for review onlyrdo.psu.ac.th/sjstweb/ar-press/58-dec/19.pdf · for review only 1 1 fresh garlic...

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Fresh garlic extract inhibits Staphylococcus aureus biofilm

formation under chemopreventive and chemotherapeutic conditions

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2015-0158.R1

Manuscript Type: Original Article

Date Submitted by the Author: 23-Nov-2015

Complete List of Authors: Ratthawongjirakul, Panan; Chulalongkorn University, Transfusion Medicine

and Clinical Microbiology Thongkerd, Vorraruthai; Chulalongkorn University,

Keyword: Chemistry and Pharmaceutical Sciences, Garlic, Staphylococcus aureus, Biofilm

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Songklanakarin Journal of Science and Technology SJST-2015-0158.R1 Ratthawongjirakul

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Fresh garlic extract inhibits Staphylococcus aureus biofilm formation under chem-1

opreventive and chemotherapeutic conditions 2

3

Panan Ratthawongjirakul1*and Vorraruthai Thongkerd

2 4

aDepartment of Transfusion Medicine and Clinical Microbiology, Faculty of Allied 5

Health Sciences, Chulalongkorn University, Bangkok, Thailand 10310 6

bBachelor of Science Program in Medical Technology, Faculty of Allied Health Scienc-7

es, Chulalongkorn University, Bangkok, Thailand 10310 8

9

*Corresponding author 10

Panan Ratthawongjirakul, Department of Transfusion Medicine and Clinical Microbiol-11

ogy, Faculty of Allied Health Sciences, Chulalongkorn University, 154 Rama I Rd, 12

Pathumwan, Bangkok, Thailand 10310 13

Tel: +662-218-1084; Fax: +662-218-1083 14

E-mail: [email protected] 15

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17

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Abstract 1

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) 2

are the leading aetiological pathogens of nosocomial infections worldwide. These bacte-3

ria form biofilms on both biotic and abiotic surfaces causing biofilm-associated infec-4

tions. Within the biofilm, these bacteria might develop persistent and antimicrobial re-5

sistant characteristics resulting in chronic infections and treatment failures. Gar-6

lic exhibits broad pharmaceutical properties and inhibitory activities against S. aureus. 7

We investigated the effects of aqueous fresh garlic extract on biofilm formation in S. 8

aureus ATCC25923 and MRSA strains under chemopreventive and chemotherapeutic 9

conditions. The viable bacteria and biofilm levels were quantified through colony count 10

and crystal violet staining, respectively. The use of fresh garlic extract under both con-11

ditions significantly inhibited biofilm formation in S. aureus strains ATCC25923 and 12

MRSA. Garlic could be developed as either a prophylactic or therapeutic agent to man-13

age S. aureus biofilm-associated infections. 14

15

Keywords: fresh garlic extract, biofilms, MRSA, Staphylococcus aureus 16

17

Introduction 18

Staphylococcus aureus is the leading causative pathogen of nosocomial infec-19

tions worldwide. It has been estimated that 50 to 70% of nosocomial S. aureus infec-20

tions result from methicillin-resistant Staphylococcus aureus (MRSA) (Palavecino, 21

2004; Lodise and McKinnon, 2005). MRSA strains are resistant to penicillinase-stable 22

pencillins mainly based on the presence of the mecA gene, which encodes a novel peni-23

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cillin-binding protein-2a (Chambers, 2001). MRSA strains have been implicated in 1

many difficult-to-treat infections, reflecting the antibiotic-resistant characteristics of 2

these bacteria. Therefore, patients infected with MRSA might be at increased risk for 3

delayed treatment, morbidity, and mortality (Lodise and McKinnon, 2005). S. aureus 4

are characterised by the formation of biofilms on biomaterials, damaged tissues, and 5

most commonly on indwelling medical devices, causing biofilm-associated infections 6

(Otto, 2008; Makino et al., 2013). These infections are becoming more common and 7

occur widely, reflecting the increased use of indwelling medical devices over the past 8

few decades (Crnich and Maki, 2002). In clinical settings, these organisms persist in 9

sessile environments, such as in biofilms, resulting in chronic infections (Gowrishankar 10

et al., 2012). Over a half of S. aureus, including MRSA, has the ability of biofilm for-11

mation in various levels (Indrawattana et al., 2013; Rezaei et al., 2013). Furthermore, S. 12

aureus also gain increased resistance to antimicrobial agents through biofilm formation 13

as a bacterial survival strategy (Donlan and Costerton, 2002; Hall-Stoodley et al., 14

2004). In addition, nosocomial MRSA infection is based on non-specific mechanism of 15

resistance, which biofilm formation is involved (Otto, 2008). Consequently, biofilms 16

formed by MRSA might become more resistant to most available antimicrobial agents, 17

resulting in treatment failures. 18

A biofilm is “a matrix enclosed bacterial population that irreversibly adheres to 19

each other and to a substratum” (Costerton et al., 1995). Bacteria initiate biofilm for-20

mation in response to environmental factors, such as nutrient and oxygen availability 21

(Thomas and Lehman, 2006). In Staphylococci, the intercellular adhesin (ica) operon 22

encodes the extracellular polymeric biofilm matrix, referred to as polysaccharide inter-23

cellular adhesin (PIA) (Gotz, 2002). The nature of the biofilm structure confers inherent 24

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antimicrobial resistance. Biofilm-associated infections are 10 to 1,000 times more re-1

sistant to conventional antimicrobial agents (Thien-Fah and O’Toole, 2001). Several 2

antibiotics have been shown to significantly affect S. aureus planktonic cells, but not the 3

bacteria in the biofilm. Rifampicin rapidly killed planktonic MRSA but did not signifi-4

cantly reduce biofilm growth, and the biofilm remained established on the Silastic sur-5

face (Jones et al., 2001). Moreover, vancomycin, quinupristin/dalfopristin, and linezolid 6

showed little effect on the viability of bacteria in biofilms in vitro, and the efficient to 7

killing of bacterial cells in biofilms was observed only at higher concentrations (50, 500 8

and 1,000 µg/ml) of these individual agents (El-Azizi et al., 2005). The occurrence of 9

penicillin resistant biofilm producing S. aureus varied from 86.3 to 88 %, followed by 10

oxacinlin resistance (72.7 to 82%), cefazolin resistance (62.6 to 63.6%) and ciprofloxa-11

cin resistance (53.7 to 54.5%). Furthermore 53.7% of biofilm producing MRSA isolates 12

were also resistant to ciprofloxacin while 17.6% of non-biofilm producing MRSA were 13

ciprofloxacin resistant(Agarwal and Jain, 2012). The antimicrobial resistance of S. au-14

reus biofilm-associated infections has become a global problem, resulting in the wide 15

spread of nosocomial infections. 16

Garlic (Allium sativum L.) and garlic extracts have been previously demonstrat-17

ed as effective in inhibiting the growth of different bacterial pathogens, including 18

Staphylococci and MRSA (Tsao and Yin, 2001; Tsao et al., 2003; Rattanachaikunsopon 19

and Phumkhachorn, 2009; Houshmand et al., 2013). The chemical analysis of garlic oil 20

extract showed that 54.5% of the total sulphides comprise diallyl monosulphide, diallyl 21

disulphide, diallyl trisulphide and diallyl tetrasulphide, and the minimum inhibitory 22

concentration of whole garlic oil extract against MRSA was 32 μl/ml, whereas the MICs 23

for the individuals sulphide compounds were 32, 12, 8 and 2 μl/ml, respectively (Law-24

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son et al., 1991). Allicin, the active compound in fresh garlic extract, also inhibited the 1

proliferation of MRSA in a dose-dependent manner (Cutler and Wilson, 2004). Recent-2

ly, garlic extract has been shown to diminish the biofilm formation of some microbes, 3

such as Staphylococcus epidermidis, Pseudomonas aeruginosa, and Candida albicans 4

(Perez-Giraldo et al., 2003; Bjarnsholt et al., 2005; Shuford et al., 2005). The aim of the 5

present study was to investigate the effects of aqueous fresh garlic extract on biofilm 6

formation in S. aureus and MRSA under two in vitro culture conditions, including a 7

chemopreventive setting, in which the bacteria were grown in the presence of garlic, 8

and a chemotherapeutic setting, in which garlic was added after the formation of the 9

mature biofilm. 10

11

Materials and methods 12

Bacterial strains 13

S. aureus strain ATCC25923 and one clinical MRSA strain isolated from the 14

Microbiology Unit of Ramathibodi Hospital, Bangkok, Thailand, were used in the pre-15

sent study. The MRSA isolate was detected with cefoxitin (30 μg, Oxoid, Hampshire, 16

UK) using the disk diffusion method, according to the Clinical and Laboratory Stand-17

ards Institute (CLSI) document M100-24 (CLSI, 2013). The bacteria were grown on 18

tryptic soy agar (TSA, Oxoid, Hampshire, UK) and incubated at 37 °C for 24 h. 19

Fresh garlic extract preparation 20

To prepare fresh garlic extract (FGE), 200 g of garlic cloves were peeled and 21

roughly crushed in sterile distilled water. A final concentration was adjusted to 20 22

mg/ml. The extract was centrifuged at 6,000 rpm for 10 min, and the supernatant was 23

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filtered through a 0.45-μm membrane. The stock solution of FGE was stored at -80 °C 1

until further use. 2

Minimum inhibitory concentration (MIC) tests 3

The MIC of FGE was determined using the broth dilution technique in accord-4

ance with the CLSI document M07A9 (CLSI, 2013). The FGE stock solution was dilut-5

ed in Mueller-Hinton broth (Oxoid, Hampshire, UK) to final concentrations of 128, 256, 6

512, 1,024, 2,048, 4,096, 8192, and 16,384 μg/ml. After culturing for 24 h, the S. aureus 7

ATCC25923 and MRSA bacterial colonies were suspended in 0.85% (w/v) sterile nor-8

mal saline, and the final bacterial concentrations were adjusted to a 0.5 McFarland den-9

sity standard (~1-2x108 CFU/ml). The bacterial suspensions were added to Mueller-10

Hinton broth containing FGE at one of the concentrations specified above. The tubes 11

were incubated at 37 °C for 16-18 h. The MIC was reported as the lowest concentration 12

of the compound required to visibly inhibit the growth of bacteria. 13

Screening of biofilm production 14

Biofilm formation in both S. aureus ATCC25923 and MRSA was preliminarily 15

evaluated through cultivation on Congo red agar (CRA) (Croes et al., 2009). Briefly, 16

CRA plates were prepared using TSA containing 0.08% (w/v) Congo red (Sigma, Mis-17

souri, USA) and 5% (w/v) sucrose. Plates were incubated at 37 °C for 24 h. The follow-18

ing descriptions were used to interpret the colony phenotypes: black colonies with 19

rough, dry surfaces were considered positive for slime (biofilm) production, whereas 20

red colonies with shiny surfaces were considered negative for slime production. 21

Biofilm formation assay 22

Biofilm formation assay was established as previously described (Stepanovic et 23

al., 2000) with some modifications. After culturing for 24 h, the S. aureus ATCC25923 24

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and MRSA bacterial colonies were suspended in tryptic soy broth (TSB, Oxoid, Hamp-1

shire, UK). For chemopreventive conditions, the bacterial suspension was inoculated 2

into flat-bottomed 96-well polystyrene microtiter plates (Nunc, New York, USA). The 3

final bacterial concentration was adjusted to 106 CFU/ml

in a total volume of 100 μl. 4

FGE was subsequently added to the wells, and the final concentration was adjusted to 5

sub-MIC. The plates were incubated at 37 °C for 24 h. For chemotherapeutic condi-6

tions, 100 μl of the bacterial suspension was inoculated into flat-bottomed 96-well poly-7

styrene microtiter plates at a final bacterial concentration of 106 CFU/ml. The plates 8

were incubated at 37 °C for 24 h to facilitate mature biofilm formation. Subsequently 9

the bacterial culture solutions were discarded, and the adherent biofilms were washed 10

twice with PBS. One hundred microliters of fresh TSB was added to the wells, followed 11

by the addition of FGE at a sub-MIC final concentration. The plates were incubated at 12

37 °C for 24 h. Bacterial biofilms without FGE treatment were used as controls in paral-13

lel experiments. Un-inoculated FGE-free and un-inoculated FGE-supplemented media 14

were established to define the background OD values. Three replicate wells for each 15

treatment were performed. 16

Colony counting 17

The viable bacteria in the biofilms were examined by determining the number of 18

CFU/ml. After incubation, the bacterial culture solutions were discarded, and the wells 19

were thoroughly washed three times with PBS. One hundred microliters of TSB was 20

added to the wells, and the adherent biofilms were scraped using sterile plastic tips. The 21

suspensions were serially diluted 10-fold in TSB and plated in duplicate onto TSA 22

plates, followed by incubation at 37 °C for 24 h. Plates with more than 25 but less than 23

250 colonies were considered. 24

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Quantification of biofilm 1

The biofilm levels were quantitated using a crystal violet staining technique. Af-2

ter incubation, the bacterial culture solutions were discarded, and the wells were thor-3

oughly washed three times with PBS. The plates were subsequently dried at 60 °C for 4

30 min. The adherent biofilms in each well were stained with 125 μl of a 0.1% (v/v) so-5

lution of crystal violet in water at room temperature for 15 min. The plates were rinsed 6

three times with water by submerging in a tub of water and tapping vigorously on a pa-7

per towel to completely remove all excess cells and dye. The plates were dried at room 8

temperature overnight. Approximately 125 μl of ethanol:acetic acid (95:5, v/v) was 9

added to each well to solubilise the crystal violet. The plates were incubated at room 10

temperature for 15 min. The solubilised crystal violet was transferred to new flat-11

bottomed 96-well polystyrene microtiter plates, and the absorbance at 570 nm was 12

measured using a spectrophotometer. The mean OD570 values for the control and tested 13

wells were subtracted from the mean OD570 values obtained from the un-inoculated 14

FGE-free and un-inoculated FGE-supplemented wells, respectively. 15

Statistical analysis 16

The data represented the average of three experiments performed in triplicate. 17

For statistical analysis, student’s t-test was used to determine differences between S. au-18

reus and MRSA. One-way analysis of variance (one-way ANOVA) was used to deter-19

mine differences among control FGE-untreated and experimental FGE-treated groups. 20

A multiple comparison between statistically significant sample pairs was determined 21

using the Fisher’s least significant difference (LSD) test. Probability values of P<0.05 22

were considered significant. 23

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Results 2

The inhibitory activity of FGE against S. aureus ATCC25923 and one clinical 3

MRSA isolate was examined in vitro using the broth dilution method. FGE inhibited the 4

growth of both strains with an MIC of 8,192 μg/ml. The sub-MIC was considered as 5

one-fold concentration lower than the MIC, i.e., 4,096 μg/ml. The bacteria were subse-6

quently cultured on CRA to detect biofilm formation. Both strains were likely to pro-7

duce biofilm according to the colony appearance shown on CRA. Black colonies with a 8

rough, dry surface were observed in S. aureus ATCC25923 and MRSA after incubation 9

for 24 h (Figure 1). 10

Next, we evaluated the effect of a sub-MIC concentration of FGE on biofilm 11

formation in S. aureus ATCC25923 and MRSA under both chemopreventive and 12

chemotherapeutic conditions. The colony count technique was used to quantitate the 13

number of viable bacteria in the scraped biofilms. FGE significantly reduced the bacte-14

rial counts in the groups treated under chemopreventive and chemotherapeutic condi-15

tions compared with the control FGE-untreated groups. The S. aureus ATCC25923 bac-16

terial counts (as expressed as log10 CFU/ml) in the control FGE-untreated, chemopre-17

ventive, and chemotherapeutic groups were 2.217±0.625, 0.039±0.034, and 18

0.109±0.084, respectively (Figure 2A). The S. aureus bacterial counts in both the chem-19

opreventive and chemotherapeutic groups were significantly lower than those in the 20

control FGE-untreated group (P<0.01). However, the difference in the bacterial count 21

between the chemopreventive and chemotherapeutic groups was not significant 22

(P>0.05). Similar bacterial counts were also observed for MRSA. The bacterial counts 23

in the control FGE-untreated, chemopreventive, and chemotherapeutic MRSA groups 24

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were 1.233±1.202, 0.135±0.131, and 0.155±0.119, respectively (Figure 2B). The bacte-1

rial counts in both the chemopreventive and chemotherapeutic MRSA groups were sig-2

nificantly lower than those in the control FGE-untreated group (P<0.05). Notably, no 3

significant difference in the bacterial counts was observed between chemopreventive 4

and chemotherapeutic conditions (P>0.05). While the bacterial counts between S. aure-5

us and MRSA in the control FGE-untreated and chemotherapeutic groups were similar 6

(P>0.05), the S. aureus bacterial counts were significantly lower than the MRSA in the 7

chemopreventive group (P<0.05). 8

The bacterial biofilms at the bottom of the well were stained with crystal violet, 9

and the absorbance was measured at 570 nm. The level of biofilm formation was ex-10

pressed as the mean OD570 value obtained for the sample subtracted by the mean OD570 11

value obtained for the blank. Figure 3 shows the mean absorbance obtained for S. aure-12

us ATCC25923 and MRSA biofilms under both chemopreventive and chemotherapeu-13

tic conditions. The mean absorbance values obtained for the control FGE-untreated, 14

chemopreventive, and chemotherapeutic S. aureus groups were 0.546±0.396, 15

0.180±0.250, and 0.380±0.185, respectively (Figure 3A), whereas the mean absorbance 16

values obtained for the untreated, chemopreventive and chemotherapeutic MRSA 17

groups were 0.403±0.573, 0.104±0.051, and 0.265±0.040, respectively (Figure 3B). 18

Thus, under chemopreventive conditions, FGE significantly reduced the levels of bio-19

film formation in both S. aureus ATCC25923 and MRSA compared with the control 20

FGE-untreated groups (P<0.05). In contrast, the statistical analysis showed no signifi-21

cant reduction in S. aureus ATCC25923 and MRSA biofilm formation under chemo-22

therapeutic conditions (P>0.05). Levels of biofilm formation in S. aureus were signifi-23

cantly higher than in MRSA under the chemopreventive group (P<0.05) and chemo-24

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therapeutic group (P<0.01), whereas no significant difference was found in both strains 1

in the control FGE-untreated groups (P>0.05). 2

3

Discussion 4

The long history of the medicinal uses of garlic has been well documented. In-5

deed garlic exhibits biological activities, such as anti-inflammatory, anti-thrombotic, 6

anti-atherosclerotic, serum lipid lowering, anti-cancer and antimicrobial activities (Har-7

ris et al., 2001; Bayan et al., 2014). Garlic also exhibits inhibitory activity against sev-8

eral common human bacterial pathogens, such as Staphylococcus aureus, Escherichia 9

coli, Staphylococcus hemolyticus, Klebsiella spp., Shigella dysenteriae, Pseudomonas 10

aeruginosa, Vibrio cholerae, Mycobacterium tuberculosis and Candida albicans (Hall-11

Stoodley et al., 2004; Nidadavolu et al., 2012; Viswanathan et al., 2014). Compared 12

with garlic powder extract, FGE has greater effects on the morphology and growth inhi-13

bition of C. albicans (Lemar et al., 2002). In the present study, we showed that FGE 14

exhibited significant inhibitory activity against S. aureus ATCC25923 and MRSA. Al-15

licin is the active compound in garlic that impacts bacterial viability (Cavallito and Bai-16

ley, 1944). The antimicrobial properties of allicin have been reported in many in vitro 17

studies (Di Paolo and Carruthers, 1960; Bouchara et al., 1986; De Pauw et al., 1995). 18

Interestingly, allicin exhibited equal LD50 on both antibiotic-sensitive and antibiotic-19

resistant bacteria (Chowdhury et al., 1991). In the present study, we observed that S. 20

aureus ATCC25923 and MRSA were inhibited by a similar concentration of FGE at an 21

MIC of 8,192 μg/ml. Thus, the antimicrobial activity of garlic is based on two principal 22

features. First, the compound must reach the potential target. Considering intracellular 23

targets, the active garlic compound must penetrate the microbial cells. Allicin has been 24

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demonstrated to readily diffuse across both artificial and natural phospholipid mem-1

branes (Miron et al., 2000). Inside the cell, the antibiotic efficiency of allicin depends 2

on reaching and reacting with targets that are important to the cell. The antimicrobial 3

effect of allicin likely reflects an interaction with the thiol-containing enzymes in mi-4

croorganisms (Cavallito and Bailey, 1944). At slightly higher concentrations of allicin, 5

other enzymes, such as dehydrogenases or thioredoxin reductases, might be affected and 6

could be lethal to microorganisms (Ankri and Mirelman, 1999). Allicin specifically in-7

hibits acetate kinase and phosphotransacetyl-CoA synthetase, which are bacterial en-8

zymes in the acetyl-CoA-forming system, and this compound also affects the DNA and 9

protein in Salmonella Typhimurium (Feldberg et al., 1988; Focke et al. 1990). 10

S. aureus and MRSA are the most frequent causes of nosocomial infections as-11

sociated with indwelling medical devices, likely involving bacterial biofilm formation 12

(Otto, 2008; Rewatkar and Wadher, 2013). S. aureus biofilm-associated infections are 13

often difficult to treat with antibiotics (Thien-Fah and O’Toole, 2001), reflecting the 14

fact that biofilm-embedded bacteria are 100 - 1000 times more resistant to antibiotics 15

than their planktonic counterparts (Widmer, 2001). However, alternative approaches to 16

overcome biofilm formation, in lieu of antibiotic treatment, have been investigated and 17

developed (Al-Adham et al., 2003; Balaban et al., 2005). Recently, various preparations 18

of garlic have been used to inhibit the biofilm formation of some bacterial pathogens. 19

Garlic ointment prevented biofilm development in burn wound bacterial pathogens, 20

such as S. epidermidis, P. aeruginosa, Acinetobacter baumannii and Klebsiella pneu-21

monia (Nidadavolu et al., 2012). At a concentration of 2 to 4 mg/ml, FGE demonstrated 22

superior inhibitory effects against mature biofilm formation in C. albicans (Shuford et 23

al., 2005). In vivo studies in a rabbit model demonstrated that allicin inhibited biofilm 24

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formation and enhanced the bactericidal effect of vancomycin against S. epidermidis on 1

the implant surfaces of prosthetic joint infections (Zhai et al., 2014). In the present 2

study, we showed that sub-MIC concentrations of aqueous FGE inhibited biofilm for-3

mation in S. aureus ATCC25923 and MRSA. Compared with the FGE-untreated 4

groups, the viable bacterial colony count obtained from scraped biofilms and the crystal 5

violet absorbance values representing the biofilm levels were significantly reduced un-6

der both chemopreventive and chemotherapeutic conditions. Allicin is exclusively re-7

sponsible for the antimicrobial activity of freshly crushed garlic (Borlinghaus et al., 8

2014). Thus, based on the results of the present study, we propose that the antibacterial 9

or anti-biofilm activities of garlic against S. aureus ATCC25923 and MRSA might pri-10

marily reflect the activity of allicin. 11

We established two in vitro culture conditions to investigate the effects of FGE 12

on biofilm formation in S. aureus ATCC25923 and MRSA. Under chemopreventive 13

conditions, the bacteria were grown in the presence of garlic. Under chemotherapeutic 14

conditions, garlic was added to the culture after formation of the mature biofilm. We 15

attempted to determine which condition would favor the inhibition of biofilm formation. 16

However, the statistical analysis demonstrated no significant difference in the reduction 17

of biofilm formation between these two conditions. In the chemopreventive setting, 18

FGE treatment resulted in slightly lower bacterial colony counts and crystal violet ab-19

sorbance values than treatment in the chemotherapeutic setting for both strains. There 20

are several possible explanations for this result, which are not mutually exclusive. In the 21

chemopreventive setting, due to the inhibitory activity of garlic against S. aureus (Ni-22

dadavolu et al., 2012), FGE might directly inhibit the growth of bacteria in the plank-23

tonic phase. S. aureus biofilm development involves in 2-step process: initial attach-24

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ment and subsequent maturation (Otto, 2008). Garlic likely interferes with bacterial ad-1

herence, determined as the primary crucial step for biofilm formation. A previous study 2

showed that sub-MIC concentrations of allicin prevented S. epidermidis adherence and 3

biofilm formation in vitro (Perez-Giraldo et al., 2003), potentially reflecting the reduced 4

level of biofilm formation observed under chemopreventive conditions compared with 5

chemotherapeutic conditions. 6

Previous studies have investigated the potential molecular mechanisms underly-7

ing the garlic-mediated inhibition of bacterial biofilm formation. In Staphylococci, the 8

biofilm comprises a matrix of extracellular polymeric substances called polysaccharide 9

intercellular adhesins (PIAs), which mediate the attachment of bacterial cells and facili-10

tate biofilm development. PIA is a β-1,6-linked N-acetylglucosamine synthesised from 11

UDP-N-acetylglucosamine through the enzyme N-acetylglucosaminyltransferase encod-12

ed by the ica operon. This operon comprises icaADBC biosynthesis genes, which are 13

tightly controlled through numerous regulatory factors. The fifth gene, icaR, is a nega-14

tive regulator of icaADBC (Gotz, 2002). Deletion of the ica locus genes presumably 15

leads to defects in biofilm formation, intracellular aggregation, and PIA synthesis in S. 16

epidermidis (Heilmann et al., 1996), suggesting that the icaADBC is necessary for the 17

maturation of bacterial biofilms. Recently, garlic has been previously shown to interfere 18

with biofilm-associated genes. Quantitative RT-PCR analyses have revealed that allicin 19

down-regulates the expression of icaA, and aap (accumulation-associated protein) in S. 20

epidermidis mature biofilms (Wu et al., 2015). These may propose a related mechanism 21

of garlic in the reduction of biofilm formation observed under chemotherapeutic condi-22

tions in this study. However, the convincing inhibitory mechanism of garlic on biofilm-23

associated genes remains unclear. 24

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Many pathogenic bacteria use the quorum-sensing (QS) system to coordinate 1

bacterial virulence expression, including biofilm development. QS is a highly specific 2

antimicrobial target that is not present in humans (Bhardwaj et al., 2013). Thus, QS 3

might be a promising strategy to control bacterial virulence. Some studies have suggest-4

ed garlic as a potential QS-interfering compound. DNA microarray analysis revealed 5

that Ajoene, a garlic-derived sulphur-containing compound, specifically inhibited QS-6

regulated gene expression in P. aeruginosa (Jakobsen et al., 2012). Allicin and FGE 7

decreased quorum-sensing signals and biofilm formation and inhibited QS-controlled 8

virulence factors in P. aeruginosa (Rasmussen et al., 2005; Lihua et al., 2013). These 9

results suggested that the reduction of biofilm formation in P. aeruginosa might reflect 10

the quorum-sensing inhibitory properties of garlic. S. aureus utilises autoinducing pep-11

tides as a signal in the QS system (Ji et al., 1995). This system is referred to as the ac-12

cessory gene regulator system, comprising two operons, RNAII and RNAIII, on the agr 13

locus (Novick, 2003; Gov et al., 2004). The up-regulation of agr has been observed in 14

biofilm-forming S. epidermidis (Batzilla et al., 2006). However, a surprising role for 15

agr in S. aureus biofilm formation has been reported (Yarwood et al., 2004). In contrast 16

to S. epidermidis, S. aureus cell dispersal from the biofilm surface has been associated 17

with agr up-regulation (Lauderdale et al., 2010). A recent microarray analysis demon-18

strated that natural compounds, such as Manuka honey, down-regulate agr expression in 19

MRSA (Jenkins et al., 2014). Inhibition of the agr system attenuates the virulence of S. 20

aureus, reducing the progression and persistence of the associated diseases (Bhardwaj et 21

al., 2013). However, whether garlic interferes with agr expression and sequentially im-22

pacts biofilm formation in S. aureus requires further investigation. 23

Because garlic displays anti-biofilm activity, this compound has been suggested 24

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as a potential therapeutic agent for controlling biofilm formation in S. aureus. Garlic has 1

been recently applied to medical innovation. Garlic ointment, containing pure garlic 2

powder in petroleum jelly, has been developed as a novel agent to prevent wound path-3

ogen biofilm formation (Nidadavolu et al., 2012). Several strategies to prevent catheter-4

associated biofilm infections have been developed, including coating the catheter with 5

antimicrobial agents, organoselenium, and silver (Tran et al., 2012; Lajcak et al., 2013; 6

Jamal et al., 2014). Based on the reduction of biofilm formation in S. aureus and MRSA 7

through FGE observed in the present study, a garlic-coated catheter could also be de-8

veloped. 9

10

Conclusion 11

In summary, we showed that aqueous FGE not only inhibited the growth of S. 12

aureus ATCC25923 and MRSA but also reduced biofilm formation in these bacteria 13

under both chemopreventive and chemotherapeutic conditions. However, the molecular 14

mechanism underlying the influence of garlic on bacterial viability and biofilm for-15

mation in S. aureus requires further in-depth study. The results obtained in the present 16

study suggest that garlic might represent a promising prophylactic or therapeutic candi-17

date for the management of S. aureus biofilms. 18

19

References 20

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biofilm in staphylococci. Indian Journal of Medicinal Research. 135, 562–564. 22

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Al-Adham, I.S., Al-Hmoud, N.D., Khalil, E., Kierans, M. and Collier, P.J. 2003. Micro-1

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in Staphylococcus aureus biofilms. Journal of Bacteriology. 186, 1838-1850. 2

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doi:10.1371/journal.pone.0102760. 6

7

8

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Figure 1. Colony appearance on CRA. The appearance of black, rough and dry colonies on 1

CRA represents biofilm formation in S. aureus ATCC25923 (A and C) and MRSA (B). 2

3

4

5

6

7

8

9

10

11

12

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Figure 2. Bacterial counts obtained from the scraped biofilms under chemopreventive and 1

chemotherapeutic conditions. The viable bacteria recovered from the scraped S. aureus 2

ATCC25923 (A) and MRSA (B) biofilms after 24 h of incubation are expressed as log10 3

CFU/ml. The control FGE-untreated biofilm, and the chemopreventive, and 4

chemotherapeutic conditions are represented in black, grey, and white, respectively. The 5

means and standard deviations were calculated from three independent experiments. The bars 6

with one and two asterisks are significantly different from the controls (ANOVA; P<0.01 and 7

P<0.05, respectively). 8

9

10

11

12

13

14

15

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Figure 3. Biofilm quantification under chemopreventive and chemotherapeutic conditions. 1

The level of S. aureus ATCC25923 (A) and MRSA (B) biofilm formation after 24 h of 2

incubation was quantified using crystal violet staining. The absorbance was measured at 570 3

nm. The control FGE-untreated biofilm and the chemopreventive and chemotherapeutic 4

conditions are represented in black, grey, and white, respectively. The means and standard 5

deviations were calculated from three independent experiments. The bars with an asterisk are 6

significantly different from the controls (ANOVA; P<0.05). 7

8

9

10

11

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