Diagnostic Microbiology and Infectious Disease 78 (2014) 487–490

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Diagnostic Microbiology and Infectious Disease

journal homepage: www.elsevier.com/locate/diagmicrobio

Molecular characterization of Streptococcus agalactiae from vaginal colonization and

neonatal infections: a 4-year multicenter study in Greece☆
Apostolos Liakopoulos a, Angeliki Mavroidi a, Sofia Vourli b, Maria Panopoulou c, Levantia Zachariadou d,
Stylianos Chatzipanagiotou e, Iris Spiliopoulou f, Loukia Zerva b, Efthymia Petinaki a, g,⁎
a
Department of Microbiology, University Hospital of Larissa, Larissa, Greece
b
Department of Clinical Microbiology, “ATTIKON” Hospital, University of Athens, Greece
c
Department of Microbiology, Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupolis, Greece
d
Aghia Sophia Children's Hospital, Athens, Greece
e
Department of Biopathology and Clinical Microbiology, Athens Medical School, Aeginition Hospital, Athens, Greece
f
Department of Microbiology, School of Medicine, University of Patras, Greece
g
Department of Microbiology, Medical School, University of Thessaly, Biopolis, Larissa, Greece

a r t i c l e i n f o a b s t r a c t

Article history: A multicenter collection comprising of 171 Streptococcus agalactiae isolates from pregnant women recovered
Received 16 October 2013 between 2007 and 2010 and 46 from unmatched neonates with invasive infections was subjected to
Received in revised form 11 December 2013 antimicrobial susceptibility testing and genetic characterization. High rates of erythromycin resistance
Accepted 15 December 2013
(20.47%) were observed only in isolates from pregnant women. ST1 was dominant in the vaginal colonization,
Available online 7 January 2014
whereas the hypervirulent ST-17 clone was detected in 67.39% of neonatal infections.
Keywords:
© 2014 Elsevier Inc. All rights reserved.
Genetic characterization
S. agalactiae
Greece
Colonization
Neonatal infections

Streptococcus agalactiae, a Group B Streptococcus, remains the
leading cause of neonatal sepsis and meningitis in many countries,
while colonizing frequently the genital track of women (Phares et al.,
2008). The estimated rate of transmission from colonized mothers to
newborns during childbirth varies from country to country and may
result in invasive infection in the newborn during the first week of life,
known as early-onset group B streptococcal infection (Phares et al.,
2008). Molecular typing methods and population genetics of S.
agalactiae have shown that the majority of neonatal infections
including meningitis are caused by a limited number of “highly
virulent clones”, such as ST17 belonging mainly to serotype III (Kong
et al., 2008; Lin et al., 2006; Manning et al., 2009; Martins et al., 2007;
Van der Mee-Marquet et al., 2009).
A previous study in Greece has demonstrated that the incidence of
S. agalactiae neonatal invasive diseases is very low (0.5%), while the
maternal and neonatal colonization rates reach approximately to 6.6%
and 2.4%, respectively (Tsolia et al., 2003). Until now, there are no data
about the prevalence of “highly virulent clones” among S. agalactiae
☆ Transparency of declaration: The authors have no conflict of interest.
⁎ Corresponding author. Tel.: +30-241-350-2517; fax: +30-241-350-1570.
E-mail address: petinaki@med.uth.gr (E. Petinaki).
strains isolated from neonatal invasive diseases and colonized
pregnant women in our country. In an effort to determine the
predominant clones among S. agalactiae isolates in Greece, molecular
characterization of 171 vaginal and 46 invasive neonatal isolates were
assessed by multilocus sequence typing (MLST) and serotyping.
The cervical isolates were recovered from 171 individual colonized
pregnant women from four tertiary hospitals located in different areas
of Greece (Northern Greece [n = 39], Southern Greece [n = 30],
Central Greece [n = 46], and Eastern Greece [n = 56]), during the
period 2007–2010. After identification, the isolates of each partici-
pating hospital were sent to the Department of Microbiology of the
University Hospital of Larissa (UHL) for further investigation. A
collection of 46 invasive S. agalactiae recovered from blood cultures
and cerebrospinal fluids, all consecutively isolated between 1995
and 2010 from equal number of neonatal invasive infections
(sepsis and meningitis) in the Paediatric Hospital of Athens “Aghia
Sofia”, were also included in this study. Ten out of 46 isolates from
neonatal infections were consecutively isolated between 2007 and
2010. We should note that the collection of the S. agalactiae
isolates recovered from the colonized pregnant women did not
include samples from the mothers of the neonates, for which no
data were available.
The identification of the isolates to species level was confirmed in
the Department of Microbiology of UHL based on Gram staining,

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488 A. Liakopoulos et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 487–490

colony morphology, haemolysis, and latex agglutination test Table 1

with specific antisera, using the Slidex Strepto Plus (BioMerieux, CCs, MLST STs, serotypes, and antimicrobial resistance patterns of 217 S. agalactiae
isolates from neonates and colonized pregnant women.
Marcy l'Etoile, France). Antimicrobial susceptibility testing to various
antimicrobial agents (penicillin, ampicillin, cefepime, ceftriaxone, CC MLST Serotypes Antimicrobial Neonatal Colonizing Total of
chloramphenicol, erythromycin, clindamycin, levofloxacin, and STa (No.) resistance isolates, isolates, isolates,
patterns (No.) No. (%) No. (%) No. (%)
tetracycline) was performed by the disk diffusion method accord-
ing to the guidelines and interpretive criteria of the CLSI, (2011). CC-1 1 IV(15), TC (23)TC, EM, 43 (25.15) 43
V(28) CM (20) (19.82)
The Double Disk Synergy Test and Gots' test were performed in all
2 V (2) TC (2) 2 (1.17) 2 (0.92)
erythromycin- and/or clindamycin-resistant isolates in order to 196 IV (2) TC (2) 2 (1.17) 2 (0.92)
distinguish MLSB-inducible, M and L phenotypes, as previously CC-10 8 Ib (21) TC (17)CC (2) 3 (6.52) 18 (10.53) 21(9.68)
recommended (Gots, 1945; Hamilton-Miller and Saroj, 2000). TC, EM, CM (2)
DNA was extracted from all isolates using the Quick-gDNA TM 10 Ib (2) TC (2) 2 (1.17) 2 (0.92)
12 Ib (15) TC (14)TC, EM, 15 (8.77) 15 (6.91)
MiniPrep kit (Zymo Research, Irvine, CA, USA), and the presence of the
CM (1)
commonly found antimicrobial resistance genes in streptococci for CC-17 17 III (35)Ia TC (35)TC, EM, 31 7 (4.09) 38
tetracycline [tet(K), tet(L), tet(M), tet(O), tet(S), tet(T)], erythromycin (3) CM (3) (67.39) (17.51)
[mef(A), erm(A), erm(B), erm(TR)] and clindamycin [vga(A), vga(B), 146 III (3) TC (3) 3 (6.52) 3 (1.38)
CC-19 19 III (27) TC (21)EM (4) 6 (13.05) 21 (12.28) 27
vga(C), vga(D), vga(E), lsa(A), lsa(B), lsa(C), lsa(E), lnu(A), lnu(B),
TC, EM, CM (2) (12.44)
lnu(C), lnu(D)] was determined by PCR with primers and conditions 28 III (6) TC (6) 6 (3.51) 6 (2.76)
as described previously (Culebras et al., 2002; Dogan et al., 2005; 106 III (2) TC, EM, CM (2) 2 (1.17) 2 (0.92)
Leclercq, 2002; Poyart et al., 2003; Roberts, 2008). 197 III (3) TC (3) 3 (1.75) 3 (1.38)
Genetic characterization of the isolates was performed by MLST, as 219 III (2) TC (2) 2 (1.17) 2 (0.92)
335 III (2) TC (2) 2 (1.17) 2 (0.92)
previously recommended (Jones et al., 2003). Sequences were
CC-22 22 III (3) TC (3) 3 (1.75) 3 (1.38)
obtained on both DNA strands; the allelic profiles were determined CC-23 23 III (34) TC (28)TC, EM, 3 (6.52) 31 (18.13) 34
and assigned to sequence types (STs), and novel alleles and STs were CM (6) (15.67)
submitted to the S. agalactiae MLST website (http://pubmlst.org/ 24 III (3) TC (3) 3 (1.75) 3 (1.38)
220 III (2) TC (2) 2 (1.17) 2 (0.92)
sagalactiae/). For each ST, the sequences of the 7 housekeeping gene
464 III (2) TC (2) 2 (1.17) 2 (0.92)
fragments were concatenated maintaining the +1 reading frame 498 III (3) TC (3) 3 (1.75) 3 (1.38)
aligned, and a neighbor-joining tree was constructed from the aligned Singleton 314 III (2) TC (2) 2 (1.17) 2 (0.92)
sequences using the MEGA software (Tamura et al., 2007). Non Subtotal 46 (100) 171 (100) 217
overlapping groups of related STs were identified using eBURST, with (100)

the default setting for the definition of groups (Feil et al., 2004). The
presence of the ST17 specific gbs2018 allele, encoding for the surface
protein HvgA was assessed by PCR with primers and conditions
as described previously (Lamy et al., 2006). In addition, serotyping
was performed with a coagglutination method using antisera
against types I to VIII (Essum, Umeå, Sweden) according to the
manufacturer's instructions.
Although S. agalactiae clinical strains with reduced sensitivity for
penicillin G have been recorded elsewhere, all 217 isolates were
sensitive to all beta-lactams tested as well as chloramphenicol and
levofloxacin (Nagano et al., 2012). The various patterns of antimicro-
bial resistance are described in Table 1; 35 isolates (16.3%) were co-
resistance to tetracycline, macrolides, and lincosamides.
Interesting finding of the present study was the high rates of
resistance to tetracycline (97.2%). In more details, 169 out 171 isolates
derived from pregnant women (98.83%) exhibited resistance to
tetracycline and carried the tet (M) gene (162 isolates), the tet (O)
gene (6 isolates), and the tet (L) gene (1 isolate). Among the 46
isolates from infected neonates, 42 (91.3%) were resistant to
tetracycline and carried the tet(M) gene (41 isolates) and the tet(O)
gene (1 isolate).
Among the 171 S. agalactiae isolates recovered from colonized
pregnant women, 33 (19.3%) exhibited cross-resistance to erythro-
mycin and clindamycin (MLSB phenotype), while 3 (1.75%) and 2
(1.17%) isolates were only resistant to clindamycin (L phenotype) and
erythromycin (M phenotype), respectively. On the other hand, among
the 46 isolates recovered from neonatal invasive diseases, 2 (4.35%)
isolates exhibited MLSB phenotype, and 1 (2.17%) exhibited M
phenotype. Of the 35 isolates exhibiting cross-resistance to erythro-
mycin and clindamycin (MLSB phenotype), 27 (77.14 %) showed the
constitutive MLSB (cMLSB) phenotype and carried the erm(B) gene,
whereas only 8 (22.86%) isolates showed the inducible MLSB (iMLSB)
phenotype and carried the erm(TR) gene. The 3 erythromycin-
resistant isolates exhibiting M phenotype carried the mef(A) gene,
but none of the resistant genes tested was found in the clindamycin-
resistant isolates exhibiting L phenotype.
TC = tetracycline; EM = erythromycin; CM = clindamycin.
a
The most prevalent STs are indicated in bold.
The distribution into STs and clonal complexes (CCs) of the
S. agalactiae isolates recovered from neonatal infections and colonized
women genotyped by MLST is shown in Table 1. We have identified
21 STs among all isolates tested, which were clustered into 6 CCs; 1 ST
was singleton (Table 1). The genetic relatedness of the 21 STs found in
the present study was assessed by the Neighbour-Joining algorithm
and is shown in Fig. 1.
STs 1, 8, 12, 17, 19, and 23 were the most prevalent ones, each
comprised of 15–43 isolates, whereas the remaining STs were
comprised of 2–6 isolates (Table 1). The distribution of the STs did
not appear any annually variability, while no differences in the
distribution of STs among the different areas of Greece were observed.
STs 8, 17, 19, and 23 were shared between colonized women and
infected neonates (Table 1). The most prevalent clone among
pregnant women was ST1 (25.15%), while among neonatal isolates,
ST17 predominated (67.39%). We note that 8 out of 10 neonatal
isolates, recovered between 2007 and 2010, belonged to ST17; the
remaining 2 isolates belonged to ST19. All ST17 S. agalactiae isolates,
either from neonates or from pregnant women, were positive for the
ST17-specific allele of the hvgA gene that confers hypervirulence and
meningeal tropism in neonates, as reported previously (Lamy et al.,
2006; Tazi et al., 2010). All erythromycin-resistant strains belonged to
several STs, mainly to STs 1 and 23, while all ST17 strains were
sensitive to macrolides and lincosamides (Table 1).
Serotyping results revealed that isolates belonging to ST8 and ST12
were classified as Ib type, ST1 as either IV or V type, ST23 and ST19 as
III type, whereas ST17 as either Ia or III type (Table 1). In our study, a
capsular switching of some STs was observed, resulting in the
appearance of new serotype-genotype combinations. This finding
emphasizes the need for the combined application of MLST and
serotyping for surveillance purposes.
 A. Liakopoulos et al. / Diagnostic Microbiology and Infectious Disease 78 (2014) 487–490
Fig. 1. Neighbor-Joining tree of the aligned concatenated gene fragments of the 7 housekeeping loci (adhP, pheS, atr, glnA, sdhA, glcK, tkt) of the 21 STs found among the 217
S. agalactiae isolates included in the present study. The origin of the isolates within each ST is indicated with different shapes: a circle for colonized pregnant women, a square for
neonatal, and a triangle for both colonizing and neonatal associated STs.
To our knowledge, this is the first report about the antimicro-
bial susceptibility patterns, the resistance genes, the serotypes, and
the molecular characterization of a multicenter collection of
S. agalactiae isolates recovered both from colonized pregnant
women and infected neonates in Greece. However, a limitation of
the present study was that the collection of S. agalactiae isolates
recovered from the neonates and colonized pregnant women was
not maternal-neonatal matched pairs; therefore, no conclusions
regarding direct transmission between mothers and neonates can
be concluded from this study.
Furthermore, high rates of erythromycin resistance (20.47%)
were detected in strains collected from colonization; this rate was
2-fold higher than that described in 2003 and could be explained
by the extensive usage of macrolides in Greece for the treatment
of skin and soft tissue infections caused by Staphylococcus aureus
mainly belonging to ST80 (Tsolia et al., 2003; E. Petinaki,
unpublished data). ST23 was the second most prevalent ST
among colonized pregnant women in this study, whereas high
rates of ST23 S. agalactiae were recently observed in invasive
infections from adults in Portugal. This finding also highlights the
importance for national surveillance of S. agalactiae from both
colonizing and causing invasive infections, in order to elucidate
the epidemiology and the transmission patterns of S. agalactiae
infections (Martins et al., 2012).
Acknowledgments
We thank A. Damani and A. Chatzimoschou for technical
assistance.
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