cristiano miguel pedroso roussado - ulisboa...cristiano miguel pedroso roussado dissertation for...

Streptococcus agalactiae in neonatal infections – a

changing population?

Cristiano Miguel Pedroso Roussado

Dissertation for obtention of Master degree in

Microbiology

Supervisors:

Professor Doctor Mário Nuno Ramos de Almeida Ramirez

Professor Doctor. Isabel Maria de Sá Correia Leite de Almeida

Examination Committee

Chairperson: Professor Doctor Jorge Humberto Gomes Leitão

Member of the Committee: Professor Doctor Constança Pomba

Member of the Committee: Professor Doctor Mário Nuno de Almeida Ramirez

October, 2015

Acknowledgments A toda a família por possibilitar uma formação de qualidade, apoiada em grandes bases educativas.

Mãe, Pai, Irmão, sem vocês nada é possível.

Aos orientadores do Instituto de Medicina Molecular, especialmente à Dr.ª Elisabete Martins e ao

Prof. Dr. Mário Ramirez por serem orientadores presentes, e com os quais enriqueci como pessoa.

À Prof.ª Dr.ª Isabel Sá Correia pela coordenação do melhor MSc em Microbiologia de Portugal. Ao Prof. Dr. José Melo Cristino pelo exemplo de profissionalismo. Ao Dr. João Carriço pelas conversas cientificamente enriquecedoras.

Ao grupo de pessoas que conheci no laboratório e com o qual se construiu uma relação profissional e

de amizade: Ana Friães, Catarina Costa, Marcos Pinho, Andreia Horácio, Catarina Pato, Jorge

Miranda, Raquel Garcia, Joana Lopes, Bruno Gonçalves, Mickael da Silva, Adriana Policarpo. E aos

mais recentes membros: Elisia, Tânia e Miguel. A todos transmito os meus votos de sucesso.

Uma palavra de agradecimento pela simpatia à Adriana, à Alice, à Antónia e à Filomena.

À Universidade de Lisboa por permitir a existência do MSc em Microbiologia, usufruindo em pleno do

processo da fusão. Também à Universidade de Lisboa agradeço o programa extracurricular levado a

cabo este ano, o qual foi fundamental para uma formação forte e multidisciplinar. Do Salão Nobre da

Reitoria, ao Complexo Interdisciplinar, e por fim à magnífica cidade de Lisboa: É vosso dever conseguir, com empenho e trabalho fiéis, que a Universidade de Lisboa se torne não

menos celebrada em todo o mundo do que a própria cidade – André de Resende, 1534

Ao Instituto de Medicina Molecular por ser um local puramente de ciência, lugar de excelência e

mérito reconhecido nacional e internacionalmente. Agradeço profundamente as condições e todo o

contexto da comunidade que tive o prazer de encontrar ao longo deste ano.

Dedico a minha tese ao meu primeiro professor universitário, ao qual devo o exemplo de conduta

académica excecional. A si levarei na memória um código em que o respeito é um valor precoce em

qualquer relação natural: Professor Doutor José Frederico Marques

iii

Abstract

Since Streptococcus agalactiae remains a leading bacterial agent of neonatal infections, we

characterized 201 group B streptococci (GBS) from newborns with invasive infection recovered in

Portugal, between 2003 and 2014. The aims of this study were to document changes in the

prevalence of serotypes, antimicrobial resistance, presence of pilus islands loci, diversity and

distribution of surface proteins encoding genes, and identify genetic lineages to check for potential

changes in the population structure. This is the first time that a serotype IX isolate was found in

Portugal, and also the first time that a serotype VIII isolate was found in babies in Portugal. Multilocus

sequence typing revealed a diverse population, with specific lineages dominating the collection

throughout the sampled years. The majority of the isolates expressed serotype III, and nearly half of

the population belonged to the ST17/III/rib/PI-1+PI-2b genetic lineage, considered to be a

hypervirulent lineage among newborns. Concomitantly with an overall increase in macrolide

resistance, the ST1/Ib/alp3 genetic lineage demonstrated an association with resistance, and this

lineage may have appeared due to capsular switching, as the most frequently found and commonly

resistant ST1/V/alp3 lineage was sparsely represented in the analyzed population. Among CC23, two

sublineages were observed, ST23/Ia/eps and ST24/Ia/bca,and they seem similar to previous studies

in Portugal. These lineages remained as an important cause of neonatal invasive infections in the

Mediterranean region in agreement with previous reports. PI-1 was found in almost the entire bacterial

collection, concordant to its potential for the development of a pilus-based vaccine.

Keywords – GBS, neonatal infectious disease, genetic lineages, molecular epidemiology

v

Resumo

Devido ao facto de Streptococcus agalactiae permanecer um importante agente de infeções

neonatais, foram caracterizados 201 estirpes estreptococcos do grupo B (GBS) isoladas em Portugal

obtidas de recém-nascidos com infeção invasiva entre 2003 e 2014. Os objetivos deste estudo foram

documentar alterações na prevalência de serótipos, na resistência antimicrobiana, na presença de

loci de ilhas codificantes de pilus, na diversidade e distribuição de genes codificantes para proteínas

de superfície, e identificar linhagens genéticas para verificar potenciais mudanças na estrutura da

população. Pela primeira vez foi detetado o serótipo IX em GBS em Portugal, e a primeira vez que o

serótipo VIII foi detetado em bebés em Portugal. A tipagem de sequência multi locus revelou uma

população diversa, com linhagens específicas a dominar a coleção ao longo dos anos amostrados. A

maioria das estirpes expressou o serótipo III, e quase metade da população pertence à linhagem

genética ST17/III/rib/PI-1+PI-2b, considerada como uma linhagem “hípervirulenta” entre recém-

nascidos. Com o aumento da resistência a macrólidos, a linhagem genética ST1/Ib/alp3 demonstrou

associação com a resistência, e esta linhagem poderá ter aparecido devido a alteração do

polissacárido capsular, pois a frequente linhagem resistente ST1/V/alp3, está pouco representada na

população. No CC23, duas sub-linhagens foram observadas, ST23/Ia/eps e ST24/Ia/bca, em

concordância com estudos prévios em Portugal. Estas linhagens permaneceram como importantes

causas de infeções invasivas neonatais na região mediterrânea, de acordo com estudos anteriores.

PI-1 foi encontrado na quase totalidade da população, de acordo com o seu potencial para o

desenvolvimento de uma vacina baseada em pilus.

Keywords – GBS, doença infecciosa neonatal, linhagens genéticas, epidemiologia molecular

vii

Table of Contents

Acknowledgments .........................................................................................................................iii Abstract ........................................................................................................................................ v Resumo ...................................................................................................................................... vii Table of Contents..........................................................................................................................ix Table List .....................................................................................................................................xi Figure List .................................................................................................................................. xiii Abbreviations ...............................................................................................................................xv Chapter 1 – General Introduction .............................................................................................. 17

1.1 Streptococcus agalactiae ................................................................................................... 17

1.2 Colonization and transmission ............................................................................................ 17

1.3 Group B streptococcal disease ........................................................................................... 18

1.3.1 Neonatal infection ....................................................................................................... 18

1.3.2 Infection in adulthood .................................................................................................. 18

1.4 Prevention guidelines ......................................................................................................... 19

1.4.1 Antibiotic prophylaxis ................................................................................................... 19

1.5 Virulence factors ................................................................................................................ 20

1.6 Capsular polysaccharide .................................................................................................... 21

1.7 Surface proteins ................................................................................................................ 22

1.8 Pilus-island ........................................................................................................................ 23

1.9 Antimicrobial resistance in GBS .......................................................................................... 24

1.9.1 β-lactams .................................................................................................................... 24

1.9.2 Macrolides and Lincosamides ...................................................................................... 24

1.9.3 Chloramphenicol ......................................................................................................... 25

1.9.4 Tetracycline ................................................................................................................ 25

1.9.5 Quinolones ................................................................................................................. 26

1.10 Vaccines ......................................................................................................................... 27

1.11 Characterization of GBS isolates ....................................................................................... 27

1.11.1 Phenotypic Methods ...................................................................................................... 27

1.11.2 Genotyping methods ..................................................................................................... 28

1.12 Epidemiology of GBS ....................................................................................................... 31

1.12.1 Molecular epidemiology ............................................................................................. 31

1.12.2 Serotype distribution .................................................................................................. 31

1.12.3 MLST-based genetic lineages .................................................................................... 32 Chapter 2 – Materials and Methods ........................................................................................... 35

2.1 Bacterial isolates ............................................................................................................... 35

2.2 Surface protein gene profile ................................................................................................ 35

2.3 Pathogenicity islands characterization ................................................................................. 35

ix

2.4 Phenotypic methods .......................................................................................................... 36

2.4.1 Serotyping .................................................................................................................. 36

2.4.2 Antimicrobial susceptibility tests ................................................................................... 36

2.5 Genotypic methods ............................................................................................................ 36

2.5.1 Multi-locus sequence typing ......................................................................................... 36

2.5.2 Macrolide-resistance genotypes ................................................................................... 36

2.5.3 Tetracycline-resistance determinants ............................................................................ 37

2.5.4 Gentamicin and streptomycin resistance determinants ................................................... 37

2.6 Statistical analysis ............................................................................................................. 37 Chapter 3 – Results ................................................................................................................... 39

3.1 Isolates ............................................................................................................................. 39

3.2 Capsular serotyping ........................................................................................................... 39

3.3 Antimicrobial susceptibility testing and resistance determinants ............................................ 40

3.4 Surface protein and pilus island gene profiling ..................................................................... 42

3.5 MLST cluster analysis ........................................................................................................ 42 Chapter 4 – Discussion ............................................................................................................. 45 Chapter 5 – Conclusions ........................................................................................................... 47 References ................................................................................................................................ 49

x

Table List

Table 1 Main virulence factors of GBS. ........................................................................................ 21 Table 2 Distribution of the 201 GBS isolates by source of isolation and sex. ................................... 39 Table 3 Serotype distribution among age groups .......................................................................... 40 Table 4 Properties of the genetic lineages found among the 201 invasive isolates. ......................... 41

xi

Figure List

Figure 1 Incidence of early- and late-onset invasive neonatal disease, 1990-2010 .......................... 20 Figura 2 Incidence of Group B streptococcus invasive disease in the Netherlands and in the UK among patients aged 3 months or younger ................................................................................... 20 Figure 3 ermB-carrying elements ................................................................................................. 26 Figure 4 Geographic distribution of studies of GBS incidence in low- and middle-income countries .... 31 Figure 5 Erythromycin and clindamycin resistance in the period 2005-2015.. .................................. 42 Figure 6 GBS MLST-based phylogenetic tree using goeBURST algorithm.. ................................... 44

xiii

Abbreviations

Alp Alpha-like proteins

AW Adjusted Wallace coefficient

bp Base pairs

CAT Chloramphenicol acetyltransferase

CPS Capsular polysaccharide

CSF Cerebral spinal fluid

DLV Double-locus variant

DNA Deoxyribonucleic acid

EOD Early-onset disease

ET Electrophoretic type

FDR False discovery rate

GBS Group B streptococci

IAP Intrapartum antibiotic prophylaxis

Kbp Kilobase pairs

LOD Late-onset disease

M Macrolide (resistance phenotype)

Mbp Megabase pairs

MLSB Macrolide-lincosamide-streptogramin B (resistance phenotype)

MLEE Multilocus enzyme electrophoresis

MLST Multilocus sequenced typing

NT Nontypeable

PCR Polymerase chain reaction

PFGE Pulse-field gel electrophoresis

PI Pilus-island

QRDR Quinolone-resistance-determining region

RFLP Restriction fragment length polymorphism

RNA Ribonucleic acid

SID Simpson’s index of biodiversity

SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

SF Synovial Fluid

SLV Single-locus variant

ST Sequence type

TLV Triple-locus variant

ULOD Ultra late-onset disease

xv

Chapter 1 – General Introduction

1.1 Streptococcus agalactiae First described in 1896 (88) Streptococcus agalactiae (Lancefield group B streptococci; GBS) was

isolated from vaginal cultures in 1935 by Rebecca Lancefield, a pioneer scientist who made important

discoveries related to the Streptococcus genus (83). GBS was later reported as a serious human

pathogen in 1938 (77), and was a relatively uncommon cause of neonatal sepsis between the1930s

and 1960s, but by 1970s it had become one of the dominant pathogens in the early period of life in UK

and in the USA (69, 136). Nevertheless, GBS is not restricted to newborns, affecting pregnant women,

non-pregnant adults, elderly people and immunocompromised adults. Streptococcus agalactiae is

responsible for considerable neonatal morbidity and mortality worldwide (54, 55). It is one of four β-

haemolytic streptococci, recognized as human pathogens. This Gram-positive species is primarily a

commensal human organism and may be seen as a Janus-faced organism since it can also invade

mucosal barriers and cause systemic infections, including sepsis and meningitis, mostly in newborns

and elderly adults (120), but also in pregnant women (102). GBS is also pathogenic for other animals,

mainly fish and bovines, causing severe economic losses in the food industry due to its mortality rates

(121).

1.2 Colonization and transmission The first and pivotal step in GBS colonization is adhesion. Adhesion factors are expressed on the

bacterial surface and allow GBS to bind to extracellular matrix proteins and epithelial cells of the colon

and the genital tract. Adhesion factors can additionally promote invasion, either by disruption of the

epithelial cell layer or by modulation of the epithelial cytoskeleton and of the assembly of junctional

proteins, which in turn allows for further translocation (84). Pathobionts are potentially pathogenic colonizers that usually reside in the intestine in coexistence

with the host, but can occasionally cause severe local or systemic disease. GBS belongs to this

category of colonizers in newborn infants, together with Escherichia coli and enterococci. Being a pathobiont, GBS primarily colonizes the gastrointestinal and genital tracts but can also be

found in the oropharynx of humans. Supporting the gastrointestinal tract as the natural reservoir of

GBS, approximately 10-30% of pregnant women were shown to be colonized in the vagina or rectum

(121). In pregnant women, GBS can traverse the placental membranes and weaken their tensile strength,

gain access to the fetus within the amniotic cavity, induce placental membrane rupture and/or trigger

premature delivery (122). The infection starts in the neonate’s lung and then proceeds into the

bloodstream of the neonate, invading multiple organs including the heart, and also GBS can penetrate

the blood-brain barrier (127). Throughout pregnancy, transmission of S. agalactiae to the fetus during or before delivery can cause

neonatal sepsis, pneumonia and meningitis (21). Normally, mothers transmit the bacteria vertically

during pregnancy, and neonatal exposure to this pathobiont occurs in utero or peripartum through

contact with vaginal fluids (47). More recently, several studies have suggested that transmission also

17

occurs postpartum through consumption of breast milk (93). Vertical transmission occur in up to 80%

of neonates born to colonized mothers depending of risk factors such as the degree of colonization,

prematurity, length of time between rupture of membranes and delivery, and route of delivery,

however, invasive disease develops with a frequency of 1-2% in these children (146). In Europe, the prevalence of GBS carriage among pregnant women varies between 6.5 and 36%, with

most countries reporting colonization rates of 15-20% (6, 158). In healthy adults, GBS predominantly

colonizes the outer mucus layer of the colon, yet may occasionally reside in the small intestine as well

(84). Of note are the studies suggesting limited interspecies GBS transmission between humans and

their livestock (121).

1.3 Group B streptococcal disease 1.3.1 Neonatal infection Group B streptococcus has the highest disease risk during the first three months of life and declines

substantially thereafter (54). GBS disease in newborns is classified as early-onset disease (EOD) or

late-onset disease (LOD), depending on the age of the infant at the time of disease manifestation (54).

EOD takes place very early in infancy, within the first week of life, and manifests as respiratory failure

and pneumonia that rapidly progresses into bacteremia and septic shock syndrome (34). Maternal

colonization is considered a probable prerequisite for EOD, (128). In EOD bacteremia results from

inhalation of infected amniotic fluid and/or genital secretions into the lungs (3). On the other hand,

LOD develops in infants up to three months of age (7-90 days), and is usually characterized by

bloodstream infection, with a high risk of progression to meningitis. About 50% of infants with LOD are

colonized at birth with the same GBS serotype as their mothers (44). Regarding LOD risk factors, they

may differ according to the gestational age at birth, being different between preterm and term infants.

However, vertical transmission, nosocomial acquisition and prematurity are recognized risk factors, but

acquisition of GBS in LOD is not completely understood (127). Ingestion of breast milk has been proposed as a possible source of GBS, although it is not clear how

commonly this is a route of LOD transmission (11, 19). Furthermore, long-term evaluation of infants

who survive GBS meningitis indicates that 30% of the cases have mild-to-moderate neurologic

sequelae and 19% have severe sequelae with global cognitive delay, cerebral palsy, cortical

blindness, and/or hearing impairment (90).

1.3.2 Infection in adulthood Despite causing life threatening invasive disease in neonates, GBS can also cause invasive disease in

pregnant women and non-pregnant adults (118). Beginning at birth, GBS colonization rates

continuously increase to 20-30% in adults (84). Specifically in pregnant women, GBS is a frequent

cause of urinary and upper genital tract infections, intra-amniotic infections, and sepsis in the USA

(84). In last decades, GBS have been gradually associated with invasive disease in nonpregnant

adults, mainly in the elderly, immunocompromised and those with diabetes mellitus and cancer (55,

18

123, 141). There is a need for developing prevention strategies against infections among adults, since

mortality in these patients is frequently higher than in newborns (55, 123).

1.4 Prevention guidelines

1.4.1 Antibiotic prophylaxis In the 1970s GBS was a common cause of invasive neonatal disease, namely sepsis and meningitis,

with >50% of mortality rate (136). Clinical trials performed in the 1980s demonstrated that giving

intrapartum intravenous ampicillin or penicillin to mothers at risk of transmitting GBS to their newborn

was highly effective at preventing invasive early-onset GBS disease (18, 133). The US consensus statement from 1996 recommended that pregnant women at 35-37 weeks of

gestation undergo routine screening (vaginal and rectal cultures) for GBS carriage, and women who

were colonized with GBS received prophylaxis with ampicillin or other antibiotics to decrease the risk

of vertical transmission (137). Women, and women who have previously given birth to neonates with

severe GBS disease, among other high-risk patients, also received antibiotics perinatally (163). Penicillin was the first line intrapartum antibiotic prophylaxis (IAP) agent recommended, with ampicillin

as an acceptable alternative. For penicillin allergic women, initial guidelines recommended

clindamycin or erythromycin for prophylaxis. However, increasing resistance among group B

streptococci to these agents (27) together with the inability of erythromycin to penetrate the amniotic

fluid, promoted the revision of the guidelines in 2002 to recommend cefazolin as the agent of choice

for penicillin-allergic women at high risk for anaphylaxis. Clindamycin is recommended only if the GBS

isolate is susceptible to both clindamycin and erythromycin, otherwise vancomycin use is

recommended (160). Antibiotic prophylaxis had a major effect on early-onset disease, which greatly declined, and was

accompanied by improvements in early detection of invasive GBS disease, and supportive care led to

a decrease in mortality rates from >50% in the 1970s to 15-25% in the 1980s and <10% by the 1990s

(138). Centers for Disease Control and Prevention (CDC) recommend that health care providers use either a

risk-based or a screening approach to identify candidates for IAP (136). Basically, in risk-based

approach women presenting at the time of labor with clinical risk factors for disease transmission are

offered IAP; in the screening approach women are screened for carriage of GBS between 35 and 37

weeks of gestation, and intrapartum chemoprophylaxis is offered to carriers. However, in both

approaches, antibiotics are given during labor to women who had group B streptococcal bacteriuria

during their current pregnancy, or who have previously had an infant with known GBS disease,

although, screening approaches seem to be 50% more effective at preventing perinatal group B

streptococcal disease (134). Since the advent of routine screening for maternal GBS colonization and antibiotic prophylaxis for

GBS carriers, the incidence of early-onset neonatal GBS disease has declined dramatically in the

USA, from 1.7 cases per 1000 neonates in 1990 to 0.34 per 1000 in 2008 (167). Despite the advances

in prevention, neonatal GBS disease has not been completely eliminated. As Figure 1 shows, the

19

incidence of early-onset disease has plateaued in the last years, and the incidence of late-onset

disease has not decreased at all since the release of the CDC guidelines (163).

Figure 1 Incidence of early- and late-onset invasive neonatal disease, 1990-2010. (Reproduced from 29).

In spite of the success in the USA and most European countries of universal screening, there are

countries in which a risk-based approach was adopted, such as the Netherlands and the UK (Figure

2).

A B

Figura 2 Incidence of Group B streptococcus invasive disease in the Netherlands and in the UK among patients aged 3 months or younger. Vertical dashed lines represents the introduction of prevention guidelines: in Netherlands (A, 1999), and in the UK (B, 2003) (Adapted from 8, 81).

1.5 Virulence factors Being a pathogenic bacterium, GBS encodes many virulence factors. These have a role in various

aspects of pathogenicity, such as host invasion, adherence, colonization and immune evasion. In spite

of the limited knowledge regarding many virulence factors, some are well characterized.

Understanding signaling responses of GBS is essential for elucidation of pathogenesis of GBS

20

infection and for the identification of novel therapeutic agents (127) (Table 1). This is crucial for future

therapeutics measures against GBS disease since vaccines are not suitable for the treatment of

infections.

Table 1 Main virulence factors of GBS. (Adapted from 47, 92, 127).

Virulence factor Mode of action Genetic basis

Promotes invasion of host cells and triggers

β-hemolysin/ cytolisin host-cell lysis

cylE and other genes in the cyl locus

Impairs cardiac and liver function

Induces inflammatory responses and apoptosis

Forms pores in host-cell membrane

CAMP factor Binds to glycosilphosphatidylinositol anchored cfb

proteins

Sialic acid capsular Prevents recognition of GBS through molecular

mimicry of host-cell surface glycoconjugates cpsA-L, neuA-D

polysaccharide

Masks pro-inflammatory cell wall components

Binds epithelial cells

Alpha-like protein (Alp) family Suffers antigenic variation as evasion eps, rib, alp2, alp3, alp4

mechanism of antibody detection

Prevents neutrophil recruitment due to cleavage

C5a peptidase of complement C5a

scpB

Promotes adherence by binding to extracellular

matrix fibronectin and epithelial cells

Cleaves fibrinogen and chemokines

Serine protease Impairs neutrophil recruitment and phagocytic cspA

killing of GBS

Promote resistance to antimicrobial peptides Pilus islands

Pili through an unknown mechanism PI-1 and PI-2

Promote adherence of GBS to host cells

Fibrinogen-binding A and B Binds extracellular matrix fibrinogen through

fbsA and fbsB

repetitive structure motifs

Hyaluronate lyase Cleaves hyaluronate and promotes spread of

hylB

GBS during infection

C protein (α and β) Binds epithelial cells

bca (α) and bac (β)

Blocks intracellular killing by neutrophils

1.6 Capsular polysaccharide The structure of capsular polysaccharides (CPS) in S. agalactiae consists of repeating units

containing four monosaccharides: glucose, galactose, N-acetylglucosamine and sialic acid. Sialic acid

is present in all but two serotypes, being the terminal on the side chain. Exceptionally, serotypes VI

and VIII have an additional rhamnose rather N-acetylglucosamine (97).

21

The flanking regions of the gene cluster responsible for CPS production are conserved across all

serotypes. This gene cluster encompasses genes that encode serotype-specific glycosyltransferases

and polymerases, flanked by upstream genes accounting for enzymes that synthesize and activate

sialic acid, and downstream genes hypothesized to have functions in the export of the polysaccharide

capsule (32). However, the amino acid sequences of proteins having similar functions in different

capsular serotypes were found to exhibit significant heterogeneity (31, 106). The genetic diversity within CPS gene clusters may be related to horizontal transfer of capsular genes

which may occur by intra- and inter-species recombination events. Moreover, changes at the capsular

locus were proposed to be driven by the equilibrium between the selective pressure imposed by host

immunity, which lead to capsular variation, and the conservation of structural elements of a particular

capsular polysaccharide that might be required for pathogenicity (22, 32). CPS is an example of

molecular mimicry, since it resembles the glycans of host vertebrate cells, also possessing sialic acid.

This mimicry allows GBS to evade host immune system, because it fails to recognize GBS as a

nonself (127). CPS is one of the best studied virulence factors of GBS. There are ten capsular serotypes

characterized: Ia, Ib and II-IX, each antigenically and structurally unique. Of these the serotypes Ia, III

and V are the most frequent in adult and neonatal invasive infections in Europe and America (127).

The capsular serotype distribution changes over time among countries and populations, being a good

epidemiological marker. There is little information related to population structure in low-income

countries (54). In spite of being a well studied virulence factor, many features of CPS regulation during

colonization and disease are still unknown (127).

1.7 Surface proteins The first surface protein described was the C antigen back in 1971 (173). This antigen is composed of

two unrelated protein components, the trypsin-resistant α protein and the trypsin-sensitive β protein,

and GBS strains may express either or both. Its importance in virulence was demonstrated in a study

showing that antibodies directed towards the C antigen conferred protective immunity in mice (82).

Ten years later, another study showed that both components of C antigen, α and β (Bca and Bac,

respectively), elicit protective immunity (13). However, the C antigen was not expressed by type III

strains and subsequent work on these strains made it possible to identify Rib, a surface protein that

elicits protective immunity and is expressed by most strains not expressing α proteins (149). The Alp (alpha-like proteins) family comprises five members: Eps, Rib, Alp2, Alp3, Alp4, and Alp5

(133). These proteins are encoded by allelic loci and each S. agalactiae strain expresses only one of

them (15, 78, 80, 149). These proteins contain large internal tandem repeats and are virulence factors

encoded by stable mosaic genes, generated by recombination of modules at the same chromosomal

locus (35). Studies of surface proteins and their corresponding genes are relevant for epidemiological

analysis of GBS infections, and may help in the development of a GBS vaccine. However, no single

gene or surface protein was sufficiently prevalent to be considered as the sole component of a

successful GBS vaccine (122).

22

The Bca protein is commonly found in strains of serotypes Ia, Ib and II, much less common in type III

and rarely found in type V (92). Some studies showed that Rib is expressed by most of serotype III

strains, by many type II strains, and by a few type V strains (93). Alp3 protein is viewed as a chimera

protein, and this finding suggests that the gene encoding this surface protein may have arisen in S.

agalactiae, followed by horizontal gene transfer from GAS (148). There is evidence of this protein in

serotypes V and VIII (92). The Alp4 protein was rarely found in GBS strains. On other hand Alp2

differs from other members in the family by having a second type of tandem repeat in the N-terminal

half of the protein (92), and Alp5 differs slightly from Alp1.

1.8 Pilus-island Group B streptococcus encodes small cell-surface appendages known as pili. These appendages are

thicker (3 to 10 nm in diameter) and longer than fibrils, typically extending from 1 to 3 µm from the

bacterial cell surface, and were first described in GBS in 2005 (85, 117). Pili are involved in adhesion,

promotion of epithelial cell surface colonization, support biofilm formation, and translocation across the

blood-brain barrier (79). GBS pili are composed of three subunits: a backbone pilin protein, two

ancillary proteins, and two pilus-specific class C sortase enzymes, which recognize the LPXTG amino

acid motif on structural proteins and facilitate covalent attachment of these subunits to each other and

to the cell wall peptidoglycan (48). Pili are encoded by two loci in different regions of the genome,

designated pilus islands 1 and 2 (PI-1 and PI-2, respectively), the later presenting two distinct variants,

PI-2a and PI-2b (130). The sequences of all three pilus-islands appear to be remarkably well

conserved, with PI-2a being the only island to show some extent of variability (102). In vitro models of GBS infection have shown that the ancillary proteins initiate adherence to various

tissues, whereas the backbone proteins facilitate invasion and paracellular translocation in the host,

where PI-2a was suggested to be more important for biofilm formation (147), and the backbone protein

of PI-2b was associated to increase intracellular survival in macrophages (28). In vivo models showed that GBS pilus components are highly immunogenic and a pilus-vaccine

containing the backbone protein genes of PI-1 and PI-2b and the ancillary protein of PI-2a has been

shown to elicit opsonophagocytic antibodies that confer protection in mice (102). Alongside the fact that all GBS strains carry pili, the sequences of the 3 pilus subunits appear to be

well conserved. This could be related to the regulation of pili expression on bacterial surface, where it

appears only transiently, thus avoiding the selective pressure of the immune system; or niche linked,

as GBS occupies environmental niches that are relatively inert from an immunological perspective,

and for that reason insufficient immune pressure may allow the relative conservation of these

structures (102).

23

1.9 Antimicrobial resistance in GBS

1.9.1 β-lactams One of the most important groups of antibiotics is the β-lactam group, which includes the medically

important penicillins, cephalosporins, and cephamycins (96). The β-lactam antibiotics are inhibitors of

cell wall synthesis, by preventing transpeptidation, the reaction that results in the cross-linking of two

glycan. Thus, penicillin and other β-lactam antibiotics bind to the transpeptidase domains of the so

called penicillin-binding proteins, resulting in weakened, self-degrading cell wall. Group B streptococcus is considered universally susceptible to penicillin and ampicillin, and these are

the first choice antibiotics for intrapartum prophylaxis as well for treatment of GBS infections in all age

groups (39). However, the identification of the first GBS isolate with reduced penicillin susceptibility

was reported in Japan, but its clinical significance has not been shown so far (76).

1.9.2 Macrolides and Lincosamides Macrolides are a group of antibiotics produced by various strains of Streptomyces and have a complex

chemical structure. They act by inhibiting protein synthesis, namely blocking the 50S ribosomal

subunit, preventing its association to tRNA. These broad spectrum antibiotics are composed of a

lactone ring of variable size. 14-membered (clarithromycin, dirithromycin, erythromycin, and

roxithomycin), 15-membered (azithromycin) and 16-membered (josamycin, midecamycin, miocamycin,

rokitamycin, and spiramycin) (139). Unlike macrolides, lincosamides (clindamycin and lincomycin) are

devoid of a lactone ring but resistance is conferred by the same gene. For treatment or prevention of

GBS disease, erythromycin and clindamycin are recommended as second-line drugs for patients with

β-lactam allergy. Currently there is concern relative to macrolide resistance in streptococcal

populations worldwide. GBS macrolide and lincosamide resistance occur mainly by two mechanisms:

1) ribosomal methylation, and 2) antibiotic efflux. In the first case, pathogenic bacteria have Erm

proteins that dimethylate a single adenine in the 23S rRNA, which is part of the large ribosomal unit

(170). A consequence of methylation, binding of erythromycin to its target is impaired (87). Among the

classes of erm genes, ermB and ermTR (a subset of the ermA class) are present in β-hemolytic

streptococci (87). Since these resistance determinants are commonly harbored by plasmids and

transposons, they are frequently self-transferable (124). Macrolide-lincosamides-stretogramin B

(MLSB) resistance can be constitutive or inducible. Inducible resistance (iMLSB) occurs when bacteria

need another antibiotic in vitro as an inducer for the proper translation of mRNA encoding the

methylase. On the other hand, in constitutive resistance (cMLSB) methylase is produced even in the

absence of an inducer (87). Usually GBS cMLSB and iMLSB isolates carry the ermB and ermA [ermTR

subclass], respectively (132). Beyond the already stated erm genes responsible for GBS macrolide

resistance, another one was identified, ermT gene, alongside ermB in one iMLSB GBS strain, sharing

97% identity with ermT gene from Lactobacillus sp. (46). The second resistance mechanism in GBS is

antibiotic efflux, leading to the M phenotype designation that consists of resistance to 14- and 15-

membered macrolides only. The mechanism consists of a proton-dependent efflux system, encoded

by the mef genes (2).

24

There are other genes conferring resistance to lincosamides in GBS, namely lnu(B) (41). Albeit not

fully understood, this gene encodes for a nucleotidyl-transferase (formerly lin), and it is responsible for

a new phenotype called L phenotype, involving low-level clindamycin resistance while remaining

susceptible to erythromycin. This phenotype was recently reported in GBS isolated in the USA,

Canada, New Zealand, Asia and Argentina (112). Another phenotype, called LSA (lincosamide-streptrogramin A) was described in New Zealand, in

which GBS strains were intermediate or resistant to clindamycin and streptogramin A, but they was

susceptible to macrolides (101). The resistance is conferred by the lsa(C) gene, similar to Enterococcus faecalis (100). 1.9.3 Chloramphenicol Chloramphenicol inhibits protein synthesis by blocking the 50S subunit of bacterial ribosomes. The

first and still predominant mechanism of bacterial resistance to chloramphenicol is enzymatic

inactivation of the drug by different chloramphenicol acetyltransferases (CATs) encoded by cat genes

(114). Among GBS strains, resistance occurs at very low percentages.

1.9.4 Tetracycline An astonishing feature of human GBS strains is their high rate of tetracycline resistance. Tetracyclines

inhibit protein synthesis by preventing the attachment of aminoacyl-tRNA to the ribosomal acceptor (A)

site (30). Extensive use of tetracycline in both animal and human therapy is explained by the

favourable antimicrobial properties and the absence of major adverse side effects, and this fact led to

high resistance levels in many commensal and pathogenic bacteria due to genetic acquisition of tet

genes. Efflux genes, tetK and tetL, and genes responsible for ribosomal protection, tetO and tetM are

the most frequent tetracycline resistance determinants in GBS. Whereas tetM is the most frequent

tetracycline resistance determinant among GBS isolates recovered from human infections, tetO is

common among bacteria isolated from bovines (46, 52). Additionally tetT and tetW ribosomal

protection genes have also been found in GBS causing human infections (132). Although tetracycline is not used to treat GBS infections, there is evidence of a frequent link between

macrolide and tetracycline resistance due to the localization of the ermB and tetM genes on the same

composite transposons, derivatives of Tn916 (Figure 3) (162).

25

Figure 3 ermB-carrying elements. The ermB gene is indicated as a checkered red arrow. Light-blue arrows indicate Tn916-related ORFs other than tetM. Dark-blue indicates the tetM gene. Pink, green, and orange arrows indicate ORFs from Tn917, the ermB element, and the MAS element, respectively. Colored areas between ORFs maps denote insertions. (Reproduced from 162).

As macrolides are commonly employed antibiotics to treat GBS infections, the suggestion of a dual

resistance spreading is tempting. Recently it was even hypothesized that acquisition of macrolide and

lincosamide resistance genes occurred after the selection of tetracycline resistance clones

contributing to the ST1 Tn916-1 lineage clonal expansion (37). However, other hypothesis show that

erythromycin and tetracycline resistance may not be linked (36).

1.9.5 Quinolones Quinolones were introduced into clinical use in the 1980s for the treatment of infections since they are

very potent broad-spectrum antibiotics. Fluoroquinolones are powerful inhibitors of bacterial type II

topoisomerases, which are essential enzymes involved in key cellular processes including DNA

replication. Fluoroquinolones target DNA gyrase and topoisomerase IV with varying efficiency,

inhibiting the bacterial control of DNA supercoiling within the cell, resulting in impaired DNA replication

and cell death (50, 51). The key target in Gram-positive microorganisms is topoisomerase IV whereas

in Gram-negative is DNA gyrase (51). Resistance involves amino acid substitutions in a region of the GyrA or ParC subunits termed “quinolone-

resistance-determining region” (QRDR). There are four main mechanisms for fluoroquinolone resistance: 1)

target-site mutation; 2) transmissible resistance mechanisms; 3) permeability related, and 4) efflux

mechanisms. The first quinolone resistant GBS strain was described in Japan back in 2003 (77). It is still

unclear if there is an association between fluoroquinolone resistance and serotype distribution. Although

80% of GBS fluoroquinolone resistant strains belong to ST19/III lineage in China (170), a report from Japan

demonstrated that fluoroquinolones resistant GBS strains were similar between infants and adults, both

expressing serotype Ib, suggesting that a single resistant clone spread rapidly through the country (115).

One survey from the USA reported 5%

26

levofloxacin resistant GBS strains in a healthcare facility related with prior quinolone therapy (172)

whereas other reports stated no association at all (174).

1.10 Vaccines The first approaches to develop an effective vaccine to GBS were based in the capsular

polysaccharides (4). However, the response was variable and low, leading to an ineffective increase in

antibody titers (4, 59). Later, it was tried the covalent conjugation of a polysaccharide and a protein,

the tetanus toxoid. Human trials were conducted in nonpregnant adults and in pregnant women and

their babies (5, 74). However, the geographic variability in the most prevalent serotypes and the

possible, but rare, capsular switching events in GBS strains make the development of conjugated

vaccines a difficult task, similarly to S. pneumoniae (24). Apart from recent failures in vaccine

construction, some researchers are trying different approaches. That is the case of reverse

vaccinology, a method for vaccine design that uses the information obtainable from whole-genome

analysis (128). Following the identification of three pilus variants whose genes are present in three

different pilus islands showing different combinations in GBS, and the fact that each pilus elicits

protective immunity in mice, a pilus-based vaccine candidate exclusively constituted by three pilus

components that is potentially capable of preventing disease by all GBS serotypes was considered

and is currently under development (102,118, 130).

1.11 Characterization of GBS isolates Bacterial epidemiology is defined as the study of the dissemination of human bacterial pathogens,

including their transmission patterns, risk-factors for and control of infectious disease in human

population (161). Concomitantly, bacterial strain typing, or identifying bacteria at the strain level, is

critical for epidemiological surveillance of bacterial infections. Moreover, strain typing has a central

application in cases of bacteria exhibiting high levels of antibiotic resistance or virulence, or those

responsible for nosocomial or pandemic infections, and even in population dynamics studies (89).

1.11.1 Phenotypic Methods Phenotyping relies on phenotypic features, which have the potential to group organisms according to

their similarity in features resulting from the expression of their genotypes. To document phenotype

markers, such as the distribution of proteins and other cell components, the morphology and behavior

of cells some approaches are employed: 1) biotyping, assesses the known biochemical variation

within each species, 2) antibiogram-based typing, used to estimate incidences of resistance to a set of

antibiotics, 3) serotyping, based in different reactions with sera corresponding to distinct surface

antigens, 4) phage and bacteriocin typing, a method which evaluate lytic patterns of test isolates that

have been exposed to a set of bacteriophages, or bacteriocins (161). Highly discriminatory power

achieved by typing methods, such as Mass Spectrometry, is helpful to better understand the

epidemiology of infections. Thus, epidemiologists can propose hypothesis regarding population

structure and spreading patterns.

27

Serotyping For many years the phenotypic method described by Lancefield was used as the standard procedure

(85). This test was based on the presence of capsular antigens extracted with hydrochloric acid (HCl)

and allowed classification of GBS strains into nine serotypes (Ia, Ib, II-VIII), while a certain percentage

remained nontypeable (NT) (144). That percentage has decreased with the improvement in growth

medium allowing better conditions for capsular production (10), and with the development of latex

agglutination assays (142). Thus, nowadays, latex agglutination is the most commonly used method

for GBS serotyping, and it is based on polyclonal antibodies specific for the 10 recognized CPS, i.e.,

serotypes Ia, Ib, and II-IX (143, 175). However, serological methods have limitations, as they may fail

to type an isolate due to the absence or low expression of CPS under the experimental conditions

(175).

Antimicrobial susceptibility testing Antimicrobial susceptibility testing, also known as antibiogram-based typing, can be performed either

by disk diffusion in solid growth media or drug dilution in liquid media, using a variety of measurement

systems (162). Methods and interpretative criteria follow recommendations from international and

independent organizations, such as Clinical and Laboratory Standards Institute (CLSI) (33). For

research purposes, susceptibility testing of selected antibiotics is usually performed by the Kirby-

Bauer disk diffusion method (7). Small disks pre-impregnated with a standard concentration of

antibiotic are placed onto a plate upon which bacteria are growing. After incubation, the diameter of

inhibition around the disk can be compared to reference tables to determine whether the bacterial

isolate is susceptible, intermediately susceptible, or resistant to the antibiotic.

1.11.2 Genotyping methods Genotypic methods assess variation in the genomes of bacterial isolates with respect to composition

(for instance, presence or absence of plasmids), overall structure (for example, restriction

endonuclease profiles, number and positions of repetitive elements), or precise nucleotide sequence

of one or more genes or intergenic regions) (161). One effective way to analyze genomes for typing purposes is performing polymerase chain reaction

(PCR). Its major advantages are flexibility, technical simplicity, wide availability of equipment and

reagents, and a fast turnover time, making PCR highly suitable for various applications in bacterial

typing. Albeit PCR nearly universally dissemination, this method cannot be seen as a library method

for fingerprinting, but it exhibits an easily adjustable level of discrimination (161). Restriction Fragments Length Polymorphism (RFLP), Pulse-field Gel Electrophoresis (PFGE) and

Multilocus Enzyme Electrophoresis (MLEE) were three common genotypic methods employed to GBS,

however, in recent years, they have been replaced by sequencing-based methods since sequencing

costs have been decreasing. RFLP generates complex banding patterns that are difficult to analyze

when the comparison of a large number of strains is intended (91). PFGE, albeit being discriminatory,

is a time-consuming and labour-intensive whole-genome based technique. MLEE examines allelic

variation of a set of housekeeping enzymes, thus providing small but detectable

28

variations in protein size and charge (126, 138). It was the precursor of Multilocus Sequence typing

(MLST), which had been used as a reference method for analyzing clonal lineages (161).

Multi-locus Sequence Typing (MLST) Multilocus sequence typing was proposed in 1998 as a portable sequence-based method for

identifying clonal relationships among bacteria (98, 99). MLST is an unambiguous sequence-based

typing method that involves sequencing approximately 500-bp fragments of seven housekeeping

genes and has been used successfully to type strains and investigate the population structure of a

number of human bacterial pathogens, including GBS (72). To the different sequences at each locus

are assigned different allele numbers, and so each strain is defined by the alleles at the seven loci,

which is called the allelic profile. Each unique allelic profile is designated a sequence type (ST), and it

represents a convenient and unambiguous descriptor for the strain or clone (57). One major

advantage of MLST is it reproducibility, thus being particularly suitable for epidemiological studies

because it provides data that can be easily compared worldwide. For that reason, alongside with the

precise, unambiguous and portable nature of the data obtained, MLST offers a valuable tool for the

characterization of bacteria strains and surpass PFGE for typing purposes (57). Like MLEE, MLST

uses alleles as the unit of comparison, rather than nucleotide sequences, and in the resulting

comparisons among isolates, each allelic change is counted as a single genetic event, regardless of

the number of nucleotide polymorphisms involved (99). Conceptually, these allele-based comparisons

provide an effective correction for the fact that in many bacteria common horizontal genetic transfer

events account for many more polymorphisms among strains than rarer point mutations (43).

Consequently, MLST is being used globally in epidemiological microbial typing and bacterial

population studies and this lead to the development of algorithms and tools to make sense of this

wealth of data in the epidemiologic, population genetics, and evolutionary contexts (63, 161). For a

more comprehensive analysis of the possible patterns of evolutionary descent, a set of rules were

proposed and implemented in the eBURST algorithm. These rules allow the division of a data set into

several clusters of related strains, dubbed clonal complexes, by implementing a simple model of clonal

expansion and diversification (63). eBURST uses an heuristic local optimization procedure, however it

may result in links within the clonal complexes that violate the rules proposed, though a global optimal

solution was proposed, goeBURST, which corrects these links by identifying the correct patterns of

descent (65). This fact is particularly relevant in GBS since it has a rich diversity within CCs

suggesting the importance of recombinational exchanges which make the relationships between STs

difficult to evaluate (23). The simplest model for the emergence of clonal complexes is that a founding genotype increases in

frequency in the population as a consequence of either a fitness advantage or of random genetic drift,

to become a predominant clone (56). Clonal complexes are the result of the diversification of the

founding genotype as it increases its frequency inside the population (57). Over time variants with

different allelic profiles will arise, by point mutations or recombination. This happens successively,

variants with one allele difference from the founder genotype, called single-locus variants (SLVs) may

diversify even further to produce variants that differ at two of the seven loci, named double-locus

29

variants (DLVs), at three of the loci, triple-locus variants (TLVs), and so on. A representation of the

level of tiebreak rule reached before deciding if a link should be drawn is implemented in goeBURST

and it helps the evaluation of the reliability of the represented hypothetical pattern of descent (64). A

software implementation of the goeBURST algorithm is available at in PHYLOViZ downloaded from

http://www.phyloviz.net (63).

Whole-genome approaches In the whole-genome era of microbiology, the need for systematic, standardized descriptions of

bacterial genotypic variation remains a priority (99). Thus, principles behind MLST can be applied to

whole-genome analysis, with schemes consisting of increasing numbers of loci. Nonetheless, this

swapping from MLST to whole-genome approaches may threaten the ordered investigation of

bacterial diversity by overloading the field due to the amount of information, but the advent of rapid

and inexpensive sequencing has removed the practical constraints that have framed the design of

MLST approaches (110). Moreover, extended multilocus sequence typing (eMLST) where it is

included gene sequences from the accessory genome, the only way to detect all the non-clonal

genetic variations that shape the fine structure of a bacterial population is by performing complete

genome approaches (110). Nowadays more than two hundred draft genomes of human-related GBS are available (37, 60, 66,

153, 154). The GBS genome is nearly 2.2 Mbp long and has over 2100 predicted coding regions.

Comparative genomics approaches show that the GBS genome has great similarity with that of other

streptococci, namely Streptococcus pyogenes and Streptococcus pneumoniae. The presence in mobile elements of many unique GBS genes expected to play a role in colonization or

disease supports the possible acquisition of virulence traits from other species. Moreover, the

presence of more than 100 genes probably duplicated suggests evolution of additional species-

specific functions, supporting the hypothesis that GBS is adapted to distinct niches in its human and

animal hosts (152). On the other hand, the sequence of a single genome does not reflect how genetic variability drives

pathogenesis and limits genome-wide screens for vaccine candidates or for antimicrobial targets, and

so there was a need to determine the global gene repertoire of the GBS bacterial species – GBS pan-

genome (153). The conserved genome encompasses the core genome that contains genes shared by

all strains within the clade (typically genes responsible for the basic aspects of the biology of the

clade), the variable genome is composed of genes shared by a subset of the strains (contributes to

the species diversity), and strain-specific genes (168). The high variability of the GBS dispensable

genome led to the concept of an open pan-genome (that including both conserved and variable

genomes) i.e., its size grows with the number of strains sequenced (23, 152). In contradiction with previous studies, a recent report stated that diversity might be primarily driven by

small genetic events rather than recombination (61). This observation suggests that GBS evolution

may be viewed as similar to the antigenic shift/antigenic drift model of influenza in which

recombination drives the emergence of new GBS subtypes, which then slowly accumulate new

genetic polymorphisms over time (60).

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1.12 Epidemiology of GBS

1.12.1 Molecular epidemiology Molecular epidemiology refers to approaches that use molecular strain-typing techniques and aims to

understand the distribution and determinants of disease occurrence among pathogens infecting

humans (62). Also it elicits the knowledge of the clone distribution and its transmission, promoting

public health and helping the development of approaches for disease prevention. In GBS

epidemiology the sort of techniques has been used to discriminate genetic lineages in order to probe

for associations between specific genotypes and disease. DNA-based typing methods such as PFGE

and MLST have been widely used in molecular typing of GBS isolates, alongside with classical

serotyping and antimicrobial susceptibility testing.

1.12.2 Serotype distribution One major issue regarding serotype surveillance is the existence of several serotypes with different

geographical distributions. Despite occasional surveillance of GBS serotype distribution causing

invasive disease, data on the serotypes that are circulating in some European countries is still missing

(112). Serotypes that cause GBS infections differ from country to country and over time, thus there is a

need for more studies to evaluate the global burden of each GBS serotype, especially in low-income

countries (Figure 4), Still, there is evidence for some clonality in spite of these geographic differences.

Interestingly, in Brazil a study from colonized patients and from symptomatic adults showed absence

of serotype III, and serotypes Ia, Ib, II and V accounted for 79% of the serotypes of the isolates

recovered (54). Additionally, similar results have been observed in China (169).

Figure 4 Geographic distribution of studies of GBS incidence in low- and middle-income countries. Black dots show the localization of the studies. OECD, Organization for Economic Cooperation and Development (Reproduced from 38).

In neonatal infections serotype III predominates irrespectively to world region, as several studies

reported, for instance in Portugal (104) and France (125). Regarding adults’ GBS epidemiology, albeit

recent efforts, limited data are available on the distribution of GBS serotypes. Although, some studies

showed that serotype III, V, Ia, IV and II were the most common serotypes in pregnant and

nonpregnant women in Germany (21), Norway (20), Sweden (67) as well in China (94), and Canada

31

(40). Moreover, the most frequent serotypes responsible for GBS invasive disease in nonpregnant

adults in Portugal were serotype Ia, followed by serotype V, III, II and Ib (110), in contrast to other

countries, such as Spain (17), Sweden (125), Norway (12), the US (141), Australia and New Zealand

(175), where serotype V is the leading cause of invasive infections in nonpregnant adults, and where

serotype Ia is much less frequent.

1.12.3 MLST-based genetic lineages In evolutionary terms, bacterial pathogens may comprise clonal lineages that disseminate in the

population as result of fitness advantage or selective forces. Even when recombination is severely

restricted it cannot be neglected, as the case of S. pneumoniae (154). Although GBS is a frequent

pathogen in neonates, it is increasing amongst older persons and among those with underlying

medical conditions (e.g. diabetes). For that reason, the recognition of genetic lineages within

serotypes is critical given the different virulence potential of some serotypes. Despite the substantial

heterogeneity within CCs regarding to capsular serotypes, there are some recognized lineages:

CC1/V, CC17/III, CC19/III and CC23/Ia (37). Of note is the claim of a bovine origin of CC17, the major “hyper” invasive neonatal clone. One report

stated that this CC grouped more closely with bovine isolate STs than with other human isolate STs

(14). On the other hand, other authors claim that bovine and human GBS isolates constitute separate

populations, showing no relatedness (145). Independently of the patient age, the major GBS genetic lineages are thought to have the following

characteristics: ST1/V/alp3, ST17/III/rib, ST19/III/rib (other serotypes may be found in this ST, such as

serotype II), ST23/Ia/eps (also with a fair number of serotype III strains) (66, 133, 152), suggesting that

the spread of clones with particular surface proteins and serotypes could reflect the selection of the

specific genetic lineages by the immune system. A recent report from Portugal revealed the

emergence of serotype IV among colonizing GBS isolates, belonging to the hypervirulent CC17

lineage (61). This serotype was also reported in other countries like France (9) and Taiwan (157), both

in invasive and colonizing isolates. As previously stated, there is evidence that GBS populations studies worldwide point to similar clonal

distribution (14, 16, 71, 73). Five major clonal complexes (CCs), i.e., clusters of genetically related

strains, are commonly found among GBS populations, namely CC1, CC10, CC17, CC19, and CC23.

Additionally, the relationship between specific CCs and pilus island, such as CC19 with PI-1 + PI-2a

and CC23 with PI-2a was also observed (105). Clonal expansion cannot be explained by the macrolide resistance, however, there is some

geographic differences regarding associations between serotypes and macrolide resistance, such as

serotype III and V in Europe (58). Of note is the association between serotype V isolates and

macrolide resistance. This association has been described in many studies (1, 65, 104, 111, 132,

159), contrasting with the association between serotype Ib and macrolides resistance in Taiwan (70). The diversification of the GBS population can include capsular switching that may play a role within

closely and divergently related clones (95, 111). Finally, continuous surveillance is crucial for a better

understanding of the dynamic nature of GBS populations, because infrequent clones may emerge and

32

expand locally with enhanced invasiveness for instance ST17 in neonatal infections or ST24 in

Europe.

33

Chapter 2 – Materials and Methods

2.1 Bacterial isolates A collection of 201 GBS isolates recovered from 2003 to 2014 in normally sterile products of neonates

(≤1 year old) in Portugal was analyzed. This was a laboratory-based surveillance program in which

microbiology laboratories of 22 Portuguese medical centers were asked to submit to a central

laboratory all non-duplicate GBS isolates recovered from cases of GBS invasive disease. The

surveillance program had the participation of the following medical centers: Centro Hospitalar do

Algarve, EPE; Centro Hospitalar do Alto Ave, EPE; Centro Hospitalar do Barlavento Algarvio, EPE;

Centro Hospitalar de Cascais, EPE; Centro Hospitalar de Coimbra; Centro Hospitalar Dr. Nélio

Mendonça, EPE; Centro Hospitalar de Entre Douro e Vouga; Centro Hospitalar da Universidade de

Coimbra; Centro Hospitalar de Leiria, EPE; Centro Hospitalar Lisboa Central; Centro Hospitalar

Lisboa Norte; Centro Hospitalar Lisboa Ocidental, EPE; Centro Hospitalar do Porto; Centro Hospitalar

de São João, EPE; Centro Hospitalar de Vila Nova de Gaia/ Espinho, EPE; Hospital Central de Vila

Real; Hospital Garcia da Orta, EPE; Hospital Dr. Fernando da Fonseca; Hospital Infante D. Pedro;

Hospital Pedro Hispano, EPE; Hospital de São Marcos, Braga; Hospital dos SAMS. A case of invasive

disease was defined as the recovery of S. agalactiae from a normally sterile body site. Whenever GBS

isolates were available from more than one sample from the same patient, only the first isolate was

included in the study. The submitted isolates included those recovered from blood (n = 174),

cerebrospinal fluid (n = 24), and synovial fluid (n = 3). The distribution among the years was the

following: n = 3 in 2003, n = 2 in 2004, n = 26 in 2005, n = 24 in 2006, n = 21 in 2007, n = 28 in 2008,

n = 17 in 2009, n = 22 in 2010, n = 18 in 2011, n = 13 in 2012, n = 16 in 2013, and n = 20 in 2014.

2.2 Surface protein gene profile To check the presence of the genes encoding surface protein associated genes of GBS strains, a

multiplex PCR assay was performed aiming at the direct identification of alpha-like protein genes, as

described elsewhere (35). Total bacterial DNA was isolated by treatment of the cells with mutanolysin

and boiling. By analyzing the amplicon size of the GBS surface protein genes given by this assay the

following allelic variants could be determined: the alpha-C protein gene (bca); epsilon protein gene

(eps); rib; alp2/3; and alp4 genes. To differentiate the alp2 and alp3 protein antigen genes another

PCR assay was performed as described elsewhere (108).

2.3 Pathogenicity islands characterization The genes encoding pili in GBS are located within two distinct loci in different regions of the genome,

designated pilus islands 1 and 2 (PI-1 and PI-2), the later presenting two distinct variants, PI-2a and

PI-2b (132). To identify the pilus islands present in each isolate and to confirm that the PI-1-negative

isolates did not carry the pilus pathogenicity islet or parts of it, another PCR assay was performed as

previously described (107).

35

2.4 Phenotypic methods

2.4.1 Serotyping Serotype classification was performed using a latex agglutination assay (Strep-B-Latex kit, Statens

Serum Institut, Denmark). In a few words, a 1-μL loopful of each latex reagent (Ia, Ib, II to IX) from the

kit was added to each drop of a saline suspension with 2 or 3 GBS colonies and mixed briefly in a

glass slide. The slide was then rotated for 15 to 30 s, and a positive reaction was indicated by

agglutination appearing within 30 s. If the reaction time exceeds 30 s, false-positive reactions may

occur. Every time that agglutination did not occur within 30 s, the strains were classified as

nontypeable (NT).

2.4.2 Antimicrobial susceptibility tests All GBS isolates were tested for susceptibility to penicillin, erythromycin, clindamycin,

chloramphenicol, tetracycline, levofloxacin, vancomycin, streptomycin and gentamicin. Antimicrobial

susceptibility was determined by the disk diffusion method according to the Clinical and Laboratory

Standards Institute (CLSI) guidelines for β-hemolytic streptococci. For the assessment of streptomycin

and gentamicin, CLSI guidelines for high level aminoglycoside resistance detection in enterococci

were applied. Moreover, the macrolide resistance phenotype was determined following the double-disk

test as previously described (113).

2.5 Genotypic methods

2.5.1 Multi-locus sequence typing MLST was performed by sequencing seven housekeeping genes as described previously (72), and

sequence types (STs) were identified by using the S. agalactiae MLST database

(http://pubmlst.org/sagalactiae) and were analyzed using the entire database and goeBURST (64).

First, total bacterial DNA was isolated by treatment of the cells with mutanolysin and boiling. Next,

PCR amplification of the seven housekeeping genes was performed, and further sequenced (GATC

Biotech, Konstanz, Germany). New alleles and sequence types were introduced at the S. agalactiae MLST database. Finally, the genetic relatedness between STs were analyzed using PHILOViZ

software (63).

2.5.2 Macrolide-resistance genotypes Isolates showing macrolide resistance were screened for the presence of resistance genes: ermB,

ermA [ermTR subclass], and mef genes, as described elsewhere (58). First, total bacterial DNA was

isolated by treatment of the cells with mutanolysin and boiling. Next, resistant genes were amplified by

multiplex PCR. To further discriminate mef genes into mefA or mefE, an additional PCR was

performed (140).

36

2.5.3 Tetracycline-resistance determinants For the detection of different resistant genes, total bacterial DNA of tetracycline-resistant S. agalactiae

strains was isolated by treatment of the cells with mutanolysin and boiling. These isolates were

screened for the presence of the tetK, tetL, tetM, and tetO genes by performing PCR, as previously

described (157).

2.5.4 Gentamicin and streptomycin resistance determinants The amplification of genes responsible for amynoglycoside resistance, aac(6)-Ie-aph(2)-Ia, aph(2)-Ib,

both for gentamycin resistance, and aph(2)-Ic, aph(2)-Id, aph(3)-IIIa, ant(4)-Ia, aadA, aadE, both for

streptomycin resistance, was performed. Briefly, total bacterial DNA was isolated by treatment of the

cells with mutanolysin and boiling, then, 1 μL of isolated DNA was used as a template in a final volume

of 50 μL of PCR mixture. The samples were amplified by heating for 3 min at 94⁰C, followed by 35

cycles of 94⁰C for 40 s, 55⁰C for 40 s, and 72⁰C for 40 s and concluding with a cycle of 72⁰C for 2 min.

The PCR products were analyzied by electrophoresis in a 1% (wt/vol) agarose gel. UV

transillumination of the bands on the agarose gel showed different band sizes, which allowed direct

identification of the resistance genes.

2.6 Statistical analysis To evaluate the population diversity among the isolates the Simpson’s index of diversity (SID) was

calculated (26) Adjusted Wallace coefficient (AW) was calculated to provide a directional

measurement of concordance between different characteristics of the isolates (26). AW calculations

were performed at the Comparing Partitions website (www.comparingpartitions.info). OR values,

confidence intervals were obtained using statistical software R, controlling the false discover rate

(FDR). Associations ≤0.05 were considered significant.

37

Chapter 3 – Results

3.1 Isolates Three subpopulations were considered in this study: EOD, defined as patients ranging in age from

birth to 6 days after birth, LOD, for patients with ages ranging from 7 days to 3 months, and ULOD

(ultra late-onset disease), which comprises patients with ages later than 3 months until 1 year of age.

GBS were more frequently recovered from male (n = 109; 54.2%) than female patients (n = 83;

41.3%), and for some patients gender information was not available (n = 6). Blood was the most

frequent source of isolation (n = 174; 86.6%), followed by CSF (n = 24; 11.9%) and synovial fluid (SF)

(n = 3; 1.5%) (Table 2). No association was found between isolate source nor sex and time of disease

presentation.

Table 2 Distribution of the 201 GBS isolates by source of isolation and sex.

No. of Isolatesa

Total

Source

EOD

LOD

ULOD

F M ND F M F M

Blood 36 55 5 34 41 2 1 174

Cerebrospinal Fluid 5 4 1 7 7 24

Synovial Fluid 1 2 3

Total 41 59 6 42 50 2 1 201

a EOD, early-onset disease; LOD, late-onset disease; ULOD, ultra late-onset disease;

F, female; M, male; ND, not determined

3.2 Capsular serotyping The results of serotyping the 201 invasive GBS isolates from neonates are summarized in Table 3. To

our knowledge, this is the first time serotype VIII and IX isolates have been identified in Portugal.

Serotype IX is rare and may have evolved because of mutation and/ or recombination between

serotype Ib and serotype V and/ or IV (144). Serotypes Ia (n = 46) and III (n = 117) were the most

frequent among the population, together accounting for 81% of the isolates. Serotypes Ia and III were

found in 25% and 51% of EOD cases, respectively, and in 22% and 66% of LOD cases, respectively.

In fact, the number of serotype III isolates found (n = 117) was higher than the sum of all other

serotypes. No significant association was found between serotype and time of disease presentation

(Table 3).

39

Table 3 Serotype distribution among age groups

Serotypea No. of Isolatesb

Total

EOD LOD ULOD

Ia 26 20 46

Ib 8 3 1 12

II 8 8

III 54 61 2 117

IV 2 2

V 4 5 9

VI 1 1

VIII 1 1

IX 1 1

NT 3 1 4

Total 106 92 3 201

a NT, nontyeable

b EOD, early-onset disease; LOD, late-onset disease; ULOD, ultra late-onset disease

3.3 Antimicrobial susceptibility testing and resistance determinants Neither resistance nor reduced susceptibility to penicillin, levofloxacin or vancomycin was detected.

Three strains were resistant to chloramphenicol, four were resistant to streptomycin, and one was

resistant to gentamicin. In the population studied here, all streptomycin resistant isolates harbored the

same resistant determinants, aph(3)-IIIa and aadE. The only gentamicin resistant strain found had

aac(6)-Ie-aph(2)-Ia. All aminoglycoside resistance strains were type III serotype, harbored rib gene,

and belonging to ST17, except one streptomycin resistant strain that belonged to ST757, a SLV of

ST17. For the total isolates in the 2003 to 2014 period, the percentage of GBS isolates that were resistant to

erythromycin and to clindamycin ranged from 15% or less between 2005 and 2009 to 40% (8/20) and

30% (6/20) in 2014, respectively (Figure 5). Of note, an increase in resistance rates for both antibiotics

was observed in the interval of the study. Among the 32/201 (16%) erythromycin resistant isolates, 20

(62.5%) had the cMLSB phenotype, 6 (18.8%) had the iMLSB, and the M phenotype was found in 5

strains (16.3%). All of the iMLSB and M resistance phenotypes were conferred by the presence of the

ermTR and mefE, respectively, whereas the cMLSB was mostly related to the presence of the ermB,

and one isolate has ermTR gene but presenrted cMLSB phenotype.

40

Table 4 Properties of the genetic lineages found among the 201 invasive isolates. Macrolide resistance Tetracycline resistance No. of isolates

CC/STa Serotype (n)b Protein (n) Pili (n)

related with

Phenotype (n) Genotype (n) Genotypec (n)

EOD/LOD/ULOD

CC1

ST1 Ib (7) ALP3 PI-1 + PI-2a cMLSB (7) ermB (7) tetM (7) 4/2/1

V (6) ALP3 PI-1 + PI-2a cMLSB (3) ermB (2), ermTR (1) tetM (2) 1/5/0

VI (1) BCA PI-1 + PI-2a 1/0/0

ST2 Ia (1) EPS PI-2a tetM (1) 0/1/0

III (1) EPS PI-2a 1/0/0

V (2) EPS PI-1 + PI-2a tetM (1) 2/0/0

ST196 Ia (1) EPS PI-1 + PI-2a tetM (1) 1/0/0

IV (1) EPS PI-1 + PI-2a tetM (1) 0/1/0

CC8

ST8 Ib (1) BCA PI-1 + PI-2a tetM (1) 1/0/0

CC10

ST10 Ib (1) EPS PI-1 + PI-2a tetM (1) 0/1/0

NT (1) EPS PI-1 + PI-2a 1/0/0

ST9 Ib BCA PI-1 + PI-2a 1/0/0

CC12

ST12 Ib (1) BCA PI-1 + PI-2a ermB (1) tetM (1) 1/0/0

II (2) BCA PI-1 + PI-2a tetM (1), tetO (1) 2/0/0

V (1) BCA PI-1 + PI-2a tetM (1) 1/0/0

CC17

ST17 III (83) RIB PI-1 + PI-2b (78), cMLSB (5), M (1) ermB (5), mefE (1) tetM (65), tetO (2), tetM+tetL (3), 36/45/2

PI-2b (5) tetM+tetO (2), Ø (1)

ST109 III (6) RIB PI-1 + PI-2b tetM (1) 2/4/0





ST482 III (1) RIB PI-1 + PI-2b tetM+tetL (1) 1/0/0




ST757 III (1) RIB PI-2b cMLSB ermB Ø (1) 0/1/0

CC19

ST19 III (14) RIB PI-1 + PI-2a (14) cMLSB (2) ermB (2) tetM (10) 8/6/0

iMLSB (5) ermTR (5)

ST28 II (5) RIB PI-1 + PI-2a tetM (1) 5/0/0

VIII (1) PI-1 + PI-2a 1/0/0

ST182 III (1) RIB PI-1 + PI-2a cMLSB ermB tetO (1) 1/0/0

ST335 III (1) RIB PI-1 + PI-2a iMLSB ermTR tetM (1) 1/0/0

ST510 III (1) RIB PI-1 + PI-2a tetM (1) 1/0/0

ST742 III (1) RIB PI-1 + PI-2a tetM (1) 1/0/0

CC22

ST22 II (1) BCA PI-2a cMLSB ermB 1/0/0

NT (1) BCA PI-1 + PI-2a tetM (1) 1/0/0

CC23

ST23 Ia (26) EPS PI-2a (26) M (4) mefE (4) tetM (26) 18/8/0

NT (1) BCA PI-1 + PI-2a Ø (1) 1/0/0

ST88 Ia (1) ALP2 PI-1 + PI-2a tetM (1) 1/0/0

ST144 Ia (1) RIB PI-2a tetM (1) 1/0/0

ST745 Ia (1) EPS PI-2a 1/0/0

ST24 Ia (10) BCA PI-2a (9) tetM (10) 3/7/0

ST452 IV (1) BCA PI-1 + PI-2a 0/1/0

ST498 Ia (6) BCA PI-2a tetM (6) 1/5/0

CC130

ST130 IX (1) BCA PI-2a 0/1/0

a CC, Clonal complex; ST, sequence type

b NT, nontypeable

c Ø, resistant strains with no amplification of either tetM, tetL, tetK, tetO, nor any combination of them.

10

Percentages

eryR clinR

45,0% 40,0% 35,0% 30,0% 25,0% 20,0% 15,0% 10,0%

5,0% 0,0%

2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 n = 26 n = 22 n = 16 n = 26 n = 16 n = 21 n = 20 n = 13 n = 16 n = 20

Year

Figure 5 Erythromycin and clindamycin resistance in the period 2005-2015. Each bar corresponds to the percentage of antibiotic resistance among all isolates isolated within each year. Note: the first two years of the study were excluded from this analysis because no resistant isolates were found and due to the low number of isolates recovered (2003, n = 3; 2004, n = 2). eryR, erythromycin resistance; clinR, clindamycin resistance.

Most of the GBS isolates were resistant to tetracycline (n = 173, 86.1%), and the most frequent

resistance determinant was tetM (n = 160, 79.6%). Four strains had tetO and, in agreement with a

previous report (71), none of the isolates showed the presence of tetK determinant. All tetL-positive

isolates also possessed the tetM gene. Two isolates possessing the tetO gene carried also the tetM.

In three isolates, the resistance determinant could not be identified (n = 3) (Table 4).

3.4 Surface protein and pilus island gene profiling All isolates were positive for the presence of only one surface protein gene. The surface protein gene

rib was the most prevalent (n = 124), followed by the eps (n = 36), bca (n = 27), alp3 (n = 13), and alp2

genes (n = 1), showing variable distributions across serotypes (Table 4). There was a significant

association between some serotypes and a particular surface protein gene, namely Ib with alp3, III

with rib, Ia and eps and V with alp3 (all OR > 30). There was a high correspondence between

serotypes and surface protein genes (AW, 0.839 [CI95%, 0.784 to 0.895]). PIs were differentially detected among GBS serotypes (Table 4). All isolates showed the presence of

at least one PI. Overall, PI-1 was detected in 73.6% (n = 148), PI-2b in 49.3% (n = 99), and PI-2a in

50.7% (n = 102) of the isolates. The most frequently PIs found were PI-1 and PI-2b (46.3%), followed

by PI-1 and PI-2a (27.4%), PI-2a alone (23.4%), and PI-2b alone (2.9%).

3.5 MLST cluster analysis To identify the genetic lineages associated with each feature, all isolates were characterized by MLST.

The goeBURST algorithm (64) implemented in PHYLOViZ (63) divided the 33 STs identified in the

data set into 7 clonal complexes (CCs). Four new STs (ST742, ST744, ST745, and ST757) were

identified among the isolates studied. The SID for the classification of the isolates according to their

10

MLST-based sequence type was 0.799 (CI95%, 0.749 to 0.849), indicating a considerably genetically

diverse population (Figure 6). MLST analysis showed that each ST was composed almost exclusively of isolates of the same

serotype and showing the same surface protein gene (AW, 0.959 [CI95%, 0.938 to 0.980] and AW,

0.982 [CI95%, 0.959 to 1.000], respectively). Thus, strong associations were found between STs and

serotypes: ST1 with Ib and V, ST17 with III, ST19 with III, ST23 with Ia, ST24 with Ia, ST28 with II,

ST498 with Ia (all OR > 42). Also between STs and surface protein genes: ST1 and alp3, ST2 and

eps, ST17 and rib, ST19 and rib, ST23 and eps, ST24 and bca, ST498 and bca (all OR > 368).

Interestingly, within CC23 there is evidence of two genetic lineages, namely ST23/Ia/eps and

ST24/Ia/bca, as discussed below. The largest group of the GBS population is the CC17 (n = 99, 49%), corresponding to the previously

identified “hypervirulent” CC among GBS neonatal invasive disease (107). Also, strains from the

ST17/III/rib genetic lineage represent 84% of the CC. Additionally, all of strains from this genetic

lineage have PI-2b (Table 3), and an association between ST17 and the combination o PI-1 and PI-2b

was found (OR = 78 [CI95%, 27 to 279]). Others major CCs are CC23 (n = 47), CC19 (n = 24), and

CC1 (n = 20). However, none of the CCs showed a significant association with time of disease

presentation. In CC1, ST1 showed an association with serotype Ib and serotype V, and both serotypes

were associated with alp3 surface protein gene. However, only serotype Ib was associated with

erythromycin resistance (OR > 10). Moreover, just the first genetic lineage showed an association with

erythromycin resistance (OR > 10), contrasting with previous reports (103, 131). Genetic lineages

ST1/Ib/alp3 and ST19/III/rib showed an association with macrolide resistance, however, only the first

one remained significant when controlled for FDR (P < 0.05), though ST19/III/rib was marginally

significant after controlled by FDR (P = 0.0512).

43

Figure 6 GBS MLST-based phylogenetic tree using goeBURST algorithm. Each node represents one ST and STs differing by only one allele are connected by a line. Node dimensions refer to the relative number of strains belonging to each ST. Colored dots represent the 7 CCs of GBS population under analysis. CC8, CC10, and CC12 were merged to facilitate visualization. Light-blue and light-green circles indicate STs that are not present in the collection analyzed but that could be found in the Pubmlst database; the latter were identified as subfounders. Within each CC, the STs present within the collection are represented in red, dark-blue, and light-green dots with the size of the circles being proportional to the number of isolates (in a logarithmic scale) . Red dots are isolates comprised in goeBURST group 0; CCs included in the dotted line circle are linked by transition STs. The figure was prepared using PHYLOViZ software (http://www.phyloviz.net/).

22

44

Chapter 4 – Discussion This study analyzed a Portuguese collection of 201 GBS strains from neonatal invasive disease from

2003 to 2014 period. In the first two years of the study there was a very small number of isolates,

preventing detailed evaluation of the temporal changes in respect to these years. The clonal diversity

of the GBS population based on each feature revealed a quite diverse population. Still the different

diversity values could be accounted by the fact that all isolates were recovered from newborns and

some characteristics, such as surface protein genes and pilus islands, are less diverse than studies in

which adults isolates were also included. Of note is the identification of the first serotypes VIII and IX GBS isolates among invasive disease in

Portugal. Serotype IX seems to have existed for at least 20 years and is widely distributed

geographically, still it is unclear what its epidemiology is (144). Regarding the capsular serotype, type

III and Ia were the most common serotypes observed. This is similar to what had been found in Spain

in 2011 (104) and in some other European countries such as Norway in the period between 1996 and

2006 (12), and France in 2008 (125). Thus indicating that these two serotypes continue to be

responsible for a great number of neonatal infections. MLST analysis shows that GBS present a straggly population, concordant with populations which

have a high recombination to mutation rate (160) (Figure 6). However, more studies might elicit the

importance of recombination in GBS diversification, since there are recent contradictory studies (23,

37, 60). The following genetic lineages were prevalent among the population ST1/alp3, ST17/rib, and ST19/rib.

Additionally, other associations were found, namely between ST24 and ST498 with bca and between

ST23 and eps. Both of these STs, ST23, ST24, and ST498 are type Ia serotype, contributing to the

importance of these lineages to neonatal infections, since serotype Ia was the second most frequent

serotype. Additionally, only two serotype Ia isolates were found outside CC23, where ST23, ST24 and

ST498 are grouped. The suggestion that ST24/bca is a clone mainly found in Europe, particularly in

the Iberian Peninsula and Mediterranean region, is supported by the results shown here (65, 95).

Additionally to the fact that serotype Ia is dominant in CC23, this dominance was also in carriage in

pregnant women, and in nonpregnant adults, revealing its widespread and its ability to colonize and

invade humans (104). Moreover, two sublineages were found within CC23, ST23/Ia/eps/PI-2a and

ST24/Ia/bca/PI-2a. In addition, six strains from another CC23 genetic lineage were found among the

population: ST498/Ia/bca/PI-2a. Since ST498 is a triple-locus variant of ST23, and a double-locus

variant of ST24, this may be evidence that these lineages are continuing to diversify. One example is

ST745, one of the several STs identified in this study, belonged to CC23. On the other hand, the number of serotype III isolates was clearly overrepresented, and the genetic

lineage ST17/III/rib accounts for near half of the GBS invasive strains among the studied population,

showing that this lineage is extremely virulent among newborns. In agreement with previous studies,

ST19 was found to be poorly represented in the population (n = 14, 7%), and it was expected since the

GBS collection analyzed included only isolates that had caused invasive neonatal infections, (104).

ST17 is mainly found associated with invasive disease in newborns, irrespectively to the age of onset

of the disease and it may be a stable “hypervirulent” clone (107).

45

Regarding macrolide resistance, an association between macrolide resistance and serotype V was not

found, contrary to previous results elsewhere (42). However, results reported here showed an

association between ST1/Ib/alp3 genetic lineage and macrolide resistance, but not with ST1/V/alp3.

This observation may lead to the suggestion that a capsular switching event may have occurred,

having been subsequently selected. Of note is the fact that macrolide resistance was found among

ST17/rib isolates, five showed the cMLSB phenotype and one showed the M phenotype, however no

significant association was observed. Up until now, both streptomycin and gentamicin resistance was

only found sporadically and it is rare among GBS (25, 116). Recently, a study from Argentina found

high-levels of resistance to aminoglycosides (13.5% to gentamicin and 16.3% to streptomycin) in GBS

isolates from pregnant women at term (168). The results here observed indicate that a vaccine including components from PI-1 and PI-2a could

provide potential coverage against 97% of isolates, supporting their use in a future vaccine as

previously suggested (102, 105). The presence of PI-2a alone was predominantly found in CC23. Nonetheless, when the distribution of

the PIs in the collection is displayed, almost exclusive correspondence between particular PI

combinations and CCs is observed (Table 4). Since the pilus island is flanked by direct repeats and

contains transposable elements (130, 147), the hypothesis of diversification of GBS clones following

the loss and acquisition of PIs might be plausible (148). However, such hypothesis lacks clear

evidence for gain and loss of PI throughout the clonal complexes evolutionary model.

46

Chapter 5 – Conclusions GBS is an important cause of neonatal sepsis and meningitis in developed countries, and it is also a

recognized and increasing cause of disease in adults in such countries, especially pregnant women,

the elderly and the immunocompromised (86). The implementation of IAP lead to a decrease in EOD incidence but LOD remains unchanged. Here

we found an increase of macrolides resistance between 2003 and 2014, mainly due to the emergence

of the ST1/Ib/alp3/ermB genetic lineage. Neonatal invasive GBS population was clustered in four major CCs: CC1, CC17, CC19, and CC23.

CC17 was the most prevalent, comprising more isolates than the sum of the other four major CCs

found in the population. Specifically, the genetic lineage ST17/III/rib/PI-1+PI-2b is extremely virulent

among neonatal infection disease, since it accounts alone for near 40% of GBS population studied.

That fact is in concordance with the association of ST17/III genetic lineage with invasive disease in

neonates reported in previous studies, where colonization of pregnant women was compared against

invasive GBS isolates recovered from neonates (110). Taken together, the results showed here reveal a stable clonal structure of the GBS causing neonatal

infections in Portugal over the period from 2003 to 2014, in spite of the limited number of medical

centers that participated in the surveillance program. Besides, the data reported here is mostly in

accordance with observations previously stated in Portugal (104), in which the presence of a particular

alpha or alpha-like surface protein gene is a clonal property. This observation can be made because of

the prevalence of a particular alpha or alpha-like protein gene in each CC, in contrast with the different

serotype distribution among CCs (Table 3). Pilus-based vaccines continue to be appealing since 97% of the considered GBS isolates share PI-1

and PI-2a, but more studies will elucidate the pili role in pathogenesis and this evolutionary pattern in

humans, as well in other animals. Finally, GBS population shows patterns supporting clonal model for its evolutionary history, although

other forces should be taken into account and continuously studied, for instance, recombination rates

within each CC and the selective pressure applied by the hosts. Further studies in developing

countries are needed to better formulate appropriate public health interventions. Upon our

understanding about the epidemiology and biology of GBS, an effective vaccine might be the best

option to relieve the disease burden caused by this emerging pathogen in both developed and

developing countries.

47

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