change in nutritional conditions. At bactericidal concentrations,

HICA was more active than Ca(OH)2 and the activity was equal to CHA or CHG

without potential inhibitors at 24–48 h. Present findings are in line with earlier

results of inhibiting effect of commonly-used antimicrobials in endodontology by

dentine (Haapasalo et al. 2000, 2007, Portenier et al. 2001, 2002, 2006). In addition,

increasing pre-incubation time of DP together with antimicrobials increases the

inhibiting effect of dentine (Haapasalo et al. 2000). Therefore, the ability of HICA

to resist dentine in spite of one hour pre-incubation indicates its potential for root

canal medication (Study IIIa).

In the experimental infection study (Study IV), antibacterial activities of

antimicrobial agents were examined from two separate perspectives by relative

comparison to negative control and by quantitative comparison of the amount of

successfully disinfected roots. Relative comparisons were based on

spectrophotometric analyses of broth cultures, in which antibacterial activity of

HICA was higher than that of the common root canal medicaments in the deeper

dentine samples (0.1–0.2 mm depth) of the root canal wall. It was similar to CHG

in the inner dentine samples (≤0.1mm depth). Quantitative comparisons were based

on both spectrophotometric analyses of broth cultures and viability testing on agar.

In these experiments and analyses, significantly more residual roots (depth of ≥0.2

mm) without bacterial growth was achieved with HICA treatment in comparison to

Ca(OH)2 medication, but not with CHG. HICA was the only agent, which

86

demonstrated significantly better antibacterial activity in comparison to sterile

saline in residual roots. HICA had long-term antibacterial activity against E.

faecalis in human dental root canals of extracted human teeth. Antibacterial activity

of HICA was comparable or better than currently-used interappointment

medicaments in endodontology. Regarding the results of Study III and IV, HICA

could be a promising interappointment antimicrobial agent for root canal treatments.

6.3.2 Calcium hydroxide (III, IV)

In the interaction study (Study IIIa), high concentration (187 mg/mL) of HA

prevented the activity of Ca(OH)2 slurry, which suggests the previous findings of

HA as main responsible substance in inactivation of Ca(OH)2 by dentine

(Haapasalo et al. 2007), whilst only lower concentrations of free HA in powder

could reflect in clinical conditions. The antibacterial activity of Ca(OH)2 slurry was

enhanced in the presence of DP in the present study, which is contrary to earlier

results (Haapasalo et al. 2000, Portenier et al. 2001). The results of Study IIIa

indicated that DP has moderate antimicrobial capacity by itself, which may be a

good explanation for the better antimicrobial effect of Ca(OH)2 with DP. In addition,

at least in clinical conditions, the water component of dentine may participate as a

source of humidity in the antimicrobial action of Ca(OH)2. It is also worth noting

that Ca(OH)2 slurry failed to sustain its antimicrobial activity with clinically-

relevant low concentrations of free DP in Study IIIa.

In the comparison of four similarly performed studies (Table 10), all studies

showed limited or no antimicrobial activity of Ca(OH)2 solution with DP in 24 h

incubation with respect to the initial bacterial counts of each study (Study IV)

(Haapasalo et al. 2000, Portenier et al. 2001, Athanassiadis et al. 2010). The

antibacterial activity of Ca(OH)2 is based on its alkalinity, and the substitution of

hydroxyl ions to the liquid phase from the slurry is needed for maintaining the high

pH. Athnassiadis and collegues (2010) showed antibacterial potential of a Ca(OH)2

slurry (Pulpdent® paste) with DP against E. faecalis at 24 h, which is in line with

present results (Study IIIa). This was also supported by a finding that the

commercial Ca(OH)2 paste sustained its high pH with 187 mg/mL of DP over a

one-hour incubation time, whilst the pH of Ca(OH)2 solution decreased from 12.5

to 9.0 in the same study.

87

Table 10. Comparison of antibacterial activity of Ca(OH)2 solution (0.55 mg/mL), Ca(OH)2

slurry (100 mg/mL) or commercial paste, CHA (0.17 mg/mL) and HICA (33.3 mg/mL) is

shown without and with 1.87–187 mg/mL of DP at 24 h (and ≥48 h) in four representative

studies in respect to initial bacterial counts of each study. In two studies only 187 mg

/mL was tested. Microbial solutions were diluted into peptone water (negative control),

except in one study to Tryptone soya broth. Final test concentrations of reagents

(mg/mL) are listed. (10 mg/mL = 1%).

Comparison Haapasalo

et al. 2000

Portenier

et al. 2001

Athanassiadis

et al. 2010

Sakko

et al. 2015

E. faecalis isolate A197A,

root canal

A197A,

root canal

ATCC29212,

urine

T-75359,

root canal

Preincubation 0 h, 1 h, 24 h 1 h 1 h 1 h

Breakpoints 1, 24 h 1, 24 h 1, 24, 72 h 1, 24, 48 h

Test agents Ca(OH)2 solution 0.55, CHA 0.17, 1.7

(NaOCl 0.33, IPI 20/40, 2/4)

Ca(OH)2

solution 0.55,

CHA 0.17

(IPI 2/4)

Ca(OH)2

solution 0.55,

Pulpdent paste®

(Ledermix®)

Ca(OH)2

solution 0.55, Ca(OH)2 slurry 100

HICA 33.3

CHA 0.17 (CHG 0.17)

Negative control PW 1.7 PW 1.7 Tryptone soya broth PW 1.7

Dentine powder 187 18.7, 187,

(75, 131)

187 1.87, 18.7, 187

Activity with no DP/ DP at 24 h (≥48 h)

Ca(OH)2 solution +++/- +++/- DP 187 +++/- (+++/-) +/+ (+/+) DP 187

+++/- DP 18.7 +/+ (+/+) DP 18.7

+/+ (+/-) DP 1.87

Ca(OH)2 slurry ND ND +++/+++ (+++/+++) ++/+++ (+/+++) DP 187

++/+++ (+/+++) DP 18.7

++/+ (+/+) DP 1.87

HICA 33.3 ND ND ND +++/- (+++/-) DP 187

+++/- (+++/ +++) DP 18.7

+++/- (+++/+++) DP 1.87

CHA 0.17 +++/+++ +++/+++ ND +++/+++ (+++/++)

+++: very good antimicrobial activity (>99% reduction in bacterial viability in comparison to initial bacterial

counts), ++: good antimicrobial activity (90–99% reduction), +: limited antimicrobial activity (50–90%

reduction), -: no antimicrobial activity (<50% reduction). PW: peptone water, NaOCl: sodium hypochlorite,

IPI: iodine potassium iodine, ND: not determined.

Present results of the antibacterial activity of Ca(OH)2 in root canals (Study IV) are

directly comparable to similar studies performed in extracted human teeth together

with three weeks E. faecalis infection and seven days medication from the direction

88

of root canal, which is assisted by embedding the root blocks into wax (Basrani et

al. 2002, 2003) (Table 11). Otherwise, the root surface may be more predisposed

for infection. The root blocks are usually cultured in the broth or without

embedding them. Three weeks infection of dentine is generally accepted for a good

practice to prepare experimental infection of dentine tubules providing E. faecalis

invasion and biofilm formation (Basrani et al. 2002, 2003, George et al. 2005,

Zapata et al. 2008, Du et al. 2015). Seven days medication could be an ideal timing

for the second appointment, which is also generally used in clinical microbiological

studies in vivo (Kim & Kim 2015).

In relative comparisons, no better antimicrobial activity of Ca(OH)2 slurry

against E. faecalis was shown in root canals compared to saline medication, which

is in line with earlier results and suggestions (Basrani et al. 2002, 2003, Zehnder

2012, Lee et al. 2013, Shokraneh et al. 2014). In the comparison of thirteen studies

with three weeks infection: two studies showed good antibacterial effect, five

studies showed limited effect and five studies showed no effect after ≤7 days of

incubation time (Table 11). The results of two studies with 14 days medication did

not deviate from the line and they showed limited or no antibacterial effect of

Ca(OH)2 in culture analyses. Method of analyses may have some influence on

detection of microbial cells. Delgado et al. (2010) showed smaller amount of living

bacterial cells after Ca(OH)2 medication by fluorescence microscopy in

comparison to culture analyses in respect to sterile saline treatment. Kim & Kim

(2014) reviewed eighteen experimental infection studies in human dental root

canals with more variable periods of infection with E. faecalis and medication times.

Twelve of them demonstrated limited activity of Ca(OH)2 and three of them good

activity, which is line with the underlying comparison (Table 11).

89

Table 11. Relative comparison of antimicrobial activity of Ca(OH)2 against E. faecalis in

infected human dental root canals of extracted teeth in respect to negative control

(saline, water, or no medication). Studies with three weeks of infection, ≤14 days of

medication and sampling methods by paper points, hand-instrumentation, or rotary-

instrumentation are accepted to this comparison. The results are based on viability

cultures on agar, spectrophotometric analyses of broth cultures, or viability analyses

of fluorescence microscopy.

Year Author N/group Control Sampling Analyses <7d 7d 14d

2001 Han et al. 12–17 No med. PF#60,90 S +

2002 Almyroudi et al. 8 Saline Round drills

016,018,021

C ++ ++ +

2002 Basrani et al. 5–15 Saline GG#4,5 S -

2003 Basrani et al. 5–15 Saline GG#4,5 S -

2007 Krithikadatta et al. 15 Saline GG#4,5 C +

2010 Delgado et al. 15 Saline GG#5,6 C (FM) - (+)

2010 Kandaswamy et al. 30 Saline GG#4,5 C +

2011 Madhubala et al. 8 Saline HI#50, PP C - +

2013 Lee et al. 12 Saline HI#90,110 S -

2014 Bhandari et al. 30 Saline GG#5 C +

2014 Javidi et al. 6–30 Water PP, GG#5 C - ++

2014 Shokraneh et al. 15 Water GG#4,5 C -

2015 Study IV 17 Saline GG#4,5 S

++: good activity with 90% reduction in bacterial viability or growth in comparison to negative control, +:

limited activity with 50–90% reduction, -: no activity with <50% reduction, PF: ProFile, PP: paper point, HI:

hand-instrument, GG: Gates-Glidden drill, C: culture, S: spectrophotometric, FM: fluoresence microscopy.

E. faecalis isolates used: Han/KCCM11814, Almyroudi/59876R, Bhandari/ATCC35550, Sakko/T-75359, all

other studies/ATCC29212.

In quantitative comparison of roots free of bacterial growth, Ca(OH)2 prevented the

bacterial growth in around 59–76% of the roots up to 0.2 mm depth, although it

was not better than sterile saline treatment (Study IV; n=17). Both Ca(OH)2 and

saline showed limited antibacterial effect in root canals in the present study. Earlier

reports based on quantitative analyses are controversial. Zehnder et al. (2006)

showed successful disinfection in 89–100% of the roots treated with Ca(OH)2 after

two weeks of E. faecalis infection and up to 0.15 mm depth of dentine (n=9). In

contrast, Basrani and collegues (2002, 2003) showed <10% of roots free of

bacterial growth to the depth of 0.2 mm (n=15). According to quantitative

comparisons in the present study, the antibacterial activity of Ca(OH)2 was limited.

90

Regarding the relative comparison to the sterile saline treatment, it was non-

existent. Therefore, a better antimicrobial activity should be sought.

6.3.3 Chlorhexidine (III, IV)

DP lacked substantial influence on the antibacterial activity of CHA or CHG in 48

h incubation (Table 12), which is in line with three earlier studies performed by DP

model (Portenier et al. 2002, 2006, Abbaszagedan et al. 2014). Similarly, no

substantial difference between the antibacterial activity of CHA and CHG was

observed in the present study. Although, delayed antibacterial effect of

chlorhexidine in the presence of DP is reported earlier at 1 h incubation (Haapasalo

et al. 2000, Portenier et al. 2001, 2002, 2006), which may also contribute to the

preincubation time with DP beforehand. At 24 h, chlorhexidine is very active

against E. faecalis even at a concentration of a thousand-fold that used in the studies

of the underlying comparison (Abbaszagedan et al. 2014) (Table 12). In the present

study, antimicrobial effect of CHA with HA or BSA, and CHG with BSA was

delayed with 187 mg/mL concentration at 1 h (Study IIIa). However, such a high

concentration of 187 mg/mL of free HA or serum albumin is hardly ever present in

root canals, but they served as technical comparators in Study IIIa. In addition, 1 h

incubation is very short in comparison to the use in interappointment medication.

In relative comparisons of Study IV, the antibacterial activity of CHG solution

was limited ≤0.1 mm depth of dentine with >50% increase of reduction in bacterial

growth and no effect in 0.1–0.2 mm depth with <50% increase of reduction with

respect to sterile saline treatment. This is in line with the results of Lee et al. (2013),

who showed that the ability of 20 mg/mL of CHG gel to eliminate bacteria is limited

up to 0.2 mm depth. In contrast, Basrani and collegues (2002, 2003) demonstrated

at least good antibacterial activity of chlorhexidine solution. They also showed that

chlorhexidine gel is less active than solution in human dental root canals ex vivo.

91

Table 12. Comparison of antimicrobial activity of HICA (33.3 mg/mL) and CHG solution

(0.00007–0.7 mg/mL) with and without DP at 24 h (≥48 h) in representative studies is

shown. In one study, the form of chlorhexidine (CHX) was not informed. The impact of

1.87–187 mg/mL of DP was examined, in three of these studies only 187 mg/mL of DP

was tested. Final test concentrations of reagents (mg/mL) are shown.

Comparison Portenier

et al. 2002

Portenier

et al. 2006

Abbaszagedan

et al. 2014

Sakko

et al. 2015

E. faecalis isolate A197A,

root canal

A197A,

root canal

AGH011,

root canal

T-75359,

root canal

Preincubation 1 h 0 h 0 h 1 h

Breakpoints 1, 24 h 1, 24 h 1, 4, 24 h 1, 24, 48 h

Test agents CHG 0.07

(IPI 1/2)

CHG 0.03,

0.07, 0.7 CHX 0.07, 0.7

(NaOCl 8,

Ag NPs)

HICA 33.3

CHG 0.17

(Ca(OH)2

solution 0.55

Ca(OH)2 slurry 100

CHA 0.17) Negative control PW 1.7 PW 1.7 PW 1.7 PW 1.7

Dentine powder 187 187 187 1.87, 18.7, 187

Activity with no DP/ DP at 24 h (≥48 h)

HICA 33.3 ND ND ND +++/- (+++/-) DP187

+++/- (+++/++) DP 18.7

+++/- (+++/+++) DP1.87

CHG

0.00007 ++/NDchx

0.03 +++/+++

0.07 +++/+++ +++/+++ +++/+++chx

0.17 +++/+++(+++/+++)

0.7 +++/+++ +++/+++chx

+++: very good antimicrobial activity (>99% reduction of bacterial viability in comparison to initial bacterial

counts), ++: good antimicrobial activity (90–99% reduction), +: limited antimicrobial activity (50–90%

reduction), -: no antimicrobial activity (<50% reduction). PW: peptone water, Aq NPs : silver nanoparticles,

NaOCl: sodium hypochlorite, ND: not determined.

In quantitative comparison of the cultures in broth and on agar, 20 mg/mL of CHG

quite effectively eliminated E. faecalis from dentine up to 0.2 mm of depth, which

was detected in spectrophotometric analyses and in viability testing. The finding of

successful disinfection in 88% of the roots is line with earlier results (Basrani et al.

2002, 2003, Bhandari et al. 2014). In relative comparison to sterile saline, only

HICA treatment could prevent all bacterial growth. This was seen in the depth of

0.1 to 0.2 mm of dentine, in the present study.

92

6.4 Future work

In the future, the activity of HICA could be evaluated against general human

pathogenic bacterial and fungal strains by using in vitro biofilm models (Ceri et al.

1999, Ramage et al. 2001). HicDH enzyme expression (Broadbent et al. 2004),

expression of HicDH regulating genes (Lerch et al. 1989), and enzyme activity in

different microbial species could be observed (Bachmann et al. 2009). The safety

of HICA for host cells such as fibroblasts and macrophages could be further

evaluated in cytotoxicity assays (Mosmann 1983).

Molecular methods will broaden the knowledge of the causative organisms of

root canal infections. So far, E. faecalis is known to be the most prevalent organism

of persisting root canal infections and could be used as a primary test organism

before anything else is shown. Suitability testings for endodontic use could be

extended by observing the potential interactions between antimicrobials and

biological factors present in root canals and their dose dependence in clinically

relevant concentrations such as 1–100 mg/mL (Haapasalo et al. 2000, Portenier et

al. 2001) and antimicrobial properties of dentine alone could be further evaluated

(Smith et al. 2012). The viability of bacterial cells in dentine tubules could be

observed in the experimental infection model of human dentine using confocal laser

scanning microscopy, also after exposure to antimicrobials (Basrani et al. 2002,

2003, Zapata et al. 2008, Du et al. 2015).

For the assessment of antimicrobial activity and efficacy of HICA as a root

canal medicament in clinical conditions randomized controlled clinical studies

would be required (Molander et al. 2007). At first, the activity of 400 mg/mL of

HICA should be compared to the most general interappointment medication, which

is Ca(OH)2. This concentration of HICA is at least ten-fold higher than the

bactericidal concentration and over five-fold higher than the fungicidal

concentration of HICA. In the clinical investigations, seven days is the most widely

accepted period of time for medication (Kim & Kim 2015). The microbiological

status of root canals is usually evaluated after the first access, immediately before

the delivery of root canal medicament after chemo-mechanical preparation and

after the interappointment medication. In several studies, paper point sampling (for

1 min) after gentle filing of root canal walls is used for the assessment of

microbiological status of the root canals (Vianna et al. 2007, Rôças & Siqueira et

al. 2011, Paiva et al. 2013, Sinha et al. 2013).

Antimicrobial activity of interappointment medication can be observed only in

root canals with detected microbial infection (Sjögren et al. 1991). On the other

93

hand, Safavi et al. (1985) observed prevention of microbial growth during

interappointment medication in root canals initially free of growth. It is worth

noting that initial disinfection with ≥0.5% of NaOCl solution together with

mechanical preparation may lead to ≥50% decrease of the amount of infected root

canals before interappointment medication (Byström & Sundqvist 1983, Sjögren et

al. 1991). Therefore, the results of chemo-mechanical preparation,

interappointment medication, and combined results of chemo-mechanical

preparation and interappointment medication should be reported separately.

Molecular methods and culture methods could be needed for appropriate analyses

of the results.

Interappointment medication is one important part of root canal treatment,

since microbiological status immediately before root canal obturation predicts

healing of AP (Molander et al. 2007). Therefore, the efficacy of HICA should also

be studied in this context. Periapical index (PAI) scoring method could be used for

following the radiological changes (Ørstavik et al. 1986). Clinical signs and

symptoms should also be followed in the meanwhile. Early and late complications

could probably be seen during three years of follow-up (Trope et al. 1999, Weiger

et al. 2000, Waltimo et al. 2005, Molander et al. 2007). Demonstration of

significant differences between interappointment medications requires large

experimental groups. Subjects of one to two hundred per group are needed

depending on the statistical methods used, if the effect of Ca(OH)2 treatment is

estimated at the level of 75% and for HICA at 90% (Study IV) and the hypothesis

would be tested at the level of 0.05 with power of 0.8, for example.

6.5 Potential of HICA for other uses (I, II)

HICA has broad-spectrum bactericidal activity for several facultative and obligate

anaerobic bacterial species, which indicates its potential for use in antisepsis and

disinfection. Bacterial HicDH activity may reflect tolerance for HICA sensitivity.

In addition to endodontic use, HICA could be a useful agent in the treatment of

superficial oral and genitourinary mycositis, skin and wound infections, and

disinfection of foreign body materials such as catheters, prostheses, and hearing

aids (Fig. 15). In particular, the high sensitivity of staphylococcal isolates for HICA

indicates its potential for the treatment of skin and foreign body infections. It could

also be a useful agent in the treatment of diabetic wounds, which are often caused

by E. faecalis. The activity for antibiotic-resistant strains of MRSA and ESBL may

strengthen its potential for the treatment of nosocomial infections.

94

HICA has fungicidal activity against several Candida and Aspergillus species,

which indicates its potential for the treatment of fungal infections. The influence of

HICA on C. albicans hyphal formation and A. fumigatus filamentation may weaken

the virulence of these human pathogens. Therefore, patients could benefit from the

use of HICA in the treatment of superficial fungal oral mycositis, sinusitis, skin

infections, or skin, mucosal and burn wound infections, or genitourinary infections,

or in the disinfection of foreign-body materials (Fig. 15). Fungicidal activity

together with bactericidal activity supports the potential of HICA for the treatment

of mixed flora infections, which is typical for superficial infections in general.

Importantly, the use of topical antiseptics with new mode of actions in the treatment

of superficial infections instead of antibiotics may restrict the development of

extensive resistance to systemic antimicrobials.

Fig. 15. Schematic illustration of potential clinical uses of antimicrobial HICA. The root

canal infection could be prevented in dental irreversible pulpitis or sterile pulpal

necrosis. The infection could be treated in primary or post-treatment apical

periodontitis (AP). HICA has potential also for the prevention or treatment of other

superficial infections, which are listed in the four background boxes.

95

7 Conclusion

Previous studies have mainly concentrated on the expression of HICA or the

HicDH enzyme in single bacterial species, primarily in nutrition. The antimicrobial

spectrum of HICA has not been defined previously. This study series aimed to gain

some knowledge of the antimicrobial spectrum of HICA and its usefulness for the

treatment of superficial infections. The interactions with potential inhibitors and

antibacterial activity of HICA against E. faecalis in experimentally-infected dental

root canals were evaluated to gain some evidence of whether HICA would be a

suitable antimicrobial agent for the treatment of root canal infections as an

interappointment medication. The hypotheses, that HICA could be a useful agent

for the treatment of topical infections as a broad-spectrum antibacterial and

antifungal agent in general and specifically it may have potential for the treatment

dental root canal infections, were accepted.

The results of the present investigation indicate that the antibacterial activity

of HICA remains high despite the presence of possible clinically-relevant dentine

concentrations. HICA also has long-term antibacterial activity against E. faecalis

in human dental root canals. At bactericidal concentrations of HICA, it was more

active than Ca(OH)2 and in ten-fold higher concentration, the activity of HICA was

at least the same as with chlorhexidine. Especially, the effect of HICA increased in

the deep parts of dentine in comparison to Ca(OH)2 or CHG. Saline treatment

resulted in considerable decrease in bacterial growth in comparison to initial growth,

which may be explained by normal bacterial growth curves and nutritional

depletion. In the present studies (I–IV), HICA and CHG were clearly microbicidal

for all tested organisms, except HICA for three A. flavus isolates. In the future,

more knowledge about the activity of HICA against microbial cells in biofilms is

needed (Nieminen et al. 2014a).

Microbial resistance to antimicrobials cause remarkable problems in the

treatment of infections. The results of the present study indicate that leucine-HICA

metabolic pathway could be a potential target in the development of new

antimicrobials. Microbial resistance to antibiotics could be prevented by managing

superficial infections with topical antimicrobials, while systemic therapy could be

saved for the treatment of deep infections. Microbial tolerance to antimicrobials is

a problem also in the treatment of root canal infections. New mechanisms of

antimicrobials could reduce the problems in both fields.

The activity of HICA against microbial biofilms, its mode of action, enzyme

expression, and enzyme activities needed for HICA-metabolism of different

96

microbial species remain to be clarified. For safety, cell reactions in the presence

HICA at microbicidal concentrations should be tested further before clinical studies,

since an optimal root canal medication must possess good biocompatibility in case

of extrusion or leakage from the root canal system to surrounding tissues. In

addition, the antimicrobial potential of dentine and the compounds responsible for

it should be further evaluated. Next generation sequencing techniques in

microbiology may provide far more knowledge about the causative organisms of

infections including root canal infections. Therefore, new strategies of

investigations will arise in the future.

Regarding the results of the susceptibility studies together with anti-

inflammatory properties (Nieminen et al. 2014b) of this relatively well-tolerated

substance (European Food Safety Authority 2012), the present investigation

provides basic evidence of HICA for general use in antisepsis and disinfection. The

results of the interaction study and experimental infection study in human dental

root canals indicate moderate evidence of antimicrobial potential of HICA for

further investigations in endodontology.

97

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Original publications

I Sakko M, Tjäderhane L, Sorsa T, Hietala P, Järvinen A, Bowyer P, Rautemaa R (2012) 2-hydroxyisocaproic acid: a new potential topical antibacterial agent. Int J Antimicr Agents 39: 539–540.

II Sakko M, Moore C, Novak-Frazer L Rautemaa V, Sorsa T, Hietala P, Järvinen A, Bowyer P, Tjäderhane L, Rautemaa R (2014) 2-hydroxyisocaproic acid (HICA) is fungicidal for Candida and Aspergillus species. Mycoses 57: 214–221.

III Sakko M, Tjäderhane L, Sorsa T, Hietala P, Rautemaa R (2015) 2-hydroxyisocaproic acid (HICA) and chlorhexidine resist inactivation by dentine. Int Endod J doi: 10.1111/iej.12465.

IV Sakko M, Tjäderhane L, Sorsa T, Hietala P, Rautemaa R (2015) 2-hydroxyisocaproic acid (HICA) is bactericidal in human dental root canals ex vivo. Manuscript.

Reprinted with permission from Elsevier (I) and John Wiley & Sons (II, III, IV).

Original publications are not included in the electronic version of the dissertation.

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Marjut Sakko

ANTIMICROBIAL ACTIVITY AND SUITABILITY OF2-HYDROXYISOCAPROIC ACID FOR THE TREATMENT OF ROOT CANAL INFECTIONS

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU, FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU;FINNISH DOCTORAL PROGRAM IN ORAL SCIENCES