Serotype distribution, antimicrobial resistance, and molecular characterization of group B Streptococcus isolates from Chinese pregnant woman

Abstract

Objective

This study was aimed to investigate the serotypes, antibiotic susceptibilities, and multi-locus sequence type (MLST) profiles of group B Streptococcus (GBS) in the Beijing area.

Methods

Lower vaginal and rectal swabs were obtained from pregnant women of 35–37 gestational weeks (GWs) who attended the Beijing Obstetrics and Gynecology Hospital. All GBS isolates were identified with Gram staining, catalase reaction assays, and CAMP tests, followed by antibiotic susceptibility testing, serotype identification, multilocus sequence typing and erythromycin resistance gene analysis (ermB and mefE).

Results

From July 2020 to June 2022, 311 (5.17%) of 6012 pregnant women that were screened for GBS colonization were detected positive. Of the eight serotypes identified (III, Ia, Ib, IV, II, VIII, V, and NT), serotypes III (43.09%), Ia (34.08%) and Ib (17.04%) were the predominant species. In the antimicrobial susceptibility experiments, the resistant rates measured for erythromycin, clindamycin, levofloxacin, and tetracycline were 76.21%, 63.99%, 50.80%, and 81.03%, respectively, and 7.6% of GBS isolates showed inducible clindamycin in resistance (D-test phenotype). Meanwhile, the multilocus sequence typing analysis showed that sequence type 19 (ST19) (30.34%) and ST10 (18.62%) were the dominant sequence types. Among the 237 erythromycin-resistant isolates, 176 harbored ermB (128, 54.00%) or mefE (48, 20.30%) gene alone.

Conclusion

The infection rates, serotypes or MSLT distribution, and antimicrobial resistance of GBS in Beijing area were investigated, which may be applied in analyses of the epidemiological characteristics of GBS. This contributes to the basic knowledge required for successful GBS vaccine development suited for disease prevention and treatment in China, as well as the implementation of effective clinical antimicrobials.

Keywords:

• GBS
• serotyping
• mLST
• susceptibility
• pregnancy

Introduction

Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a β-hemolytic, Gram-positive bacterium that commonly colonizes the lower gastrointestinal and genital tracts. It is a leading cause of infection during pregnancy, preterm birth, and neonatal infection [1–3]. Furthermore, some research reports that colonized mothers are more likely to transmit GBS to their offspring, making infants develop early-onset disease GBS (EOD-GBS, developing within the first week after birth and usually manifesting as pneumonia and sepsis) [4,5]. Since the mid-1980s, clinical trials and observational studies have demonstrated that EOD-GBS may be prevented by the administration of antibiotics during labor in women colonized with GBS [6]. In China and many other countries, pregnant women are routinely screened at 35–37 weeks for GBS colonization by rectovaginal swab and subsequent culturing [6]. According to the intrapartum antibiotic prophylaxis (IAP) procedure implemented at our institute, those GBS colonized pregnant women are administered with penicillin or cefazolin/clindamycin, depending upon their penicillin allergy status [7]. The earlier reports pointed that the incidence of EOD-GBS has decreased significantly after active prevention, although GBS-introduced infection remained as the main disease of neonatal morbidity and mortality [6].

GBS can be divided into 10 different serotypes according to its capsular polysaccharides (Ia, Ib, and II to IX) or multiple sequence types (STs, isolates with the same allelic profile) based on the multilocus sequence typing (MLST) results [5]. Several studies suggest that some serotypes and/or STs are associated with specific disease phenotypes [8,9,]. For example, serotype III strain with ST17 may have an enhanced likelihood of causing EOD-GBS [10]. In addition, maternal antibodies against type-specific GBS capsular polysaccharides must potentially prevent invasive GBS disease to be protective, and immunization of pregnant women with polysaccharide conjugate vaccine formulations is expected to further reduce the burden of neonatal disease in GBS infection [11]. However, the epidemiology of maternal GBS colonization is variable and can be influenced geographically, over time or ethnically [12,13]. Therefore, it is essential to actively monitor the colonization rate, serotype/MSLT distribution, and antimicrobial resistance of GBS strains for local pregnant women to improve the outcome of infection.

Methods

Detection and confirmation of GBS

Lower vaginal and rectal swabs were obtained from the pregnant women of 35–37 gestational weeks (GWs) who attended the Beijing Obstetrics and Genecology Hospital from July 2020 to June 2022. Participants with history of using antibiotic(s) within two weeks prior to recruitment were excluded from the study. The swabs were inoculated onto Colombian plates supplemented with 5% sheep blood and were incubated at 35 °C under 5% CO2 and examined after 18–24 h growth. To confirm morphologically suspected GBS isolates, Gram staining, catalase reaction assays (3% hydrogen peroxide was used to demonstrate the presence of catalase, Haiderun, China), and CAMP tests were performed. The suspected isolates from culture plates were alternatively identified by the Vitek 2 compact system (bioMerieux, France). Finally, all confirmed GBS isolates were stored at −80 °C with strain preservation tubes (Hopebio, China).

Reagents and instruments

Chromosomal DNA was extracted from overnight-grown cultures of GBS isolates using a DNA Mini Kit (SBS Genetech, China). All the primers were synthesized by Eurogentec (Eurogentec, Liège Belgium). Polymerase chain reaction (PCR) was performed using a Light Cycler 480 (LC480) instrument (Roche Diagnostics, Mannheim. Every batch of samples tested included negative (PCR-grade distilled, de-ionized water) and positive controls (plasmids containing target genes) (Techpool Biosciences, China). A positive PCR signal was defined as a quantification cycle (Ct) ≤40.

Serotype identification

PCR was applied to serotype identification in all GBS isolates using the primers (Supplementary Table 1) and reaction conditions described previously [14]. The isolates were defined as non-typeable (NT) if the method failed in serotype identification. The following thermal cycling program was applied in the serotyping experiments. The samples were amplified by a denaturation step for 5 min at 95 °C, followed by 15 cycles of 95 °C for 60 s, 54 °C for 60 s, and 72 °C for 2 min and then by additional 25 cycles of 95 °C for 60 s, 56 °C for 60 s, and 72 °C for 2 min and a final cycle of 72 °C for 10 min. Then, the PCR products from each GBS isolate were applied to 1.5% high-resolution agarose-1000 gel (Life Technologies) for electrophoresis analysis, which showed two or three bands pattern by UV transillumination of the amplified products on the agarose gel. A 688 base pairs band corresponding to the amplification of a conserved fragment of the cpsL gene was used as the internal positive control.

Antimicrobial susceptibility testing

For IAP, erythromycin, clindamycin, levofloxacin, and tetracycline are important alternative options for individuals that are allergic to penicillin [7,15,16]. Moreover, GBS was highly resistant to tetracycline and erythromycin and displayed significant regional differences in resistance rates to clindamycin and levofloxacin [15,17]. The susceptibility of erythromycin, clindamycin, levofloxacin, and tetracycline were determined using the Kirby-Bauer method. The break-points adopted complied with the 2020 Clinical and Laboratory Standards Institute (CLSI) criteria, with the results reported as susceptible, intermediate or resistant [18]. The detection of inducible clindamycin resistance was done by using the D test method as previously described [19–21]. Briefly, erythromycin (15 µg) and clindamycin (2 µg) disks (Oxoid, Davies-Diagnostics, Pretoria, South Africa) were placed 12–15 mm apart edge to edge. Blunting was defined as growth within the clindamycin zone of inhibition proximal to the erythromycin disk, indicating inducible macrolide-lincosamide-streptogramin B (iMLSB) resistance. Resistance to both erythromycin and clindamycin indicated constitutive macrolide-lincosamide-streptogramin B (cMLSB). Resistance to erythromycin but susceptibility to clindamycin without blunting indicated an efflux mechanism (M phenotype). ATCC49619 was used as a reference strain for antimicrobial susceptibility testing [18].

Analysis of erythromycin resistance genes

The macrolide resistance genes ermB and mefE were analyzed by PCR, which was performed on the erythromycin-resistant strains. The primers (Supplementary Table 2) and the PCR procedure were implemented as described by Lopardo et al. [22]. All of the PCRs were carried out by denaturation for 10 min at 94 °C, followed by 35 amplification cycles and a final elongation at 72 °C. Specifically, for ermB (639 bp), the 35 amplification cycles consisted of 1 min at 94 °C, 1 min at 52 °C, 55 °C, 46 °C, 50 °C, and 48 °C, respectively, and 1 min at 72 °C. For mefE, the amplification step consisted of 35 cycles of 1 min at 94 °C, 1 min at 50 °C, and 1 min at 72 °C.

Multilocus sequence typing (MLST)

The internal fragments within seven housekeeping genes (adhP, pheS, atr, glnA, sdhA, glcK, and tkt) were simultaneously amplified and sequenced. The primers corresponding to these genes were designed as described previously [23]. PCR conditions included a 10 min step at 94 °C; 35 cycles of 92 °C for 1 min, 53 °C for 1 min, and 72 °C for 30 s; and a final 5 min elongation at 72 °C. Five-microliter PCR products from each GBS isolates was applied to 2% agarose gel for electrophoresis analysis, followed by amplification fragments purification and two-way sequencing conducted by Shanghai Shenggong Biological Engineering Technology Service Co., Ltd. Sequencing results were submitted to the GBS MLST website (http://pubmlst.Org/sagalactiae/) to allow for allelic assignment and to determine STs.

Statistical analysis

The correlation between serotypes and MLST types of GBS was tested using Fisher’s exact test by SPSS 25 (SPSS, Inc., Chicago, IL, USA), and p < .05 were considered statistically significant.

Ethical approvement

The study protocol was approved by the Ethics Committee of Beijing Obstetrics and Gynecology Hospital (2016-KY-015-01). The ethics committee waived the requirement for informed consent from participants because all GBS screening tests were part of routine antenatal care during pregnancy. All the laboratory data were anonymous before use.

Results

Colonization rate and serotypes of GBS

Of the 6012 pregnant women that were screened, 311 were detected positive for GBS colonization, with a positive rate of 5.17%. Using the aforementioned PCR method, eight serotypes were identified among the 311 colonized GBS strains. As shown in Figure 1(A), the predominant serotypes were III (43.09%), Ia (34.08%), and Ib (17.04%), followed by IV (1.93%), II (1.61%), VIII (0.96%), V (0.64%), and NT (0.64%).

Figure 1. Distributions of serotyping and erythromycin-resistant genes in GBS isolates. (A) Serotype distribution of 311 GBS isolates; (B) distribution of erythromycin resistance genes in 237 GBS isolates.

Antimicrobial susceptibility tests (AST)

The antimicrobial susceptibility tests were carried out with erythromycin, clindamycin, levofloxacin, and tetracycline, and the detection of inducible clindamycin resistance was conducted using the D test method. As summarized in Table 1, the majority of GBS isolates were found to be resistant to tetracycline (n = 252, 81.03%), erythromycin (n = 237, 76.21%) or clindamycin (n = 199, 63.99%), followed by levofloxacin with a resistance rate of 50.80% (n = 158). Among the resistant isolates to erythromycin and clindamycin, the prevalence of cMLSB, iMLSB, and M phenotypes was 81.90%, 7.60% and 10.50% respectively.

Table 1. GBS antimicrobial susceptibility patterns.

	Susceptibility
Antibiotics	S (%)	I (%)	R (%)
Erythromycin	16.72% (52/311)	7.07% (22/311)	76.21% (237/311)
Clindamycin	23.47% (73/311)	12.54% (39/311)	63.99% (199/311)
Levofloxacin	41.16% (128/311)	8.04% (25/311)	50.80% (158/311)
Tetracycline	12.54% (39/311)	6.43% (20/311)	81.03% (252/311)

S: susceptible; I: intermediate; R: resistant.

Genes analysis of erythromycin-resistant strains

To make a full comparison with the findings by Wang et al. [24], the ermB and mefE genes that were highly associated with the resistance to erythromycin in previous studies [25–27] were investigated in our experiments. Among the 237 GBS isolate that were resistant to erythromycin, 36 (15.20%) isolates harbored both ermB and mefE genes; 176 isolates were found to carry ermB (128, 54.00%) or mefE (48, 20.30%) gene alone, and 25 (10.50%) isolates harbored neither ermB nor mefE genes, as shown in Figure 1(B).

MLST

Totally 145 GBS isolates were randomly selected and subjected to MLST analysis. As shown in Figure 2, ST19 (30.34%) and ST10 (18.62%) were the dominant sequence types, followed by ST12, ST1, and ST17, accounting for 12.41%, 6.90%, and 6.90% of the sequenced GBS strains, respectively. In addition, ST27, ST23, ST8, ST921, ST485, ST862 and so on were relatively rare (accounting for 0.69% to 5.52% of the sequenced GBS isolates). In the main serotype-sequence types (STs) association analysis (Table 2), serotypes Ia and III showed significant differences between the isolates of different STs (p < .01). Specifically, serotype III strains were predominantly observed for ST19 (83.33%) and ST17 (100.00%), while serotype Ia. Strains were predominantly associated with ST1 (62.50%), ST10 (62.96%) and ST12 (66.67%).

Figure 2. Distributions of sequence types in 145 GBS isolates.

Table 2. The relationship between main serotypes and STs of GBS.

Serotypes	STs					P
	ST1 (n = 8)	ST10 (n = 27)	ST12 (n = 18)	ST17 (n = 9)	ST19 (n = 42)
Ia	5 (62.5%)	17 (62.96%)	12 (66.67%)	0	1 (2.38%)	<.01
Ib	3 (37.5%)	9 (33.33%)	5 (27.78%)	0	6 (14.29%)	.092
III	0	1 (3.70%)	1 (5.56%)	9 (100.00%)	35 (83.33%)	<.01

Due to the small number of remaining genotypes, only the main serotypes and sequence types are listed in the table. STs: sequence types.

Discussion

In women, colonized GBS isolates serve as a reservoir of serotype diversity, virulence factors, and antibiotic resistance. The pathogenic ability of GBS is related to virulence factors, including sialic acid-rich capsular polysaccharide (CPS), perforin, lipoteichoic acid, neuraminidase and so on. For example, CPS is widely distributed on the terminal glycan structures present on host cell surfaces, one of the most important virulence factors promoting, beneficial for GBS innate immune evasion by molecular mimicry, developing diseases [5]. As we know, determining the population structure of colonized isolates is key to improve clinical treatment and outcomes of GBS colonization for local pregnant women. At our institute, the GBS colonization rate in the screened pregnant women was 5.17%, which was close to that reported by Wang et al. in the same geographical area (Beijing City) [24], but was significantly lower than the global overall prevalence [28]. The distinct difference in GBS positive rate from different published reports may be attributed to a few factors. For instance, the prevalence of maternal colonization in GBS may vary by region [13], ethnicity and socioeconomic status [29]. In addition, the differences of GBS positivity in pregnancy may be introduced by methodological issues, such as time of GBS screening (during pregnancy or at delivery [30]), sampling site (in particular, whether rectal samples were performed [31–34]), and laboratory culture techniques. The 2020 American College of Obstetricians and Gynecologists (ACOG) committee opinion for GBS prevention recommended the application of vaginal–rectal cultures to increase the detection rates of GBS [35].

In this investigation, it was found that the GBS colonization in pregnant women was most frequently caused by serotypes III and Ia, which was highly consistent with previous reports from the USA and Canada [12,36]. Other studies [37–39] have shown that the serotype III was the most common type in Chinese infants. While in a local report by Shen et al. serotypes II and III were found as the predominant serotypes in both pregnant and non-pregnant women [40]. There are several factors that may have impacts on the epidemiological distribution of GBS serotypes, such as geographical regions, ethnicity and age of study population [24]. For example, Lopardo et al.’s [22] study found that serotypes II, Ia/c, III, and IV were commonly found in adults, with serotype II prevalent in younger adults (18 to 69 years old) and serotype Ia/c prevalent in elderly adults (>70 years old). Additionally, the distribution of serotypes was variable over time [8]. For example, compared with the study of Wang et al. in 2015[24], the incidence of the serotypes III and Ia were increased and the serotypes IV, and VIII were identified in our study but not in Wang’s. A recent laboratory evidence showed that there was a capsular switching between the polysaccharide type III and type IV in this lineage due to the exchange of a 35.5-kb DNA fragment containing the entire cps operon [41], causing GBS serotype variation and suggesting that the genetic recombination of GBS leads to the GBS serotype variation. Thus, ongoing close surveillance of GBS from both molecular and epidemiological levels is required for the implementation of appropriate preventative strategies and GBS vaccine development [42,43].

In our genetic diversity analysis with the tools of MLST, the top five STs (ST19, ST10, ST12, ST1 and ST17) accounted for more than three-quarters of GBS isolates. Further, our study reported that serotype III/ST17 was the dominant association, followed by serotype III/ST19, Ia/ST12, Ia/ST10, and Ia/ST1. Similar findings were reported by Gao et al. [15] who documented that the most common ST strain of maternal infection was ST19 and it accounted for the major species of serotype III. According to Wang’s study [24] with a much smaller number of GBS isolates (n = 56) of local pregnant women (Beijing area), ST19 was predominant, followed by the serotypes of ST23, ST12, and ST1, which were different from the ST findings in our study. The difference in the prevalence order of predominant ST species between the two similar studies substantiate the importance of sample size in GBS-related epidemiological study.

The preventive application of antibiotics to pregnant women has greatly reduced the risk of GBS infection in infants, while bacterial resistance increases with the misuse of antibiotics [44]. In the antimicrobial susceptibility tests study with our pregnant women, 76.2% and 64.0% of the GBS isolates were resistant to erythromycin and clindamycin, respectively. Similar antimicrobial susceptibility patterns were reported in various studies [14,17,24], implying that erythromycin or clindamycin should not be considered a primary antibiotic choice for intrapartum GBS chemoprophylaxis. In addition, the prevalence of inducible and constitutive methylation and efflux in GBS were different from those previously reported [21,45]. These differences emphasize the need for laboratories to understand the mechanisms of macrolide resistance in reporting clindamycin susceptibility with appropriate approaches. Highly variable resistance rates against levofloxacin have been observed in a previous study [17], which were ranged from 24.0% in South China to 67.7% in Northeast China. The resistance rate of GBS against tetracycline was also high in different regions of China [27] or Africa [46], which was highly consistent with our study. Among the erythromycin-resistant GBS strains, the prevalence of the species carrying the ermB gene alone was predominant (54.00%) and higher than that reported by a study of Wang et al. in 2015, suggesting increased resistance of erythromycin over time. It’s in line with the resistance mechanism of erythromycin, in which the methylation of 23S ribosomal RNA introduced by erythromycin ribosome methylase (encoded by the erm genes) prevents erythromycin from binding to the ribosome, allowing the bacteria to continue protein synthesis [47].

This was a relatively large-sized population-based study (n = 6012), rendering ample statistical power for the subsequent prevention and treatment of maternal GBS colonization. Ongoing GBS monitoring in serotype fitness, herd immunity changes, and the spread of antibiotic-resistant clones are important with respect to vaccine development [8]. Our study showed that a considerable number of GBS isolates were resistant to clindamycin and erythromycin, thus decreasing the options for prophylaxis in pregnant women who are allergic to penicillin [27]. However, limitations still exit in the present study. Firstly, as this was a single-center study with narrow representation of pregnant women, the molecular and epidemiological information of GBS may not be directly applied to non-Chinese population. Secondly, the enrichment broth was not used for GBS testing in our experiment, leading to a lower GBS colonization rate when compared with the global overall prevalence. Last but not least, there was a lack of information on invasive GBS isolates recovered from neonates (such as EOD-GBS). Thus, a future multicentric study involving wider gene profiles of resistant strains and neonate infection information is warranted to boost the clinical significance of this type of study.

Conclusion

In summary, the data from this study provide important epidemiological information on GBS colonization isolates recovered from pregnant women in China, which contributes to the basic knowledge required for GBS vaccine development as well as implementation of effective clinical antimicrobials.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Raw data were generated at Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Derived data supporting the findings of this study are available from the corresponding author ZC and YZ on request.

Additional information

Funding

This work was supported by Beijing Health Technologies Promotion Program (BHTPP2022017), Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing Maternal and Child Health Care Hospital ‘Discipline Backbone’ Plan Special Funds (XKGG201802) and Beijing Hospitals Authority Clinical Medicine Development of Special Funding Support (ZYLX202120). The funding body did not take part in the design of the study, the collection, analysis and interpretation of the data, or manuscript writing.