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Prevention of early invasive infections with group B streptococci in the newborn
Copyright (c) 2022 Slovenian Medical Journal. This work is licensed under a
Creative Commons Attribution-NonCommercial 4.0 International License.
Prevention of early invasive infections with group B 
streptococci in the newborn – microbiological methods 
for detection of collonization before and at birth
Preprečevanja zgodnjih invazivnih okužb novorojenčka zaradi streptokoka skupine B – 
mikrobiološke metode za ugotavljanje nosilstva pred in ob porodu
Ajda Kovačič,1 Gal Kranjc,1 Miha Lučovnik,1,2 Petra Vovko,3 Samo Jeverica3
Abstract
At the end of last year, the Health Council of the Republic of Slovenia adopted a programme for the prevention of early inva-
sive neonatal infections caused by group B streptococci, which includes universal screening of pregnant women between the 
35th and 37th week of pregnancy. In this article, we provide an overview of the different diagnostic modalities for screening 
for collonization and the factors that significantly influence the success of screening, both in gynaecological practise and in 
the microbiology laboratory. We instruct the reader on the proper collection and transport of specimens. We also present the 
chosen testing strategy, using a combination of enriched culture and molecular testing, and provide the reader with a list of 
registered molecular tests suitable for screening. In the last part of the article, we discuss the importance of the hypervirulent 
clone CC -17, which causes most invasive neonatal infections in Slovenia, and the methods by which it can be detected.
Izvleček
Ob koncu lanskega leta je Zdravstveni svet Republike Slovenije potrdil program preprečevanja zgodnjih invazivnih okužb 
novorojenčkov, povzročenih s streptokokom skupine B, ki vključuje univerzalno presejanje nosečnic v 35.–37. tednu noseč-
nosti. V prispevku smo pregledali različne diagnostične metode presejanja in dejavnike, ki pomembno vplivajo na njihovo 
uspešnost tako v ginekološki ambulanti kakor tudi v mikrobiološkem laboratoriju. Bralca opozarjamo na pravilnost odvze-
ma kužnine, mu predstavimo izbrano strategijo testiranja s kombinacijo obogatene kulture in molekularnega testiranja ter 
mu ponujamo seznam registriranih molekularnih testov, primernih za testiranje. V zadnjem delu prispevka razpravljamo 
o pomenu hipervirulentnega klona CC-17, ki povzroča večino invazivnih okužb novorojenčkov v Sloveniji, in o metodah, s 
katerimi ga prepoznamo.
1 Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
2 Division of Gynaecology, University Medical Centre Ljubljana, Ljubljana, Slovenia
3 National Laboratory of Health, Environment and Food, Maribor, Slovenia
Correspondence / Korespondenca: Samo Jeverica, e: samo.jeverica@nlzoh.si
Key words: pregnancy; neonatal infections; streptococcus agalactiae; diagnostic methods; molecular detection; screening
Ključne besede: nosečnost; neonatalne okužbe; Streptococcus agalactiae; diagnostične metode; molekularna diagnostika; presejanje
Received / Prispelo: 20. 12. 2020 | Accepted / Sprejeto: 5. 4. 2021
Cite as / Citirajte kot: Kovačič A, Kranjc G, Lučovnik M, Vovko P, Jeverica S. Prevention of early invasive infections with group B streptococci 
in the newborn – microbiological methods for detection of collonization before and at birth. Zdrav Vestn. 2022;91(7–8):309–18. DOI: https://
doi.org/10.6016/ZdravVestn.3202
eng slo element
en article-lang
10.6016/ZdravVestn.3202 doi
20.12.2020 date-received
5.4.2021 date-accepted
Infections Infekcije discipline
Review article Pregledni znanstveni članek article-type
Prevention of early invasive group B strep-
tococcal infections in the newborn – micro-
biological methods for pre- and post-natal 
carriage detection
Preprečevanja zgodnjih invazivnih okužb novo-
rojenčka zaradi streptokoka skupine B – mikrobi-
ološke metode za ugotavljanje nosilstva pred in 
ob porodu
article-title
Prevention of early invasive group B strepto-
coccal infections in the newborn
Preprečevanja zgodnjih invazivnih okužb novoro-
jenčka zaradi streptokoka skupine B alt-title
pregnancy, neonatal infections, streptococcus 
agalactiae, diagnostic methods, molecular 
detection, screening
nosečnost, neonatalne okužbe, Streptococcus 
agalactiae, diagnostične metode, molekularna 
diagnostika, presejanje
kwd-group
The authors declare that there are no conflicts 
of interest present.
Avtorji so izjavili, da ne obstajajo nobeni 
konkurenčni interesi. conflict
year volume first month last month first page last page
2022 91 7 8 309 318
name surname aff email
Samo Jeverica 3 samo.jeverica@nlzoh.si
name surname aff
Ajda Kovačič 1
Gal Kranjc 1
Miha Lučovnik 1,2
Petra Vovko 3
eng slo aff-id
Faculty of Medicine, University 
of Ljubljana, Ljubljana, Slovenia
Medicinska fakulteta, Univerza v 
Ljubljani, Ljubljana, Slovenija 1
Division of Gynaecology, 
University Medical Centre 
Ljubljana, Ljubljana, Slovenia
Ginekološka klinika, Univerzitetni 
klinični center Ljubljana, 
Ljubljana, Slovenija
2
National Laboratory of Health, 
Environment and Food, Maribor, 
Slovenia
Nacionalni laboratorij za 
zdravje, okolje in hrano, Maribor, 
Slovenija
3
Slovenian Medical Journallovenian Medical Journal
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1 Introduction
The proportion of invasive infections (sepsis, men-
ingitis and pneumonia) in the morbidity and mortality 
of newborns and children up to the third month of age 
is significant (1). In Slovenia, about a fifth of neonatal 
deaths are caused by invasive infections (2). As in the 
developed world, the most common causative agent in 
Slovenia is the group B streptococcus (GBS), causing up 
to 50% of invasive infections (2,3). These are classified as 
early-onset (0–7 days of age) and late-onset (8–90 days of 
age). Early neonatal GBS infections result from vertical 
transmission of the bacteria from the mother at the time 
of delivery. Their incidence in Slovenia is >30% higher 
than the global and European estimates, and amounts 
to 0.53/1,000 births (3,4). With advances in treatment, 
mortality has decreased below 10%, but a large propor-
tion of children can have permanent sequelae in mental 
and motor development (20–30%), representing a sig-
nificant disease and economic burden on society (4-6).
The proportion of Slovenian pregnant women who 
are carriers of GBS in the intestine or vagina was deter-
mined in two studies, and was found to be 17% and 23% 
respectively (7,8). The transmission of bacteria from 
mother to child during vaginal delivery occurs in ap-
proximately half of pregnant women, and invasive neo-
natal infection occurs in 1–2% (9). In the 1980s, it was 
found that early invasive infections can be effectively 
(>90%) prevented by intrapartum antibiotic prophylax-
is, in which the mother receives an antibiotic intrave-
nously during childbirth (9).
Pregnant women can be selected for antibiotic pro-
phylaxis in three ways: a) by the presence of perina-
tal risk factors (premature birth, prolonged rupture of 
membranes, fever, presence of GBS in the urine during 
pregnancy or neonatal GBS infection during a previous 
pregnancy), b) in the case of previously established GBS 
colonization in the third trimester of pregnancy, typi-
cally between the 35th and 37th week, or c) in the case 
of established perinatal GBS colonization of the mother 
(10). Numerous studies have established that screening 
between the 35th and 37th weeks of pregnancy is more 
effective than simply identifying risk factors (11,12). In a 
recent national study, perinatal risk factors were absent 
in 52% of affected children, which confirms the insuf-
ficiency of prevention based solely on this type of risk 
assessment (4).
The Health Council of the Republic of Slovenia 
recently adopted a proposal to introduce universal 
screening for GBS in the 35th to 37th week of pregnancy. 
In this paper, we present the natural history of GBS colo-
nization and both new and already established methods 
for determining GBS colonization in pregnant women.
2 The natural history of GBS colonization
Infection and colonization of the mother are neces-
sary prerequisites for the transmission of bacteria to the 
child in early-onset infections. It is assumed that the 
mother is colonized via the faecal-oral route from anoth-
er person (close contact, including sexual contact), from 
the environment (due to poor hygiene) or through food 
(dairy products), and then from the intestine, GBS colo-
nization spreads to different parts of the body, particu-
larly to the urinary and reproductive tracts. Concurrent 
colonization of several body regions is frequently present 
(13-15). The dynamics of colonization are probably more 
complex than is commonly believed. In a Danish study, 
pregnant women were monitored during and after preg-
nancy. They found that just over half of the women (53%) 
were not colonized at the time of the study. The rest were 
either permanent (28%) or intermittent carriers (19%). 
The population of bacteria was genetically homogeneous 
in each woman and the bacterial strain’s genotype did not 
change over time. They concluded that the GBS propor-
tion of the intestinal microbiota at the time of sampling is 
important for their successful detection (16).
Colonization status in the late third trimester of preg-
nancy is a predictive factor of actual colonization at the 
time of delivery. As the time from testing to delivery 
increases, the predictive value of such testing decreas-
es. The negative predictive value of a sample taken <5 
weeks before delivery is a satisfactory at 95–98%, but 
before this period it drops rather steeply to 80% (17,18). 
The poorer negative predictive value is the reason that 
in environments that perform screening, most early-on-
set invasive neonatal infections are observed in pregnant 
women with a negative antenatal screening test result. 
Such cases may be the result of a false-negative test or 
new colonization of the pregnant woman during the pe-
riod from testing to delivery (11).
In the latest guidelines, the American College of Ob-
stetricians and Gynaecologists (ACOG) has changed its 
recommendation for taking vaginal-rectal cultures in 
the period >5 weeks before delivery to determine GBS 
carrier status due to the poorer negative predictive value 
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Prevention of early invasive infections with group B streptococci in the newborn
of testing. Now, instead of performing screening after 35 
0/7 weeks of pregnancy, they recommend performing 
screening between 36 0/7 and 37 6/7 weeks of pregnancy 
(19). In this way, the window until delivery at term was 
shortened to five weeks (36 0/7 to 41 0/7). If the delivery 
takes place >5 weeks after screening, repeat GBS screen-
ing is reasonable.
The opinion of the article’s authors is that such a 
solution eliminates some shortcomings in determining 
antenatal colonization. However, we would like to point 
out that after the 37th week of pregnancy, the risk that 
delivery will start before we receive the screening results 
begins to increase. Given that our system of antenatal 
care does not provide for weekly examinations until the 
37th week, we suggest that our recommendation remains 
unchanged for now and for screening to be performed 
between the 35th and 37th weeks of pregnancy; it should 
be noted that screening between the 36th and 37th weeks 
is more appropriate, when possible. In the future, based 
on data from individual periods, we will change the rec-
ommendation accordingly.
3 Antenatal colonization detection
In the following text, we present the basic microbi-
ological method for determining antenatal coloniza-
tion between the 35th and 37th week of pregnancy, i.e. 
enrichment culture, and list some of the more import-
ant improvements that increase the detection method’s 
sensitivity.
3.1 Enrichment culture
Use of an enrichment culture is the standard for de-
termining GBS colonization; one or two vaginal-rectal 
specimens are first incubated in a selective liquid broth 
(enrichment). After 16–24 hours, the incubated broth 
is subcultured to an appropriate agar plate. For enrich-
ment, the Todd-Hewitt broth supplemented with gen-
tamicin (8 µg/ml) and nalidixic acid (15 µg/ml) or the 
Lim broth with colistin (10 µg/ml) and nalidixic acid 
(15 µg/ml) are used. The antibiotics inhibit the growth 
of Gram-negative bacteria. After overnight incubation, 
the enrichment broth is subcultured to one of the agar 
plates used to detect GBS colonies, such as blood agar, 
blood agar supplemented with colistin and nalidixic acid 
(CNA agar) or one of the specialized commercial chro-
mogenic agars. The subcultured agar plates should be 
incubated to an additional 48 hours (12,20).
The identification of typical GBS colonies, char-
acterized by beta haemolysis, on solid culture media 
has recently been mostly performed by MALDI-TOF 
(Matrix Assisted Laser Desorption/Ionisation Time of 
Flight) mass spectrometry. It can also be performed us-
ing one of the older identification methods, either with 
latex agglutination or biochemical tests. Bacterial culti-
vation is currently the only way to test the antimicrobial 
susceptibility of GBS to antibiotics, which is particularly 
important in cases of beta-lactam allergy and for resis-
tance control (12,20).
Nonhaemolytic and nonpigmented (NH/NP) strains, 
which appear in 2–5% of positive samples and can just 
as easily cause invasive infections, are a problem in GBS 
screening using bacterial cultures (21). The absence of 
colony haemolysis and pigmentation mainly affects 
phenotypic identification, which requires more staff ex-
perience and a greater number of additional tests used 
for identification, and can be the cause of false-nega-
tive results when using enrichment broth. Similarly, the 
competitive growth of Enterococcus faecalis, which is a 
frequent intestinal colonizer and grows together with 
streptococcus in enrichment broth, can inhibit GBS 
growth on selective media (22).
The following two factors play an important role in 
the successful GBS detection in an enriched culture: a) 
using selective enrichment broth in b) collecting com-
bined vaginal-rectal specimens (Figure 1). Using enrich-
ment broth has a significantly higher sensitivity than us-
ing only direct agar plating. It is estimated that without 
enrichment broth, up to 50% of tests are falsely negative 
(12,23). Furthermore, collecting combined vaginal-rec-
tal specimens is also very important. This is primarily a 
reflection of the natural colonization process, in which 
the intestine is colonized first, followed by bacterial 
growth and vaginal colonization. In most studies, GBS 
was more frequently found in the rectum than the va-
gina, up to a ratio of 2:1 (24,25). Combined specimen 
sampling from both anatomical sites improves detection 
rates by approximately 30% (23).
We must emphasize that the gynaecologist is respon-
sible for specimen collection and that suboptimal speci-
men collection, as is often the case in microbiology, has 
a significant impact on the validity of the results. There-
fore, the data from the Slovenian 2016 study, in which 
only a minority (17%) of specimens were collected cor-
rectly, is worrying (8).
When using enrichment culture, live bacterial 
cells must be transported to the laboratory. Therefore, 
high-quality transport systems consisting of swabs and 
a transport medium are important. We have tradition-
ally used cotton-tipped swabs and a semi-solid trans-
port medium. Today, flocked swabs with an improved 
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Figure 1: Specimen collection instructions for determining GBS colonization of pregnant women. Adapted from the Centres 
for Disease Control and Prevention (CDC) instructions (12). First, a vaginal specimen at the depth of two centimetres is 
collected (1), followed by rectal specimen collection (with the same swab) (2).
Emphasis: sample collection and transport.
For vaginal-rectal specimen collection, a swab with a transport medium (e.g. Stuart, Amies) is required, which is first inserted 
into the lower third of the vagina (2 cm deep) and then into distal rectum (1 cm deep). The swab is rotated in both anatomical 
places for about five seconds to allow adequate specimen collection. The specimen is then placed in a transport medium and 
sent to the microbiological laboratory within 48 hours, preferably as soon as possible. Specimen transport takes place at room 
temperature. When the specimen cannot be sent to the laboratory on the same day, it should be kept in the refrigerator (+ 4 ºC) 
for up to four days (Figure 1) (12).
1. Vaginal swab
(depth of two 
centimetres)
2. Rectal swab
(depth of one 
centimetre)
(1) (2)
composition and topography of the synthetic fibre tip 
are available, preventing bacterial containment in the 
tip and improving their release into the transport me-
dia (26). Bacteria remain viable in transport media for 
at least 48 hours at room temperature. When specimens 
cannot be sent to the laboratory immediately, they can 
be stored in a refrigerator (4 °C) for up to four days (12). 
Some manufacturers combine swabs with enrichment 
broth (THBS, Lim, Carrot), which means that incuba-
tion in the laboratory can start without additional spec-
imen manipulation.
3.2 Improving enrichment culture
3.2.1 Chromogenic media
In this paper, chromogenic media are defined as all 
media in which positivity is equated with a change in 
the colour of either the media or bacterial colony. This 
category includes both liquid chromogenic media used 
in the enrichment phase (Carrot broth) and solid chro-
mogenic media used for direct plating or plating after 
enrichment. Based on the mechanism of the colour reac-
tion development, we distinguish between chromogenic 
agars and Granada-type media.
In recent years, there has been rapid expansion in the 
availability and use of chromogenic media for the detec-
tion of various pathogenic bacteria, including GBS (22). 
Chromogenic agars contain enzyme substrates specific 
for individual bacteria linked to chromogenic molecules 
(indoxyl compounds), which are released into the en-
vironment after an enzymatic reaction, leading to pre-
cipitation within and typical staining of bacterial colo-
nies (27). Unlike blood agar and Granada media, only 
chromogenic agars can detect NH/NP GBS strains. They 
are characteristically cultured under aerobic conditions, 
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Prevention of early invasive infections with group B streptococci in the newborn
which are optimal for substrate degradation and colour 
reaction development. Since chromogenic compounds 
degrade in light, they must be stored in the dark (22).
Chromogenic agars can be used both for direct plat-
ing and plating after enrichment. The ability of chromo-
genic agars to stain NH/NP strains is the basic reason 
for their improved sensitivity. Compared to other cul-
ture media, approximately 5% more positive cultures are 
detected, reflecting the proportion of NH/NP strains in 
the population (28). However, colour change detection 
is not always straightforward. GBS colonies can stain 
poorly, particularly when commensal flora is abundant 
and significantly exceeds the GBS proportion. On the 
other side of the spectrum, colonies of bacteria related 
to GBS can also be stained, including other streptococci 
or even some enterococci and staphylococci. An experi-
enced examiner will recognize such non-specific colo-
nies macroscopically, but the authors advise caution and 
recommend controlling the identification of all stained 
and suspicious colonies with the MALDI-TOF mass 
spectrometry method, if possible (22).
Many chromogenic media from different manufac-
turers are available on the market, including: bioMérieux 
(ChomID Strepto B), Bio-Rad (StreptoB Select), Oxoid 
(Brilliance GBS) and others. If swabs are inoculated di-
rectly onto them and incubated for one day, a sensitivity 
of about 85% (77–96%) is achieved. If the swabs are first 
inoculated into liquid enrichment broth and then into 
chromogenic media, which are incubated for an addi-
tional two days, the sensitivity approaches 100% (93–
100%) (29-34). Of course, it should be emphasized that 
the sensitivity of 100% in this case reflects the fact that 
this method was used as the gold standard.
The second type of chromogenic media (Grana-
da-type) are media that stimulate natural pigment for-
mation in the vast majority of GBS strains. This property 
is linked to the formation of haemolysins, so NH/NP 
strains are not detected using these culture media. Pig-
ment formation is enhanced under anaerobic conditions 
(20). Compared to chromogenic agars, the sensitivity of 
Granada-type media is expectedly worse and is around 
95% after enrichment (34). Pigment formation is also 
used in some liquid enrichment media (Carrot broth, 
liquid biphasic Granada medium), but their sensitivity 
is worse than established liquid media. Their populari-
ty is mainly attributed to the high specificity of the or-
ange pigment and the fact that stained liquid cultures 
do not need additional confirmation of GBS detection. 
The declared sensitivity of one of the registered liquid 
culture media (Strep B Carrot Broth) is 88%. In the ab-
sence of visual colour production, the broth should be 
subcultured to solid medium which is observed for 48 
hours for GBS growth (35).
3.2.2 Molecular assays
The use of molecular methods has greatly changed 
diagnostic microbiology. In the field of determining GBS 
colonization of pregnant women, many molecular tests 
have been developed, among which, at the time of writ-
ing this article, 12 were registered with the US Food and 
Drug Administration (FDA) (reviewed on 21.11. 2020). 
Among them, 11 tests are intended for GBS confirma-
tion after prior enrichment, and only one is for testing 
directly from the specimens taken during delivery (Ta-
ble 1).
GBS detection by molecular assays after overnight 
enrichment in liquid culture media greatly improves 
sensitivity and shortens testing time. With such testing 
methods, 20–40% more colonized pregnant women are 
detected on average compared to the standard method 
described in the guidelines of the American Centres for 
Disease Control and Prevention (CDC) (12,28,36-41). 
Such testing is also faster and can be completed within 
24 hours, i.e. 1–2 days faster than the standard method. 
Because of the above, some believe that the combina-
tion of enrichment and molecular GBS detection should 
be considered as the new gold standard for screening. 
(22,41). This type of testing was also confirmed in the 
Slovenian programme for the prevention of early-onset 
invasive neonatal infections caused by GBS. Briefly, af-
ter admission to the laboratory, combined vaginal-rec-
tal specimens are first inoculated in THBS enrichment 
broth for overnight incubation (16–18 hours). The next 
day, the broth is tested for GBS with one of the registered 
molecular tests according to the test manufacturer’s in-
structions. There is an increasing number of commer-
cial molecular test manufacturers for GBS detection on 
the market, which will undoubtedly reduce prices and 
increase the availability of this type of testing method 
(Table 1).
3.2.3 GBS antigen detection
In the past, a number of antigenic tests for urinary 
and vaginal GBS detection were developed, but regard-
less of the detection method (latex agglutination, enzyme 
immunoassay, optical immunoassay), they proved to be 
insufficiently sensitive when used directly on the collect-
ed specimens. The bacterial burden and the associated 
number of antigens can be very small during coloniza-
tion. Compared to enrichment, the sensitivity of antigen 
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Legend: rtPCR – real-time polimerase chain reaction; LAMP – loop-mediated isotermal amplification; HDA – helicase-dependant 
amplification; ND – unknown; GBS – I. group B streptococcus.
1 Sensitivity and specificity as listed in FDA documentation.
* The only diagnostic test that is currently also registered for intrapartum screening and has a lower sensitivity compared to 
the others.
Molecular test Registration 
year
Testing 
method
Method Target 
(gene)
Sensitivity1 
% (95% CI)
Specificity1 
% (95% CI)
Xpert GBS* 2006 Direct rtPCR cfb 89 (83-93) 97 (95-98)
Xpert GBS LB 2012 After enrichment rtPCR cfb 99 (96-100) 92 (90-94)
BD Max GBS 2012 After enrichment rtPCR cfb 95 (90–98) 97 (95–98)
Alethia GBS 2012 After enrichment LAMP ND 99 (97-100) 93 (92-95)
AmpliVue GBS 2013 After enrichment HDA atoB 100 (97-100) 93 (91-94)
Aries GBS 2016 After enrichment rtPCR cfb 96 (91-98) 91 (89-94)
Great Basin Portrait 
GBS 
2016 After enrichment rtPCR cfb 98 (93-99) 96 (94-98)
Solana GBS 2017 After enrichment HDA atoB 100 (98-100) 96 (94-97)
GenePOC GBS LB 2017 After enrichment rtPCR cfb 96 (92-98) 96 (94-97)
NeuMoDx GBS 2018 After enrichment rtPCR pcsB 97 (94-98) 96 (95-97)
Simplexa GBS 2018 After enrichment rtPCR cfb 97 (92-99) 96 (93-98)
Panther Fusion GBS 2020 After enrichment rtPCR cfb,sip 100 (98-100) 97 (95-98)
Table 1: Molecular tests that are registered with the US Food and Drug Administration (FDA).
tests is 15–74%, so most professional associations do not 
recommend them for detecting colonization (12,22,42).
Due to the high specificity of these types of tests, nev-
ertheless, and their ease of use, there have been attempts 
to use antigen tests after enrichment, particularly recent-
ly. Such use of rapid antigen tests could possibly become 
comparable to the molecular GBS detection after enrich-
ment (43,44,45).
4 Intrapartum GBS screening
The development of rapid and simple molecular 
methods which do not require an advanced molecular 
laboratory and experienced technical staff has made it 
possible to detect colonization in pregnant women di-
rectly upon admission to the maternity ward or at la-
bour onset. Such a method of testing could improve 
some of the shortcomings of antenatal testing, namely: 
a) the problem of premature birth before the 35th week of 
pregnancy, b) the problem of intermittent colonization, 
in which the carriage status can change from the time 
of testing to delivery, c) the logistical problem of testing 
between the 35th and 37th weeks of pregnancy, which 
could be a problem particularly in less developed coun-
tries, and ultimately, d) the problem of unnecessary an-
tibiotic prophylaxis in pregnant women who are not de-
monstrably colonized with GBS during childbirth (42).
However, intrapartum testing also has some disad-
vantages. The existing rapid molecular tests are complet-
ed at the fastest in 1–2 hours from specimen collection. 
In many cases, this is not fast enough for all pregnant 
women, some of whom may give birth before the test is 
complete (46). Subsequently, many studies have shown 
that the sensitivity of molecular tests without prior en-
richment is 10–20% lower than with tests with prior en-
richment (47-49). In this way, with a less sensitive test, 
colonized pregnant women could be missed and there-
fore wouldn’t receive antibiotic prophylaxis. In most 
studies to date, it has been shown that technical prob-
lems appear in approximately 10% of test, which then re-
quires repeat testing; again, it becomes an issue of time. 
Finally, at the time of writing, there is only one registered 
test used for this purpose (Xpert GBS).
Because of all this, intrapartum screening is not 
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Prevention of early invasive infections with group B streptococci in the newborn
1 In most comparative studies, the enriched culture (CDC-recommended method) was considered as the gold standard (11). In 
studies comparing molecular tests after enrichment, such testing detected 20-40% more colonized pregnant women (see text).
2 The indicated prices are approximations.
# With concurrent direct chromogenic agar plating, antibiotic susceptibility testing can normally be performed.
* Suitable for prenatal testing (35th to 37th weeks of pregnancy).
** Suitable for intrapartum screening. In most studies, such testing was about 10% less sensitive.
Type of test Duration Relative 
sensitivity1
Relative 
specificity
Antibiotic 
susceptibility 
testing
Relative 
cost2 (€)
Enrichment culture* 48-72 h 90-95% 90-100% Yes ≈15-20
Molecular (after enrichment) * 24 h 95-100% 95-100% Conditional# ≈50-60
Molecular (direct)** 1-2 h 85-95% 95-100% No ≈45-55
Antigen (direct) Up to 1 h 15-75% up to 75% No ≈10
Table 2: NMicrobiological testing methods and their basic characteristics for determining colonization of pregnant women. 
Adapted from Rosa-Fraile M., et al., 2017 (22).
recommended by any professional association for the 
time being. On the contrary, such testing is expressly 
discouraged by the American Society for Microbiol-
ogy (ASM), as testing without enrichment is not suffi-
ciently sensitive and has a low negative predictive value 
(50). Nevertheless, recent individual pilot studies have 
demonstrated both the advantages and disadvantages 
of intrapartum screening (51-53). In any case, it is nec-
essary to regularly monitor innovations in the field of 
molecular diagnostics, which in the future could be the 
basis for intrapartum screening; if possible, such testing 
in local environment is preferable. 
5 Hypervirulent GBS clone (serotype III, 
clonal complex 17)
The polysaccharide capsule is the most important 
GBS virulence factor. According to the capsular poly-
saccharide, GBS is divided into 10 different serotypes 
(Ia, Ib, II-IX), which differ from each other antigenical-
ly and structurally (54). In Europe, the most common 
serotypes are Ia, II, III and V, with serotype III causing 
most invasive infections (3). Colonization success and 
infection development are also related to the presence of 
other virulence factors, among which various adhesive 
molecules and structures that allow adhesion either to 
the epithelial surface (pili, HvgA) or molecules of the ex-
tracellular matrix (FbsA-C, Srr1-2, Lmb, ScbP and oth-
ers) are very important (55).
GBS is a commensal bacterium. It is therefore sur-
prising that, at the genomic level, most of the human 
colonization and clinical isolates belong to only five clon-
al complexes (CC – CC-1, CC-12, CC-17, CC-19 and 
CC-23). Among them, the clonal complex CC-17 is con-
sidered to be hypervirulent as it causes the vast majority 
(>80%) of late-onset invasive infections and dominates 
among the causative agents (50%) of early-onset invasive 
infections. Most strains of the CC-17 clone belong to the 
capsular serotype III and have the hypervirulent adhesin 
A (hvgA) gene. HvgA contributes to adhesion to intes-
tinal epithelial cells, vascular endothelium and choroid 
plexus cells in the brain (55).
From an evolutionary point of view, the main event 
in the formation of such a homogeneous GBS popula-
tion structure is considered to be the beginning of med-
ical use of tetracycline in 1948, and the acquisition of 
the tetracycline resistance gene (tetM) (56). These two 
events initiated the selection of a very limited number of 
human adapted tetracycline resistant GBS clones. Coin-
ciding with this was the increase in GBS infections in the 
1960s, including invasive infections of pregnant women 
and neonates. In several longitudinal studies that mon-
itor the incidence of invasive neonatal infections at the 
clonal level, it is concluded that it is the replacement of 
clones within the GBS population, and particularly the 
emergence and spread of the hypervirulent CC-17 clone, 
that is the reason for the reduced effectiveness of pro-
grammes to prevent neonatal sepsis (57).
In Slovenia, we have recently genomically charac-
terized all available invasive neonatal GBS isolates and 
compared them with colonization isolates. As expected, 
we found that serotype III, CC-17 isolates predominated 
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References
1. Liu L, Oza S, Hogan D, Perin J, Rudan I, Lawn JE, et al. Global, regional, 
and national causes of child mortality in 2000-13, with projections to 
inform post-2015 priorities: an updated systematic analysis. Lancet. 
2015;385(9966):430-40. DOI: 10.1016/S0140-6736(14)61698-6 PMID: 
25280870
2. Lučovnik M. Je napočil čas a uvedbo presejanja za kolonizacijo s 
strepotkokom skupine B v slovensko predporodno varstvo? In: Trojner 
Bregar A, Lučovnik M. 18. Novakovi dnevi. Zbornik prispevkov. Maj 2017; 
Slovenj Gradec, Slovenija. V Ljubljani: Združenje za perinatalno medicino 
SZD; 2017.
3. Edmond KM, Kortsalioudaki C, Scott S, Schrag SJ, Zaidi AK, Cousens S, et 
al. Group B streptococcal disease in infants aged younger than 3 months: 
systematic review and meta-analysis. Lancet. 2012;379(9815):547-56. 
DOI: 10.1016/S0140-6736(11)61651-6 PMID: 22226047
4. Lasič M, Lučovnik M, Pavčnik M, Kaparič T, Ciringer M, Krivec JL, et al. 
Invazivne okužbe novorojenčkov z bakterijo Streptococcus agalactiae v 
Sloveniji, 2003–2013. Zdrav Vestn. 2017;86(11-12):493-506.
5. Ouchenir L, Renaud C, Khan S, Bitnun A, Boisvert AA, McDonald J, et al. 
The epidemiology, management, and outcomes of bacterial meningitis 
in infants. Pediatrics. 2017;140(1):e20170476. DOI: 10.1542/peds.2017-
0476 PMID: 28600447
6. Stoll BJ, Hansen NI, Sánchez PJ, Faix RG, Poindexter BB, Van Meurs KP, et 
al.; Eunice Kennedy Shriver National Institute of Child Health and Human 
Development Neonatal Research Network. Early onset neonatal sepsis: 
the burden of group B Streptococcal and E. coli disease continues. 
Pediatrics. 2011;127(5):817-26. DOI: 10.1542/peds.2010-2217 PMID: 
21518717
7. Fišer J, Špacapan S, Prinčič D, Frelih T. Odkrivanje kolonizacije nosečnic 
z bakterijo Streptococcus agalactiae v severnoprimorski regiji. Zdrav 
Vestn. 2001;70:623-6.
8. Lučovnik M, Tul Mandić N, Lozar Krivec J, Kolenc U, Jeverica S. Prevalenca 
kolonizacije z bakterijo Streptococcus agalactiae pri nosečnicah v 
Sloveniji v obdobju 2013–2014. Zdrav Vestn. 2016;85(7-8):393-400.
9. Boyer KM, Gotoff SP. Prevention of early-onset neonatal group B 
Streptococcal disease with selective intrapartum chemoprophylaxis. 
NEJM. 1986;314(26):1665-9. DOI: 10.1056/NEJM198606263142603 PMID: 
3520319
10. Fabjan Vodušek V, Cerar LK, Mole H, Šajina Stritar B, Pokorn M, Lučovnik 
M, et al. Antibiotično zdravljenje ob porodu – priporočila. Zdrav Vestn. 
2019;88(1):93-102. DOI: 10.6016/ZdravVestn.2916
11. Van Dyke MK, Phares CR, Lynfield R, Thomas AR, Arnold KE, Craig AS, et 
al. Evaluation of universal antenatal screening for group B streptococcus. 
N Engl J Med. 2009;360(25):2626-36. DOI: 10.1056/NEJMoa0806820 PMID: 
19535801
12. Verani JR, McGee L, Schrag SJ; Division of Bacterial Diseases, National 
Center for Immunization and Respiratory Diseases, Centers for 
Disease Control and Prevention (CDC). Prevention of Perinatal Group 
B Streptococcal Disease: Revised Guidelines from CDC, 2010. MMWR 
Recomm Rep. 2010;59(RR10):1-32. PMID: 21088663
13. Bliss SJ, Manning SD, Tallman P, Baker CJ, Pearlman MD, Marrs CF, et al. 
Group B Streptococcus colonization in male and nonpregnant female 
university students: a cross-sectional prevalence study. Clin Infect Dis. 
2002;34(2):184-90. DOI: 10.1086/338258 PMID: 11740706
among invasive (67%) and non-invasive isolates (33%). 
Their predominance was particularly high in late-onset 
invasive infections (81%) compared to early-onset infec-
tions (48%) (58).
From a microbiological point of view, in addition to 
traditional typing methods based on sequencing part 
of the genome, there are also some simpler and faster 
methods for determining the CC-17 clone. On the one 
hand, there is the specific presence of the hvgA gene, 
which can be used as a genetic and diagnostic marker of 
infection with this clone (59). On the other hand, CC-
17 also differs from other GBS clones in the presence 
of specific peaks in MALDI-TOF mass spectrometry, a 
method that is commonly used to identify bacteria in re-
cent times (60). Whether hypervirulent CC-17 clone de-
tection could contribute to disease prognosis is difficult 
to say for the time being, as no such clinical data exists.
6 Conclusion
With the adoption of the GBS screening programme 
in pregnant women in 2020, much has been achieved 
to reduce the incidence of early-onset invasive neonatal 
GBS infections, which is currently higher in Slovenia 
than in comparable countries. We decided to use the 
currently most sensitive diagnostic methodology, i.e. 
combinations of enrichment and molecular testing (Ta-
ble 2). In this way, we combined the advantages of both 
methods, achieved higher sensitivity, enabled the possi-
bility of determining antibiotic resistance, and shortened 
the diagnostic procedure duration; higher screening 
sensitivity and specificity was achieved with molecular 
testing, but only with appropriate specimen collection, 
namely, combined vaginal-rectal swabbing. In the fu-
ture, it will be necessary to routinely include universal 
screening for GBS colonization in pregnancy in the Slo-
venian antenatal care system, for which the The Health 
Insurance Institute of Slovenia (ZZZS) funds will have 
to be provided. It will also be necessary to monitor the 
correct and efficient implementation of the programme 
and continue the search for new improvements.
Conflict of interest
None declared.
317
REVIEW ARTICLE
Prevention of early invasive infections with group B streptococci in the newborn
14. Manning SD, Neighbors K, Tallman PA, Gillespie B, Marrs CF, Borchardt 
SM, et al. Prevalence of group B streptococcus colonization and potential 
for transmission by casual contact in healthy young men and women. 
Clin Infect Dis. 2004;39(3):380-8. DOI: 10.1086/422321 PMID: 15307006
15. Manning SD, Tallman P, Baker CJ, Gillespie B, Marrs CF, Foxman B. 
Determinants of co-colonization with group B streptococcus among 
heterosexual college couples. Epidemiology. 2002;13(5):533-9. DOI: 
10.1097/00001648-200209000-00008 PMID: 12192222
16. Hansen SM, Uldbjerg N, Kilian M, Sørensen UB. Dynamics of Streptococcus 
agalactiae colonization in women during and after pregnancy and in 
their infants. J Clin Microbiol. 2004;42(1):83-9. DOI: 10.1128/JCM.42.1.83-
89.2004 PMID: 14715736
17. Regan JA, Klebanoff MA, Nugent RP, Eschenbach DA, Blackwelder WC, 
Lou Y, et al.; VIP Study Group. Colonization with group B streptococci in 
pregnancy and adverse outcome. Am J Obstet Gynecol. 1996;174(4):1354-
60. DOI: 10.1016/S0002-9378(96)70684-1 PMID: 8623869
18. Yancey MK, Schuchat A, Brown LK, Ventura VL, Markenson GR. The 
accuracy of late antenatal screening cultures in predicting genital group 
B streptococcal colonization at delivery. Obstet Gynecol. 1996;88(5):811-
5. DOI: 10.1016/0029-7844(96)00320-1 PMID: 8885919
19. American College of Obstetricians and Gynecologists Committee on 
Obstetric Practice. Prevention of group B streptococcal early-onset 
disease in newborns: ACOG committee opinion, Number 797. Vol. 135. 
Obstet Gynecol. 2020;135:e51-72. DOI: 10.1097/AOG.0000000000003668 
PMID: 31977795
20. Vovko P. Assesment of laboratory procedures for group B streptococci 
screening in pregnant women. Zdrav Vestn. 2007;76:33-9.
21. Six A, Firon A, Plainvert C, Caplain C, Bouaboud A, Touak G, et al. 
Molecular characterization of nonhemolytic and nonpigmented group B 
streptococci responsible for human invasive infections. J Clin Microbiol. 
2016;54(1):75-82. DOI: 10.1128/JCM.02177-15 PMID: 26491182
22. Rosa-Fraile M, Spellerberg B. Reliable Detection of group B Streptococcus 
in the clinical laboratory. J Clin Microbiol. 2017;55(9):2590-8. DOI: 
10.1128/JCM.00582-17 PMID: 28659318
23. Platt MW, McLaughlin JC, Gilson GJ, Wellhoner MF, Nims LJ. Increased 
recovery of group B Streptococcus by the inclusion of rectal culturing 
and enrichment. Diagn Microbiol Infect Dis. 1995;21(2):65-8. DOI: 
10.1016/0732-8893(95)00022-3 PMID: 7628194
24. Badri MS, Zawaneh S, Cruz AC, Mantilla G, Baer H, Spellacy WN, et al. 
Rectal colonization with group B streptococcus: relation to vaginal 
colonization of pregnant women. J Infect Dis. 1977;135(2):308-12. DOI: 
10.1093/infdis/135.2.308 PMID: 320278
25. Boyer KM, Gadzala CA, Kelly PD, Burd LI, Gotoff SP. Selective intrapartum 
chemoprophylaxis of neonatal group B streptococcal early-onset disease. 
II. Predictive value of prenatal cultures. J Infect Dis. 1983;148(5):802-9. 
DOI: 10.1093/infdis/148.5.802 PMID: 6355317
26. Buchan BW, Olson WJ, Mackey TL, Ledeboer NA. Clinical evaluation of the 
walk-away specimen processor and ESwab for recovery of Streptococcus 
agalactiae isolates in prenatal screening specimens. J Clin Microbiol. 
2014;52(6):2166-8. DOI: 10.1128/JCM.00374-14 PMID: 24622104
27. Orenga S, James AL, Manafi M, Perry JD, Pincus DH. Enzymatic substrates 
in microbiology. J Microbiol Methods. 2009;79(2):139-55. DOI: 10.1016/j.
mimet.2009.08.001 PMID: 19679151
28. Church DL, Baxter H, Lloyd T, Larios O, Gregson DB. Evaluation of StrepB 
select chromagar and the ast Track Diagnostics group B Streptococcus 
(GBS) real-time polymerase chain reaction assay compared to routine 
culture for detection of GBS during antepartum screening. J Clin 
Microbiol. 2017;55(7):2137-42. DOI: 10.1128/JCM.00043-17 PMID: 
28446575
29. Craven RR, Weber CJ, Jennemann RA, Dunne WM. Evaluation of a 
chromogenic agar for detection of group B streptococcus in pregnant 
women. J Clin Microbiol. 2010;48(9):3370-1. DOI: 10.1128/JCM.00221-10 
PMID: 20592154
30. Louie L, Kotowich L, Meaney H, Vearncombe M, Simor AE. Evaluation 
of a new chromogenic medium (StrepB select) for detection of group 
B Streptococcus from vaginal-rectal specimens. J Clin Microbiol. 
2010;48(12):4602-3. DOI: 10.1128/JCM.01168-10 PMID: 20962144
31. Morita T, Feng D, Kamio Y, Kanno I, Somaya T, Imai K, et al. Evaluation 
of chromID strepto B as a screening media for Streptococcus agalactiae. 
BMC Infect Dis. 2014;14(1):46. DOI: 10.1186/1471-2334-14-46 PMID: 
24479795
32. Poisson DM, Chandemerle M, Guinard J, Evrard ML, Naydenova D, 
Mesnard L. Evaluation of CHROMagar StrepB: a new chromogenic agar 
medium for aerobic detection of Group B Streptococci in perinatal 
samples. J Microbiol Methods. 2010;82(3):238-42. DOI: 10.1016/j.
mimet.2010.06.008 PMID: 20600363
33. Salem N, Anderson JJ. Evaluation of four chromogenic media for 
the isolation of Group B Streptococcus from vaginal specimens 
in pregnant women. Pathology. 2015;47(6):580-2. DOI: 10.1097/
PAT.0000000000000299 PMID: 26308132
34. Verhoeven PO, Noyel P, Bonneau J, Carricajo A, Fonsale N, Ros A, et al. 
Evaluation of the new brilliance GBS chromogenic medium for screening 
of Streptococcus agalactiae vaginal colonization in pregnant women. 
J Clin Microbiol. 2014;52(3):991-3. DOI: 10.1128/JCM.02926-13 PMID: 
24403300
35. Block T, Munson E, Culver A, Vaughan K, Hryciuk JE. Comparison of 
carrot broth- and selective Todd-Hewitt broth-enhanced PCR protocols 
for real-time detection of Streptococcus agalactiae in prenatal vaginal/
anorectal specimens. J Clin Microbiol. 2008;46(11):3615-20. DOI: 10.1128/
JCM.01262-08 PMID: 18799703
36. El Aila NA, Tency I, Claeys G, Verstraelen H, Deschaght P, Decat E, et al. 
Comparison of culture with two different qPCR assays for detection of 
rectovaginal carriage of Streptococcus agalactiae (group B streptococci) 
in pregnant women. Res Microbiol. 2011;162(5):499-505. DOI: 10.1016/j.
resmic.2011.04.001 PMID: 21514378
37. Couturier BA, Weight T, Elmer H, Schlaberg R. Antepartum screening 
for group B Streptococcus by three FDA-cleared molecular tests and 
effect of shortened enrichment culture on molecular detection rates. 
J Clin Microbiol. 2014;52(9):3429-32. DOI: 10.1128/JCM.01081-14 PMID: 
25009049
38. Hernandez DR, Wolk DM, Walker KL, Young S, Dunn R, Dunbar SA, et al. 
Multicenter diagnostic accuracy evaluation of the Luminex Aries real-time 
PCR assay for group B Streptococcus detection in lim broth-enriched 
samples. J Clin Microbiol. 2018;56(8):1-9. DOI: 10.1128/JCM.01768-17 
PMID: 29848562
39. Jeverica S, Kotnik E, Lučovnik M, Tul Mandić N. Ugotavljanje nosilstva 
bakterije Streptococcus agalactiae v nosečnosti – ali moramo v 
diagnostiki narediti naslednji korak? Zdrav Vestn. 2016;85:15-23.
40. Miller SA, Deak E, Humphries R. Comparison of the AmpliVue, BD MAX 
System, and Illumigene molecular assays for the detection of group 
B Streptococcus in antenatal screening specimens. J Clin Microbiol. 
2015;53(6):1938-41. DOI: 10.1128/JCM.00261-15 PMID: 25788551
41. Shin JH, Pride DT. Comparison of three nucleic acid amplification tests 
and culture for detection of group B Streptococcus from enrichment 
broth. J Clin Microbiol. 2019;57(6):e01958-18. DOI: 10.1128/JCM.01958-18 
PMID: 30944190
42. Di Renzo GC, Melin P, Berardi A, Blennow M, Carbonell-Estrany X, 
Donzelli GP, et al. Intrapartum GBS screening and antibiotic prophylaxis: 
a European consensus conference. J Matern Fetal Neonatal Med. 
2015;28(7):766-82. DOI: 10.3109/14767058.2014.934804 PMID: 25162923
43. El Shahaway AA, El Maghraby HM, Mohammed HA, Abd Elhady RR, 
Abdelrhman AA. Diagnostic performance of direct latex agglutination, 
post-enrichment latex agglutination and culture methods in screening 
of group B streptococci in late pregnancy: a comparative study. Infect 
Drug Resist. 2019;12:2583-8. DOI: 10.2147/IDR.S203543 PMID: 31692504
318
INFECTIONS
Zdrav Vestn | July – August 2022 | Volume 91 | https://doi.org/10.6016/ZdravVestn.3202
44. Takayama Y, Matsui H, Adachi Y, Nihonyanagi S, Wada T, Mochizuki J, et 
al. Detection of Streptococcus agalactiae by immunochromatography 
with group B streptococcus-specific surface immunogenic protein in 
pregnant women. J Infect Chemother. 2017;23(10):678-82. DOI: 10.1016/j.
jiac.2017.07.001 PMID: 28779876
45. Park CJ, Vandel NM, Ruprai DK, Martin EA, Gates KM, Coker D. Detection of 
group B streptococcal colonization in pregnant women using direct latex 
agglutination testing of selective broth. J Clin Microbiol. 2001;39(1):408-
9. DOI: 10.1128/JCM.39.1.408-409.2001 PMID: 11191227
46. Subramaniam A, Blanchard CT, Ngek ES, Mbah R, Welty E, Welty T, et 
al.; Cameroon Health Initiative. Prevalence of group B streptococcus 
anogenital colonization and feasibility of an intrapartum screening 
and antibiotic prophylaxis protocol in Cameroon, Africa. Int J Gynaecol 
Obstet. 2019;146(2):238-43. DOI: 10.1002/ijgo.12870 PMID: 31127871
47. Furfaro LL, Chang BJ, Payne MS. Detection of group B Streptococcus 
during antenatal screening in Western Australia: a comparison of culture 
and molecular methods. J Appl Microbiol. 2019;127(2):598-604. DOI: 
10.1111/jam.14331 PMID: 31120589
48. Silbert S, Rocchetti TT, Gostnell A, Kubasek C, Widen R. Detection of 
group B Streptococcus directly from collected ESwab samples by use of 
the BD Max GBS assay. J Clin Microbiol. 2016;54(6):1660-3. DOI: 10.1128/
JCM.00445-16 PMID: 27053670
49. Virranniemi M, Raudaskoski T, Haapsamo M, Kauppila J, Renko M, 
Peltola J, et al. The effect of screening-to-labor interval on the sensitivity 
of late-pregnancy culture in the prediction of group B streptococcus 
colonization at labor: A prospective multicenter cohort study. Acta 
Obstet Gynecol Scand. 2019;98(4):494-9. DOI: 10.1111/aogs.13522 PMID: 
30578547
50. Filkins L, Hauser JR, Robinson-Dunn B, Tibbetts R, Boyanton BL, Revell 
P. American Society for Microbiology provides 2020 guidelines for 
detection and identification of group B streptococcus. J Clin Microbiol. 
2020;59(1):e19-2. DOI: 10.1128/JCM.01230-20 PMID: 33115849
51. Helmig RB, Gertsen JB. Diagnostic accuracy of polymerase chain reaction 
for intrapartum detection of group B streptococcus colonization. Acta 
Obstet Gynecol Scand. 2017;96(9):1070-4. DOI: 10.1111/aogs.13169 
PMID: 28504863
52. Picchiassi E, Coata G, Babucci G, Giardina I, Summa V, Tarquini F, et al. 
Intrapartum test for detection of Group B Streptococcus colonization 
during labor. J Matern Fetal Neonatal Med. 2018;31(24):3293-300. DOI: 
10.1080/14767058.2017.1369041 PMID: 28817995
53. Ramesh Babu S, McDermott R, Farooq I, Le Blanc D, Ferguson W, 
McCallion N, et al. Screening for group B Streptococcus (GBS) at labour 
onset using PCR: accuracy and potential impact - a pilot study. J Obstet 
Gynaecol. 2018;38(1):49-54. DOI: 10.1080/01443615.2017.1328490 PMID: 
28764569
54. Furfaro LL, Chang BJ, Payne MS. Perinatal Streptococcus agalactiae 
epidemiology and surveillance targets. Clin Microbiol Rev. 2018;31(4):1-
18. DOI: 10.1128/CMR.00049-18 PMID: 30111577
55. Shabayek S, Spellerberg B, Group B. Streptococcal colonization, 
molecular characteristics, and epidemiology. Front Microbiol. 2018;9:437. 
DOI: 10.3389/fmicb.2018.00437 PMID: 29593684
56. Da Cunha V, Davies MR, Douarre PE, Rosinski-Chupin I, Margarit I, 
Spinali S, et al.; DEVANI Consortium. Streptococcus agalactiae clones 
infecting humans were selected and fixed through the extensive use of 
tetracycline. Nat Commun. 2014;5(1):4544. DOI: 10.1038/ncomms5544 
PMID: 25088811
57. Bekker V, Bijlsma MW, van de Beek D, Kuijpers TW, van der Ende A. 
Incidence of invasive group B streptococcal disease and pathogen 
genotype distribution in newborn babies in the Netherlands over 25 years: 
a nationwide surveillance study. Lancet Infect Dis. 2014;14(11):1083-9. 
DOI: 10.1016/S1473-3099(14)70919-3 PMID: 25444407
58. Perme T, Golparian D, Bombek Ihan M, Rojnik A, Lučovnik M, Kornhauser 
Cerar L, et al. Genomic and phenotypic characterisation of invasive 
neonatal and colonising group B Streptococcus isolates from Slovenia, 
2001-2018. BMC Infect Dis. 2020;20(1):958. DOI: 10.1186/s12879-020-
05599-y PMID: 33327946
59. Lamy MC, Dramsi S, Billoët A, Réglier-Poupet H, Tazi A, Raymond J, et 
al. Rapid detection of the “highly virulent” group B Streptococcus 
ST-17 clone. Microbes Infect. 2006;8(7):1714-22. DOI: 10.1016/j.
micinf.2006.02.008 PMID: 16822689
60. Lartigue MF, Kostrzewa M, Salloum M, Haguenoer E, Héry-Arnaud 
G, Domelier AS, et al. Rapid detection of “highly virulent” Group B 
Streptococcus ST-17 and emerging ST-1 clones by MALDI-TOF mass 
spectrometry. J Microbiol Methods. 2011;86(2):262-5. DOI: 10.1016/j.
mimet.2011.05.017 PMID: 21663770