Molecular characteristics of group B Streptococcus isolates from infants in southern mainland China

Group B Streptococcus (GBS), also called Streptococcus agalactiae, is a main causative pathogen of invasive neonatal bacterial infection, causing high morbidity and mortality [1‐3]. A meta-analysis by Madrid et al. found that the incidence of invasive GBS disease in infants worldwide was 0.49 per 1000 live births (95% confidence interval [CI], 0.43–0.56), and was highest in Africa (1.12) and lowest in Asia (0.30) [4]. Some potential factors which may contribute to the apparent lower incidence in Asia include the low overall prevalence of maternal colonization, incomplete case ascertainment, and the lack of well-trained clinicians and appropriate laboratory facilities to identify the disease, especially in primary healthcare centers [5]. In this meta-analysis, early-onset disease (EOD) and late-onset disease (LOD) were defined as invasive GBS infections occurring 0–6 days and 7–89 days after birth, respectively [6]. GBS is a highly diverse organism and commonly colonizes the human gastrointestinal and genitourinary tracts. Based on capsular polysaccharides (CPS), GBS can be classified into 10 serotypes including Ia, Ib, and II through IX. However, only limited subtypes have been identified in infants with invasive infection, indicating that variation in expressed surface molecules is related to pathogenicity of strains. The Madrid et al. meta-analysis found that serotype III accounted for the highest proportion of serotypes globally (61.5%) [4], while our group’s recent study found the proportion of serotype III in Guangdong province was 77.9% [7], much higher than the global level. Jones et al. analyzed GBS isolates with multilocus sequence typing (MLST) and reported that serotype III strains have been categorized to ST17 and belong to complex clone 17 (CC17) [8]. In the CC17 lineage, which is strongly associated with neonatal meningitis, the hvgA gene encoding a surface-anchored protein named hypervirulent GBS adhesin (HvgA) has been identified [9]. The HvgA surface-anchored protein not only acts as an important adhesion medium for destroying and penetrating the intestinal and blood-brain barriers, but also mediates the migration of bacteria into the bloodstream and the central nervous system, which leads to infection [9, 10].

In addition to expressing many factors which help it evade immune detection and clearance, acquiring antimicrobial resistance genes is another strategy observed in GBS to improve survival within the host. Penicillin G and ampicillin are recommended by U.S. CDC as the first-line drugs for prophylaxis and treatment of neonatal GBS infection, while clindamycin was the recommended alternative for individuals with a β-lactam allergy [6, 11]. Unfortunately, the proportion of isolates reported to be resistant to erythromycin and clindamycin has increased in recent years, and especially high macrolide resistance in GBS have been detected among isolates circulating in both infected infants and adult female carriers in China [11‐13].

Although invasive GBS infection in Chinese infants has gradually gained attention in recent years, no standardized or official guidelines for GBS prevention and control exists and the molecular epidemiology of the pathogen remains not well known. Furthermore, published studies have shown that geographical and ethnic differences in hosts effect the molecular epidemiology of invasive GBS [9, 14‐17]. By determining serotypes, sequence types (STs), hvgA expression and antibiotic resistance of GBS isolates from infants with invasive infections in four hospitals in Guangdong province, China between January 1, 2013 and June 30, 2016, this study aims to elucidate the molecular characteristics of neonatal GBS disease prevailing in southern mainland China. This work will be helpful for exploring preventive and treatment strategies for GBS infections in southern mainland China and for the development of serotype-based vaccines in the future.

A total of 93 non-duplicate GBS isolates which caused invasive disease in infants were recovered; among them were 65 from Guangzhou Women and Children’s Medical Center, 12 from Dongguan Tungwah Hospital, 8 from Guangdong Women and Children’s Hospital and Health Institute, and 8 from Zhongshan Boai Hospital. GBS identification by the Vitken Compact 2 system were confirmed for all isolates using mass spectrometry. A total of 34 cases (36.6%) were diagnosed as EOD and 59 (63.4%) were diagnosed as LOD (Table 1). There were 49 GBS cases which were diagnosed with meningitis, 47% (16/34) of which were observed in EOD cases and 55.9% (33/59) in LOD cases. Overall, there were 43 GBS cases which were diagnosed with sepsis, and one case with arthritis.

GBS remains a leading cause of infant sepsis and meningitis worldwide within the first 90 days of life. The recent meta-analysis by Madrid et al. showed the incidence of invasive GBS disease in infants was 0.49 per 1000 live births worldwide, with serotype III (61.5%) being the dominant type and with 97% of cases caused by just five serotypes (Ia, Ib, II, III, and V) [4]. In the present study, serotypes Ia, Ib, III, and V were detected, and serotype III was the dominant serotype observed (79.6%), which is higher than the global average of 61.5% cited above, but similar to estimates from France [9], Italy [20] and other parts of China [7, 13].GBS vaccines covering five serotypes (Ia, Ib, II, III and V) have already entered Phase III clinical trials [16]; an approved vaccine would cover 95% of global newborn GBS infection [2, 5, 21], and would protect from all serotypes detected in this study. Additionally, in contrast to the approximately 50% of GBS-related meningitis cases caused by serotype III in infants worldwide [21, 22], serotype III accounted for 62.5 and 81.8% in EOD and LOD cases with meningitis in this study group, respectively.

In the present study, we obtained 15 STs from 93 invasive GBS isolates, of which 61.3% were caused by the highly pathogenic genotype ST17. The five types of CCs detected in this study (CC17, CC1, CC10, CC19, and CC23) are also the most common clones globally. Among 93 isolates, more than half of neonatal GBS infection were caused by the highly pathogenic strain CC17 (ST17, ST357, ST188). We found serotype III was more commonly associated with the most virulent clone, clonal complex (CC17). CC17 accounted for 78.4% of serotype III strains, and mainly caused the observed meningitis. Studies have shown that less than 12% of GBS strains with ST17 colonize adults, whereas CC17, in contrast, presents as highly pathogenic and virulent among both infants with under-developing immune systems and immunocompromised patients [10]. In the process of occurrence and development of neonatal invasive infection, CC17 strains enhance the pathogenicity and invasiveness of GBS through a special virulence mechanism, which is the main cause of meningitis in these patients (> 80%) [9, 10, 23, 24]. CC17 colonization is the main risk factor for neonatal GBS infection [25, 26]. A study from the Netherlands found that the main contributing factor to the increase in incidence of GBS infection between 1987 and 2011 was the increase in prevalence of CC17 [25]. Manning SD et al. [23] found ST17 and ST19 were the local dominating genotypes of neonatal invasive infections in Canada, and that ST1, ST12, and ST23 were the primary genotypes among pregnant women. In China, similar to the results of that study, ST17 was found as the main genotype for newborns infected with GBS, and ST19 for pregnant women [27]. CC17 and CC19 are the most common important gene pedigrees of GBS serotype III, and likely for that reason serotype III is the most common serotype causing GBS invasive disease in infants.

Our findings showed around 95% of serotype-III/CC17 isolates harbouring the hvgA gene, which suggested that hvgA is involved in the pathogenesis of invasive GBS infectious diseases. The ST17 strain without the selective hvgA gene presents low adhesion to the host cell, which is comparable to that of the non-ST17 strain [10]. The hvgA gene was previously known to be carried only by ST17, while our study found the hvgA gene was mainly detected from strains with CC17 (92%,57/62), while CC10, CC19, and CC23 strains also carried the hvgA gene. Moreover, a strain with serotype-Ib/ST357/CC17 had the hvgA gene as well. Our findings also indicated that most of the hvgA genes have been detected in serotype-III/ST17 strains, which is consistent with other reported findings [18, 28].

Antimicrobial susceptibility testing of GBS isolates is critical for selecting appropriate antibiotic treatment. We found all isolates were susceptible to β-lactam antibiotics over the study period. Some studies have found that there was a decrease in the susceptibility of GBS to β-lactam (such as penicillin and cephalosporin) [29‐31]. This study found that 93.5% of GBS isolates showed resistance to tetracycline, comparable to France (90, 95.5%) [24, 26], Italy (93.3%) [20], and Beijing (100%) [13]. The rate of erythromycin resistance was 60.2%, which is higher than previous results from France (16.7, 13.8%) [24, 26], Italy (12%) [20], and the United States (32%) [2]. This high resistance to macrolide antibiotics including erythromycin and clindamycin is possibly due to the overuse of antibiotics in animal husbandry and humans in China in the past decades. We found 85.7% of the erythromycin-resistant strains carried the resistance gene ermB. In this study, tetracycline resistance was mainly mediated by tetO and tetM, with carrier gene rates of 75 and 46%, respectively, whereas European countries were mainly dominated by tetM, with gene rates of 95% in France [24, 26] and 97.1% in Italy [20]. Furthermore, it was observed that a small number of antibiotic-sensitive GBS strains carried resistant genes, such as tetracycline-sensitive strains and erythromycin-sensitive strains. We also found linB is an important reason for clindamycin resistance; among 61 strains of clindamycin-resistant strains, 15 (24.6%) strains had the linB gene. This is lower than observations in South Africa where 38% (11/29) of strains had linB [32]. In contrast, linB was not detected in some investigations from Korean [33] and Iran [34]. Our group’s previous work revealed multi-drug resistance clusters (tetracycline, aminoglycoside, macrolide/lincomycin, and others) in GBS CC17 hypervirulent isolates by using genomic analysis [35]. This suggests that the antibiotics recommended by the intrapartum antibiotic prophylaxis program to prevent neonatal GBS in southern China are in need of updating. On the other hand, in addition to strengthening the rational use of antibiotics, and taking into account the higher proportion of CC17 of neonatal infection at the local level, we suggest more attention should be put on GBS multi-drug resistance control.

Until now, many studies have reported on the resistance spectrum of GBS strains among colonized pregnant women, but by contrast, fewer studies have been performed on the resistance spectrum of neonatal GBS infection [2, 13, 20, 24, 26]. In this study, genotypes exhibiting resistance to tetracycline and erythromycin were significantly correlated with a drug-resistant phenotype. However, antibiotic resistance was not limited to a specific serotype or sequence type. There was no correlation between GBS resistance and serotype, gene sequence type, and type of disease presentation. A study by Poyart C. et al. demonstrated similar results [24], but other studies have found the opposite. Joubrel, C. et al. reported that GBS serotype had a correlation with resistance, with the serotype-III strain having a low resistance rate and a high resistance rate to serotype-V strain [26]. One possible reason might be due to differences in the study population.

Our study has several limitations. GBS isolates were only from one province in China, Guangdong, and the sample size is relatively small. Therefore, these findings may not uniformly represent the whole China. We also did not collect data of population-based incidence rate and maternal information, which prevented limited further population-level analysis.

The molecular epidemiologic characteristics of GBS neonatal invasive infections in southern China were mainly comprised of serotype-III/CC17. In Guangdong, resistance to tetracycline was mainly associated with tetO and tetM genes, erythromycin resistance was associated with the presence of ermB, and clindamycin resistance was associated with linB. It is recommended to establish corresponding prevention and control measures accordingly. Further large scale studies and routine GBS surveillance are required to learn more about this infection and to prevent it from occurring.