Microbial Pathogenesis 99 (2016) 119e122

Contents lists available at ScienceDirect

Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Characterization of virulence factors, antimicrobial resistance pattern

and clonal complexes of group B streptococci isolated from neonates
Mohammad Emaneini a, Fereshteh Jabalameli a, Akbar Mirsalehian a, Amir Ghasemi b,
Reza Beigverdi a, *
a
Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran
b
Department of Microbiology and Immunology, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran

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

Article history: Between January and December 2013, swab samples were taken for the throat and external ear canals of
Received 8 March 2016 1037 newborns for screening of Group B Streptococcus (GBS or S. agalactiae). Isolates were analyzed form
Received in revised form Multilocus sequence typing (MLST), capsular type, virulence genes and antibiotic susceptibility. The
16 July 2016
MLST analysis of 19 GBS isolates showed 8 sequence types (STs). Overall the most common STs were ST19
Accepted 18 August 2016
Available online 20 August 2016
and ST28. Other STs were ST1, ST4, ST8, ST12, ST335 and ST734 (a new ST). The most common clonal
complexes (CCs) were CC19 (68.4%) and CC10 (21%). The scpB, hlyB and bca virulence genes were detected
in all STS, while the bac gene was predominant in ST12 with capsular type (CT) Ib. The IS1548 and the rib
Keywords:
GBS
genes were particularly prevalent in CTIII and were detected in isolates belong to ST19, ST335 and ST734
CC19 and were grouped in CC19. All isolates were susceptible to penicillin, vancomycin, linezolid and
Neonates quinupristin-dalfopristin. Resistance to tetracycline was observed in all 19 (100%) strains and was
Resistance correlated with presence of the tetM gene except for one isolate with ST12. All the ST8 and ST12 isolates
Virulence gene were resistant to macrolide carrying two resistance genes; the ermTR and the ermB, respectively. The
results of this study showed that the CC19 was a major clone in the neonatal intensive care unit (NICU) of
Imam Khomeini hospital which can cause severe infections in susceptible neonates (particularly in
premature infants). As a result, an intensive infection control policy is needed to prevent the spread of
this clone.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction bacteremia are more common in LOD [3,4]. In order to understand

Group B Streptococcus (GBS or S. agalactiae) is known as a
leading cause of neonatal sepsis and meningitis [1,2]. It is estimated
that 10e30% of pregnant women are rectovaginal colonized by GBS
and maternal carriage is the major source of neonatal infections [3].
In the absence of any intervention, between 1 and 2% of neonates
born from GBS-colonized mothers develop severe diseases [3].
Neonatal disease results from vertical transmission from mothers
to neonates during childbirth or via horizontal transfer by nursery
personnel [4]. GBS infections are separated into Early-Onset Dis-
ease (EOD) occurring in newborns 0e6 days of age and Late-Onset
Disease (LOD) occurring in newborns 7e90 days of age [4]. Sepsis
and pneumonia are more common in EOD, while meningitis and
* Corresponding author. Department of Microbiology, School of Medicine, Tehran
University of Medical Sciences, 100 Poursina St., Keshavarz Blvd., Tehran, Iran.
E-mail addresses: r-beigverdi@tums.ac.ir, rbeigverdi@gmail.com (R. Beigverdi).
the population structure of GBS, a variety of techniques have been
developed among which multilocus sequence typing (MLST) was
introduced as the standard approach for typing [5]. MLST of GBS
isolates from different countries have shown that only limited
numbers of clonal complexes (CCs) including CC1, CC10, CC17, CC19
and CC23 were associated with colonizing or invasive isolates [6,7].
Among these CCs, CC17 is a hypervirulent clone, mostly associated
with invasive disease in neonate, whereas CC19 causes invasive
diseases among either neonates or adults [8,9]. However, more
recent studies have shown that CC1, CC19 and CC23 were respon-
sible for a high proportion of colonizing isolates [5,10]. Several
studies have revealed that CC17 is relatively a homogenous group of
capsular type (CT) III isolates and other CCs are heterogeneous
groups which express different CTs [11,12]. The polysaccharide
capsule is the most important virulence factor and, based on the
differences in the structure of surface polysaccharides, 10 CTs (Ia, Ib,
IIeIX) have been described [13,14]. Moreover, GBS has several

http://dx.doi.org/10.1016/j.micpath.2016.08.016
0882-4010/© 2016 Elsevier Ltd. All rights reserved.
120 M. Emaneini et al. / Microbial Pathogenesis 99 (2016) 119e122
mobile genetic elements (MGEs) in the genome, among which
GBSi1 and IS1548 can serve as genetic markers for lineages CC17
and CC19, respectively [15]. There are several reports from different
areas of Iran on the prevalence of GBS among pregnant women
[16,17]; however, to our knowledge no data exist on the molecular
characteristics of GBS strains isolated from neonates. The goal of
current study was to determine phenotypic and genotypic char-
acteristics of GBS isolated from neonates in Tehran, Iran.
2. Methods
2.1. Patients sampling
Between January and December 2013, swab samples were taken
from the throat and external ear canals of 1037 newborns that did
not develop EOD or LOD, and were hospitalized in the neonatal
intensive care unit (NICU) of Imam Khomeini hospital, Tehran-Iran
due to various clinical conditions. Sample processing was per-
formed according to the recommendations of the Centers of Dis-
ease Control and Prevention (CDC) guidelines [3].
2.2. Bacterial strains
Briefly, after sampling, swabs were inserted directly into Todd-
Hewitt broth with 8 mg/ml gentamicin and 15 mg/ml nalidixic acid
and were transferred to the microbiology laboratory. After over-
night incubation at 35  C, the incubated broth was then sub
cultured on 5% sheep blood agar (SBA) for 18e24 h at 35  C with 5%
CO2. Presumptive GBS colonies on the SBA plate were identified to
the species level by a combination of standard tests [13]. To confirm
the identity of isolate as GBS, the dltS gene was amplified by po-
lymerase chain reaction (PCR) [13].
2.3. Ethical approval
The study was approved by the Ethics Committee of Tehran
University of Medical Sciences.
2.4. Antimicrobial susceptibility testing
Antibiotic susceptibilities were determined against clindamycin
(2 mg), vancomycin (30 mg), erythromycin (15 mg), linezolid (30 mg),
penicillin (10 units), quinupristin-dalfopristin (synercid; 15 mg) and
tetracycline (30 mg) using disc diffusion method according to the
Clinical and Laboratory Standards Institute (CLSI) guidelines [18].
Antibiotic discs were purchased from Mast Company, UK.
2.5. DNA extraction
For DNA isolation, the bacterial DNA extraction kit (Gene All
Exgene™ Cell SV) was used according to the manufacturer's pro-
tocol (Gene ALL, Seoul, Korea). All DNA preparations were stored at
4  C until they were used.
2.6. Detection of antimicrobial resistance, virulence and capsular
genes
The genes encoding resistance to the macrolides and lincosa-
mides (ermA, ermB, ermC, ermTR, mefA and linB), tetracyclines (tetM,
tetL, tetK and tetO), virulence factors (bca, bac, rib, hylB and scpB)
and mobile genetic elements (IS1548 and GBSi1) were investigated
by PCR as described previously and validated using the control
strains [13]. The identification of CTs (Ia, Ib, IIeVIII) of all GBS iso-
lates were carried out by multiplex PCR assay as previously
described [13].
2.7. MLST
MLST was carried out as described by Jones et al. [19]. Briefly, 7
housekeeping genes (adhP, atr, glcK, glnA, pheS, sdhA, tkt) were
amplified by PCR and were sequenced in both directions by Mac-
rogen Inc. (Korea). The unique sequence of each gene was then
uploaded to the GBS database at http://pubmlst.org/sagalactiae/ to
provide a unique allele number, and the combination of the allele
numbers of the seven loci was given a sequence type (ST). New
allelic and ST numbers were confirmed by the MLST website
curator. The eBURST v3 software (http://eburst.mlst.net/) was
applied used to determine the relationships between isolates and
isolates grouping to group isolates into CCs based on five out of
seven shared alleles, otherwise, an ST was considered as singleton.
3. Results
Of the 1037 studied neonates, 19 (2%) were found to be colo-
nized with GBS. The phenotypic and genotypic characteristics of
GBS isolates are shown in Table 1. The MLST analysis of 19 GBS
isolates showed 8 STs. Overall the most common STs were ST19
(34.6%; 6/19), followed by ST28 (21%; 4/19), ST335 (10.5%; 2/19),
ST8 (10.5%; 2/19) and ST12 (10.5%; 2/19). Other STs, including ST1,
ST4, and ST734 (a new ST) were represented by a single isolate.
Using eBURST, the STs were grouped in three CCs and one singleton.
The most common CC was CC19 (68.4%, 13/19). The most common
CT was type III, found in 52.6% of isolates, followed by type II
(31.6%), Ib (10.5%), and V (5.2%). No strains of CTIa, IV, VIeVIII were
identified. All isolates harbored the scpB, hlyB, and bca virulence
genes, whereas the rib, IS1548 and bac genes were detected in
73.7%, 47.4% and 10.5% of the isolates, respectively. The GBSi1 was
not detected. The scpB, hlyB and bca virulence genes were detected
in STS, while the bac gene was predominant in ST12 with CTIb. The
IS1548 and the rib genes were particularly prevalent in CTIII and
were detected in isolates belong to ST19, ST335 and ST734 and were
grouped in CC19.
All isolates were susceptible to penicillin, vancomycin, linezolid
and quinupristin-dalfopristin. Resistance to erythromycin and
clindamycin were found in 5 (26.3%) and 6 (31.6%) isolates
respectively. Considering the CC and antibiotic susceptibility, the
CC10 revealed the highest resistance rate to erythromycin and
clindamycin. Resistance to tetracycline was observed in all 19
(100%) strains and was correlated with presence of the tetM gene
except for one isolate with ST12. All the ST8 and ST12 isolates were
resistant to macrolide carrying two resistance genes; the ermTR and
the ermB, respectively. Of 19 studied isolates, 14 isolates with 6
various STs showed one resistant genotype while the other 5 iso-
lates, consisted of 4 STs, revealed two resistant genotypes (Table 1).
Regarding tetracycline and macrolide resistant genes, 18 (98%) had
tetM, 4 (21%) and 2 (10.5%) had ermTR and ermB gene respectively.
None of the isolates were positive for ermA, ermC, mefA, linB, tetL,
tetK and tetO in the PCR assay.
4. Discussion
Our investigation revealed that the most prevalent STs in the
studied neonatal in intensive care units were ST-19, ST-28, ST-335,
ST-12 and ST-8. The majority of STs recognized in this study have
also been reported previously as common STs for strains isolated
from neonatal and/or adult infections [9,11,20]; however, despite
the small number of strains, we found one new ST (ST-734) that was
present in the MLST database. Associations between CT and STs
have been previously reported by many authors [9,11,19]. The
number of strains examined in our study was too small to confirm
the association between CTs and STs. However, our results suggest
 M. Emaneini et al. / Microbial Pathogenesis 99 (2016) 119e122 121

Table 1
The molecular characteristics, antimicrobial resistance and virulence gene profiles of GBS isolates.

Isolate No Source Resistance pattern Resistance gene Virulence gene Capsular type ST CC*

1 Ear T¥ tetM scpB, hlyB,bca V 1 1

2 Throat T, CD, E tetM, ermTR scpB, hlyB,bca II 8 10
3 Ear T, CD, E tetM, ermTR scpB, hlyB,bca II 8
4 Throat T, CD, E tetM, ermB scpB, hlyB,bca, bac Ib 12
5 Throat T, CD, E ermB scpB, hlyB,bca, bac Ib 12
6 Throat T tetM scpB, hlyB, IS1548, rib,bca III 19 19
7 Ear T tetM scpB, hlyB, IS1548, rib,bca III 19
8 Ear T, CD, E tetM, ermTR scpB, hlyB, IS1548, rib,bca III 19
9 Throat T tetM scpB, hlyB, IS1548, rib,bca III 19
10 Ear T tetM scpB, hlyB, IS1548, rib,bca III 19
11 Ear T tetM scpB, hlyB, IS1548, rib,bca III 19
12 Ear T tetM scpB, hlyB, IS1548, rib,bca III 335
13 Ear T tetM scpB, hlyB, IS1548, rib,bca III 335
14 Throat T tetM scpB, hlyB, rib,bca II 28
15 Ear T tetM scpB, hlyB, rib,bca II 28
16 Ear T tetM scpB, hlyB, rib,bca II 28
17 Throat T tetM scpB, hlyB, rib,bca II 28
18 Ear T tetM scpB, hlyB, IS1548, rib,bca III 734
19 Throat T, CD tetM, ermTR scpB, hlyB, rib,bca III 4 Singleton
¥
T:Tetracycline, CD:Clindamycin, E: Erythromycin, *CC:Clonal Complex, ST: Sequence Type.

that there is a correlation between CTIII with ST-19 and CTII with
ST-28 [9,21]. In contrast to the study of Usein et al. [12] our data did
not support any association between CTV with ST-19. Results of the
current study supports the idea that strains with the same ST could
belong to different CTS [5,19,20]. It has been suggested that hori-
zontal transfer of capsular genes may occur in GBS population
[11,19]. We observed that strains isolated from the throat mostly
belonged to the STs 8 and 12 suggesting that throat may be an
important portal of entry for invasive GBS strains; however, due to
our small sample size, this issue requires confirmation by further
investigations including larger numbers of throat carriage strains.
In the present study, seven STs were clustered in three CCs, namely,
CC1, CC10 and CC19 among them the CC19 was the predominant CC.
Our findings support the data published by Fluegge et al. in which
the CC19 [50% (23/46)] was more frequent among colonizing
neonatal strains [10]. On the other hand, Morozumi et al. reported
that CC19 has been mostly isolated from neonates with invasive
diseases [9]. In the current study, similar to reports from Italy and
France, all CC19 isolates were resistant to tetracycline and harbored
the tetM gene and approximately 70% of these isolates had capsular
types III and carried the rib and IS1548 elements [15,22]. Similar to
different reports, our finding showed that IS1548 serves a genetic
marker for CC19 of CTIII isolates [11,15]. Dissemination of the CC19
in many regions including United States, Europe, Africa and Asia
may show that the CC19 is an international clone distributed
worldwide [5,9,11,20,23]. In this study, the majorities of macrolide-
resistant isolates were CTIb and CTII, ermTR and ermB positive and
belonged to the CC10. However this is not constant, for example
Ryu et al. reported that CC1 was the predominant clone among
erythromycin-resistant in Korea [24] and Gherardi et al. observed
that CTV harboring ermB and belonged to the CC1 was the major
clone among erythromycin-resistant in Italy [22]. These differences
among CCs may be due to the horizontal transfer of macrolide
resistant genes, which has been shown to occur between distinct
clones [25]. According to our observations, tetracycline resistance
was not limited to either a specific capsular or sequence type but
was present in four different CT (Ib, II, III and V) and 8 different STs.
Our results were similar to those reported in Poland and Italy
[22,26]. In the current study, all isolates were susceptible to peni-
cillin, vancomycin, linezolid and quinupristin-dalfopristin. These
findings are in agreement with reports from other authors [27e29]
and confirm that application of penicillin as a first-line agents for
treatment of GBS infections is suitable; however, several studies
have demonstrated the reduced effect of penicillin against GBS
infections [30,31]. In our study, all GBS isolates were resistant to
tetracycline which was slightly higher than reports from Tunisia
(97.3%) and Egypt (98%) [32,33]. The tetM gene was the most
common tetracycline resistance gene in this population. These
finding is in agreement with other studies [32e34]. In the current
study, high rates of resistance were seen to both erythromycin and
clindamycin (26.3% and 31.6%, respectively). Several studies
revealed that macrolide resistance rates vary greatly among
different parts of the world [32e35], which may reflect differences
in doses and dosing schedules used, differences in the public health
policies and spread of particular clones [21,34,36,37]. The most
prevalent gene encoding macrolide resistance was ermTR (21%)
followed by ermB (15.8%). These results are similar to those from
the USA and Canada [38,39]. Similar to previous reports, combi-
nations of macrolide resistance genes were not observed in this
study [26,33,40]. We observed the coexistence of erythromycin and
tetracycline resistance genes among our GBS isolates that was in
accordance with previous reports by Hraoui and Shabayek et al.
[32,33]. Acquisition of resistance genes to erythromycin and
tetracycline is associated with the presence of mobile elements
such as transposons, conjugative transposons, plasmid and inte-
gron [13]. All of the studied GBS isolates in the current study carried
the scpB, hlyB, bca genes which is in accordance with results of our
prior study in pregnant women in which we identified that 97% of
isolates harbored these genes [13]; however, it was higher than
similar reports from Malaysia and Kuwait [41,42]. This variation
may be related to the geographical differences. In agreement with
other studies [43,44], a correlation between CTs and virulence
genes was found, namely, rib with CTIII and bac with CTIb. In the
current study four different CTs (Ib, II, III and V) isolated from ne-
onates and more than half were CTIII. Similar findings have been
observed in Poland and Taiwan [6,20] where CTIII was shown to be
common in neonates, but differs from Lebanon in which CTV has
been introduced as a predominant type [45]. The diversity in CTs
distribution might be related to the source of isolations, ethnic
origins, and diagnostic techniques as well as differences in the
spreading of GBS isolates worldwide [13,21]. Moreover, several
studies show that the CTIII is associated with invasive disease in
neonates therefore the high prevalence of CTIII is a major concern
[9,11,13,20]. In conclusion, this study as a first report about
122 M. Emaneini et al. / Microbial Pathogenesis 99 (2016) 119e122
phenotypic and genotypic characterizations of GBS isolated from
neonates in Iran showed that CC19 is a major clone in the NICU of
Imam Khomeini hospital over the study period. Dissemination of
CC19 is a matter of considerable concern, since this clone has the
ability to create severe infections in susceptible neonates (partic-
ularly in premature infants) and needs changes in the infection
control policy as well as implementation of additional surveillance
programs to prevent the spread of this clone.
Conflict of interest
There are no conflicts of interest.
Acknowledgements
This research has been supported by Tehran University of
Medical Sciences and Health Services grant 92-01-30-20839.
References
[1] B. Percha, M.E. Newman, B. Foxman, Transmission probabilities and durations
of immunity for three pathogenic group B Streptococcus serotypes, Infect.
Genet. Evol. 11 (2011) 1407e1412.
[2] M. Emaneini, A. Mirsalehian, R. Beigvierdi, et al., High incidence of macrolide
and tetracycline resistance among Streptococcus agalactiae strains isolated
from clinical samples in Tehran, Iran, Maedica. (Buchar) 9 (2014) 157e161.
[3] J.R. Verani, L. McGee, S.J. Schrag, Prevention of perinatal group B streptococcal
diseaseerevised guidelines from CDC, 2010, MMWR Recomm. Rep. 59 (2010)
1e36.
[4] G. Lindahl, M. Stalhammar-Carlemalm, T. Areschoug, Surface proteins of
Streptococcus agalactiae and related proteins in other bacterial pathogens,
Clin. Microbiol. Rev. 18 (2005) 102e127.
[5] J.F. Bohnsack, A. Whiting, M. Gottschalk, et al., Population structure of invasive
and colonizing strains of Streptococcus agalactiae from neonates of six U.S.
Academic Centers from 1995 to 1999, J. Clin. Microbiol. 46 (2008) 1285e1291.
[6] M. Brzychczy-Wloch, T. Gosiewski, M. Bulanda, Multilocus sequence types of
invasive and colonizing neonatal group B streptococci in Poland, Med. Princ.
Pract. 23 (2014) 323e330.
[7] V. Da Cunha, M.R. Davies, P.E. Douarre, et al., Streptococcus agalactiae clones
infecting humans were selected and fixed through the extensive use of
tetracycline, Nat. Commun. 5 (2014) 4544.
[8] M. Salloum, N. van der Mee-Marquet, A.S. Valentin-Domelier, et al., Diversity
of prophage DNA regions of Streptococcus agalactiae clonal lineages from
adults and neonates with invasive infectious disease, PLoS One 6 (2011)
e20256.
[9] M. Morozumi, T. Wajima, Y. Kuwata, et al., Associations between capsular
serotype, multilocus sequence type, and macrolide resistance in Streptococcus
agalactiae isolates from Japanese infants with invasive infections, Epidemiol.
Infect. 142 (2014) 812e819.
[10] K. Fluegge, J. Wons, B. Spellerberg, et al., Genetic differences between invasive
and noninvasive neonatal group B streptococcal isolates, Pediatr. Infect. Dis. J.
30 (2011) 1027e1031.
[11] S.L. Luan, M. Granlund, M. Sellin, et al., Multilocus sequence typing of Swedish
invasive group B streptococcus isolates indicates a neonatally associated ge-
netic lineage and capsule switching, J. Clin. Microbiol. 43 (2005) 3727e3733.
[12] C.R. Usein, M. Militaru, V. Cristea, et al., Genetic diversity and antimicrobial
resistance in Streptococcus agalactiae strains recovered from female carriers in
the Bucharest area, Mem. Inst. Oswaldo Cruz 109 (2014) 189e196.
[13] R. Beigverdi, F. Jabalameli, A. Mirsalehian, et al., Virulence factors, antimi-
crobial susceptibility and molecular characterization of Streptococcus aga-
lactiae isolated from pregnant women, Acta. Microbiol. Immunol. Hung. 61
(2014) 425e434.
[14] M. Emaneini, B. Khoramian, F. Jabalameli, et al., Comparison of virulence
factors and capsular types of Streptococcus agalactiae isolated from human
and bovine infections, Microb. Pathog. 91 (2016) 1e4.
[15] R. Al Safadi, S. Amor, G. Hery-Arnaud, et al., Enhanced expression of lmb gene
encoding laminin-binding protein in Streptococcus agalactiae strains
harboring IS1548 in scpB-lmb intergenic region, PLoS One 5 (2010) e10794.
[16] S. Tajbakhsh, M. Norouzi Esfahani, M. Emaneini, et al., Identification of
Streptococcus agalactiae by fluorescent in situ hybridization compared to
culturing and the determination of prevalence of Streptococcus agalactiae
colonization among pregnant women in Bushehr, Iran, BMC Infect. Dis. 13
(2013) 420.
[17] M. Shirazi, E. Abbariki, A. Hafizi, et al., The prevalence of group B streptococcus
colonization in Iranian pregnant women and its subsequent outcome, Int. J.
Fertil. Steril. 7 (2014) 267e270.
[18] Clinical and Laboratory Standards Institute, Performance standards for anti-
microbial susceptibility testing; twenty-fourth informational supplement,
CLSI 34 (1) (2014) 68e75. M100eS24.
[19] N. Jones, J.F. Bohnsack, S. Takahashi, et al., Multilocus sequence typing system
for group B streptococcus, J. Clin. Microbiol. 41 (2003) 2530e2536.
[20] N. Tien, C.M. Ho, H.J. Lin, et al., Multilocus sequence typing of invasive group B
Streptococcus in central area of Taiwan, J. Microbiol. Immunol. Infect. 44
(2011) 430e434.
[21] P. Wang, J.J. Tong, X.H. Ma, et al., Serotypes, antibiotic susceptibilities, and
multi-locus sequence type profiles of Streptococcus agalactiae isolates circu-
lating in Beijing, China, PLoS One 10 (2015) e0120035.
[22] G. Gherardi, M. Imperi, L. Baldassarri, et al., Molecular epidemiology and
distribution of serotypes, surface proteins, and antibiotic resistance among
group B streptococci in Italy, J. Clin. Microbiol. 45 (2007) 2909e2916.
[23] C.A. Huber, F. McOdimba, V. Pflueger, et al., Characterization of invasive and
colonizing isolates of Streptococcus agalactiae in East African adults, J. Clin.
Microbiol. 49 (2011) 3652e3655.
[24] H. Ryu, Y.J. Park, Y.K. Kim, et al., Dominance of clonal complex 10 among the
levofloxacin-resistant Streptococcus agalactiae isolated from bacteremic pa-
tients in a Korean hospital, J. Infect. Chemother. 20 (2014) 509e511.
[25] A.S. Domelier, N. van der Mee-Marquet, L. Arnault, et al., Molecular charac-
terization of erythromycin-resistant Streptococcus agalactiae strains,
J. Antimicrob. Chemother. 62 (2008) 1227e1233.
[26] E. Sadowy, B. Matynia, W. Hryniewicz, Population structure, virulence factors
and resistance determinants of invasive, non-invasive and colonizing Strep-
tococcus agalactiae in Poland, J. Antimicrob. Chemother. 65 (2010)
1907e1914.
[27] K. Fluegge, S. Supper, A. Siedler, et al., Antibiotic susceptibility in neonatal
invasive isolates of Streptococcus agalactiae in a 2-year nationwide surveil-
lance study in Germany, Antimicrob. Agents Chemother. 48 (2004)
4444e4446.
[28] S.M. Borchardt, J.H. DeBusscher, P.A. Tallman, et al., Frequency of antimicro-
bial resistance among invasive and colonizing Group B streptococcal isolates,
BMC Infect. Dis. 6 (2006) 57.
[29] G. Piccinelli, V. Biscaro, F. Gargiulo, et al., Characterization and antibiotic
susceptibility of Streptococcus agalactiae isolates causing urinary tract in-
fections, Infect. Genet. Evol. 34 (2015) 1e6.
[30] S. Dahesh, M.E. Hensler, N.M. Van Sorge, et al., Point mutation in the group B
streptococcal pbp2x gene conferring decreased susceptibility to beta-lactam
antibiotics, Antimicrob. Agents. Chemother. 52 (2008) 2915e2918.
[31] K. Kimura, S. Suzuki, J. Wachino, et al., First molecular characterization of
group B streptococci with reduced penicillin susceptibility, Antimicrob.
Agents. Chemother. 52 (2008) 2890e2897.
[32] M. Hraoui, I. Boutiba-Ben Boubaker, M. Rachdi, et al., Macrolide and tetracy-
cline resistance in clinical strains of Streptococcus agalactiae isolated in
Tunisia, J. Med. Microbiol. 61 (2012) 1109e1113.
[33] S. Shabayek, S. Abdalla, Macrolide- and tetracycline-resistance determinants
of colonizing group B streptococcus in women in Egypt, J. Med. Microbiol. 63
(2014) 1324e1327.
[34] S.S. Boswihi, E.E. Udo, N. Al-Sweih, Serotypes and antibiotic resistance in
Group B streptococcus isolated from patients at the Maternity Hospital,
Kuwait, J. Med. Microbiol. 61 (2012) 126e131.
[35] S.E. Gygax, J.A. Schuyler, L.E. Kimmel, et al., Erythromycin and clindamycin
resistance in group B streptococcal clinical isolates, Antimicrob. Agents.
Chemother. 50 (2006) 1875e1877.
[36] W.C. Ko, J.J. Yan, N.Y. Lee, et al., Polyclonal spread of erythromycin-resistant
Streptococcus agalactiae in southern Taiwan, Microb. Drug Resist. 10 (2004)
306e312.
[37] M.A. De Francesco, S. Caracciolo, F. Gargiulo, et al., Phenotypes, genotypes,
serotypes and molecular epidemiology of erythromycin-resistant Strepto-
coccus agalactiae in Italy, Eur. J. Clin. Microbiol. Infect. Dis. 31 (2012)
1741e1747.
[38] J.C. de Azavedo, M. McGavin, C. Duncan, et al., Prevalence and mechanisms of
macrolide resistance in invasive and noninvasive group B streptococcus iso-
lates from Ontario, Canada, Antimicrob. Agents. Chemother. 45 (2001)
3504e3508.
[39] B. Dogan, Y.H. Schukken, C. Santisteban, et al., Distribution of serotypes and
antimicrobial resistance genes among Streptococcus agalactiae isolates from
bovine and human hosts, J. Clin. Microbiol. 43 (2005) 5899e5906.
[40] M. Brzychczy-Wloch, T. Gosiewski, M. Bodaszewska, et al., Genetic charac-
terization and diversity of Streptococcus agalactiae isolates with macrolide
resistance, J. Med. Microbiol. 59 (2010) 780e786.
[41] E.E. Udo, S.S. Boswihi, N. Al-Sweih, Genotypes and virulence genes in group B
streptococcus isolated in the maternity hospital, Kuwait, Med. Princ. Pract. 22
(2013) 453e457.
[42] N. Eskandarian, Z. Ismail, V. Neela, et al., Antimicrobial susceptibility profiles,
serotype distribution and virulence determinants among invasive, non-
invasive and colonizing Streptococcus agalactiae (group B streptococcus)
from Malaysian patients, Eur. J. Clin. Microbiol. Infect. Dis. 34 (2015) 579e584.
[43] C.S. Lachenauer, R. Creti, J.L. Michel, et al., Mosaicism in the alpha-like protein
genes of group B streptococci, Proc. Natl. ACad. Sci. U. S. A. 97 (2000)
9630e9635.
[44] F. Kong, S. Gowan, D. Martin, et al., Molecular profiles of group B streptococcal
surface protein antigen genes: relationship to molecular serotypes, J. Clin.
Microbiol. 40 (2002) 620e626.
[45] M. Seoud, A.H. Nassar, P. Zalloua, et al., Prenatal and neonatal Group B
Streptococcus screening and serotyping in Lebanon: incidence and implica-
tions, Acta. Obstet. Gynecol. Scand. 89 (2010) 399e403.