Introduction
Streptococcus agalactiae, the group B streptococcus (GBS), is a gram-positive and encapsulated bacterium, which displays beta-hemolytic activity on blood agar. It is part of the commensal flora in the genital and lower gastrointestinal tracts in 10–40 % of healthy adults [
1]. However, vaginal colonization by GBS during pregnancy may be clinically significant because it is associated with neonatal meningitis and septicemia [
2]. During recent decades, GBS infection has increasingly emerged in adults, particularly those with underlying illness such as diabetes [
3].
Penicillin is the first-line agent for prevention and treatment of GBS infections [
1,
4]; however, since 1994, GBS isolates with reduced susceptibility to penicillin have been reported periodically [
5]. In case of penicillin allergy, clindamycin and erythromycin are the most recommended alternatives. Consequently, the increasing use of these drugs, consumed by around 20 % of all GBS carriers, resulted in elevated rates of clindamycin and erythromycin resistance worldwide [
6,
7]. Capsular serotype contributes to disease severity and defines vaccine development [
8]. Ten GBS capsular types have been identified (Ia, Ib and II-IX) and vaccination against five serotypes (Ia, Ib, II, III and V) is in development [
7,
9]. Several investigations revealed that strains harboring the CPS types Ia, II, III, and V are the most significant causes of GBS invasive disease in neonates and other patients [
10,
11]. PCR can identify serologically non-typable strains since these generally still carry the required genes [
12]. Therefore, molecular serotyping techniques have an elevated discriminatory power for epidemiological studies [
13].
GBS has many virulence factors including surface proteins, toxins and hydrolytic enzymes [
14]. Surface proteins mostly function as adhesins, which may also contribute to immune evasion. These include C5a peptidase (ScpB), laminin-binding protein (Lmb), the α and β-subunits of C protein (Bca and Bac) and Rib protein. GBS secrete a variety of toxins such as β-hemolysin/cytolysin (cylE), hyaluronidase (hylE) and the CAMP factor (cfb) which facilitate entry of GBS into host cells and promote its intra-cellular survival [
15].
Epidemiological studies on GBS, assessment of their antibiotic susceptibility and conventional serotyping are sparse in Malaysia. Molecular serotyping and virulence determinants of Malaysian GBS are published for the first time in this study.
Results
Clinical data of all patients are summarized in Table
1. Among the 103 GBS isolates, 11 (10.7 %) were from neonates, all of whom presented with early-onset disease (<7 days old) and more than half of the neonates (64 %) were known to have GBS bacteremia. Ninety-two isolates (89.3 %) were recovered from adults (age range 20–87). In non-pregnant adults (
n =43), GBS isolates were often isolated from blood (37.3 %), vagina (35 %), wound and abscess specimens (26 %). Urine sample yielded only one isolate (1.7 %). Among pregnant women, most of the GBS isolates were recovered from vaginal specimens (90 %). Blood, urine and placenta swabs yielded a few additional isolates (10 %).
Table 1
Distribution of 103 GBS isolates among patient groups
Neonate | 11 (10.7) | 7 | 4 | 0 | 11 |
Pregnant adults | 49 (47.6) | 0 | 3 | 46 | 49 |
Non-pregnant adults | 43 (41.7) | 15 | 14 | 14 | 43 |
Total | 103 (100) | 22 | 21 | 60 | 103 |
Comparison of conventional serotyping (CS) with molecular serotyping (MS)
CS was able to identify the capsular serotypes of 80.6 % (83/103) of the GBS isolates studied and 20 (19.4 %) isolates remained non-typable. All GBS isolates were also tested by MS in order to confirm the accuracy of CS. MS was in accordance with CS for the CS-typeable isolates. MS was able to detect capsular serotype genes in all 20 (19.4 %) serologically non-typable isolates. Six isolates showed serotype III while the remaining 14 isolates were of serotype VII. Overall, taking CS and MS together, serotype VI was the most common capsular type (n =23, 22.3 %) followed by VII (n =22, 21.4 %), III (n =21, 20.4 %), Ia (n =18, 17.5 %), V (n =10, 9.7 %), II (n =8, 7.7 %) and IV (n =1, 1 %). No serotype Ib isolates were found in the present study.
Antimicrobial susceptibility profiles
All GBS strains were susceptible to penicillin G, ampicilin, cefuroxime, ceftriaxone, levofloxacin, vancomycin and chloramphenicol. A hundred percent susceptibility to penicillin was also confirmed by E-test where the MIC ranged from 0.023 to 0.064 μg/ml. The AST results showed that the majority of isolates (n =74, 71.8 %) were resistant to tetracycline. Twenty-four isolates (23.3 %) were resistant to erythromycin and 18 (17.5 %) to clindamycin. Only one case of the 18 resistant isolates showed inducible clindamycin resistance.
PCR amplification of the virulence genes
All isolates were positive for the cfb gene, which helped to confirm the species identity of GBS. Genes encoding the β-hemolysin/cytolysin (βH/C), laminin binding protein, C5a peptidase, and hyaluronate lyase (cylE, lmb, scpB and hylB) were present in 97.1 %, 97.1 %, 96.1 % and 94.2 % of isolates, respectively, making them the predominant virulence genes. The rib, bca, and bac genes were found in 29.1 %, 14.6 % and 9.7 % of the isolates, respectively.
Association of genotypic traits with molecular serotyping
The distribution of three genotypic traits (
bac,
bca,
rib) and various serotypes are presented in Table
2. As
cylE,
lmb,
scpB,
hylB were detected in almost all GBS isolates (97.1 %, 97.1 %, 96.1 %, 94.2 %, respectively), these were excluded from the analysis. Serotype IV was detected only once and was also excluded. The
rib gene is significantly associated with serotype Ia, VI (
p < 0.05), II and III (
p < 0.01). The
bca gene was more common among serotype II (
p < 0.001) and III (
p < 0.01) strains. A significant association was also found between the
bac gene and serotype II (
p < 0.001) and III (
p < 0.05) (Table
2).
Table 2
Association between capsular serotype and virulence genes in 103 isolates of group B streptococcus
Ia | 1 (5.0 %) | < 0.05 | 0 (0.0 %) | > 0.05 | 0 (0.0 %) | > 0.05 |
II | 7 (87.5 %) | < 0.01 | 7 (87.5 %) | < 0.001 | 5 (62.5 %) | < 0.001 |
III | 12 (63.2) | < 0.01 | 8 (42.1 %) | < 0.001 | 5 (26.3 %) | < 0.01 |
V | 3 (37.5 %) | > 0.05 | 0 (0.0 %) | > 0.05 | 0 (0.0 %) | > 0.05 |
VI | 2 (7.7 %) | < 0.05 | 0 (0.0 %) | > 0.05 | 0 (0.0 %) | > 0.05 |
VII | 5 (23.8 %) | > 0.05 | 0 (0.0 %) | > 0.05 | 0 (0.0 %) | > 0.05 |
Total | 30 (29.4) | | 15 (14.7 %) | | 10 (9.8 %) | |
Although all serotypes were observed among the three populations studied, serotype Ia was significantly more common among patients with invasive infections (
p < 0.01) and serotype VI isolates were significantly more common among carriers (
p < 0.05) (Table
3).
Table 3
Distribution of the serotypes within the studied GBS populations
Ia | 10 (45.5)** | 6 (28.6) | 2 (3.4) |
II | 1 (4.5) | 0 (0.0) | 7 (11.8) |
III | 2 (9.1) | 4 (19.0) | 15 (25.0) |
V | 1 (4.5) | 2 (9.5) | 7 (11.8) |
VI | 3 (13.7) | 3 (14.3) | 17 (30.0)* |
VII | 5 (22.7) | 6 (28.6) | 11 (18.0) |
No statistically significant associations were found between the virulence genes and the isolate status or age of patients in the present study (p >0.05).
Discussion
In Malaysia, data on detection of GBS virulence genes and molecular serotyping are non-existent. In the present research, GBS isolates were serotyped by both phenotypic and genotypic approaches and further characterized for their virulence patterns and antibiotic susceptibility profiles.
Serotype distribution of GBS isolates has previously been reported to vary geographically [
9,
22]. In the present study, MS results showed serotype VI (22.2 %) to be overrepresented in the Malaysian population. In contrast, an older study found lower numbers of serotype VI isolates among adults and neonates [
23]. Serotype VII is rarely reported worldwide [
7,
24,
25]; however, we found a relatively high number of strains with serotype VII (
n =22, 21.3 %) among Malaysians. Only eight GBS isolates from our study were CPS type II while a cross-sectional study in South East Asia reported this type as predominant among pregnant women [
22]. Serotype Ia was significantly more common among patients with invasive infections (
p < 0.01) and serotype VI isolates were significantly more common among carriers (
p < 0.05). All isolates were typable by MS, while a considerable proportion (19.4 %) of isolates could not be typed by CS, as also observed in previous studies [
9,
26,
27].
The overall beta-lactam susceptibility of Malaysian GBS was confirmed here [
1,
4,
22]. Neither penicillin-resistant nor penicillin intermediate GBS strains were detected. Some investigations have identified GBS isolates with reduced sensitivity [
5,
28,
29]. Hence, penicillin could still be used as a first-line drug for intra-partum prophylaxis and treatment of GBS infections in Malaysia.
The rates of resistance to erythromycin and clindamycin were lower than those in the studies from Japan, Europe and the United States [
30,
31], but can be considered high in comparison to the studies from Asian countries [
32,
33]. The resistance documented for erythromycin and clindamycin provides a warning that these drugs need to be prescribed with care since also non-susceptible GBS were observed in the United States and Japan [
28,
34].
The frequency of virulence gene
rib presence is similar to other studies from Europe, the United States and Lebanon [
15,
18,
35] and higher than those reported among pregnant women in Poland and Kuwait [
14,
36]. The
bca encodes
α-C surface protein (ACP) was observed in a lower frequency than in previous reports, where its presence varied from 42 to 85 % [
15,
36,
37]. Another study has identified the
bca gene in 100 % of strains; however, in this case only potentially clonal type Ia strains were tested [
38]. The
bac gene was found at a lower frequency (9.7 %) as compared to other studies from the United States, Europe and New Zealand [
7,
18,
39]. A recent study among pregnant women in Kuwait detected
bac in only 3.2 % of isolates [
37].
Certain virulence genes were significantly more prevalent in given CPS types. For instance,
rib was associated significantly with serotypes Ia, II, III and VI. Previous data from South East Asian countries and Europe confirmed the association between
rib gene and serotype Ia, II and III [
39‐
42]. Moreover, our results indicated a significant association between serotypes II and III with
bca and
bac, while other studies demonstrated association between
bca and
bac with serotype Ib and II [
35,
39,
41]. According to Duarte et al. (2005) around 30–55 % of serotype Ia GBS isolates in humans harbored the
bca gene [
20]. In another study reported by Dore et al. around 50 % of serotype III isolates harbored the
bca or
bac or both [
42]. This difference may be caused by geographical differences in prevalence or may indicate the appearance of silent
bca and
bac genes in these serotypes.
The present study could not demonstrate any correlation between the virulence genes and clinical status of the patients from whom the isolates were obtained (
p >0.05); however, as pointed out by Maning et al. invasive isolates showed a tendency to have
rib and
bac [
18]. No significant associations were found between the occurrence of virulence genes and the age of patients in the present study (
p >0.05). However, based on data presented by Ho et al. the majority of
bca genes (85.4 %) in GBS isolates were observed in strains from adults, whereas 59.2 % of
rib gene was harbored among isolates from neonates [
40]. Given the lack of correlation we describe above, our data do not confirm the virulence potential of the genes detected.
In conclusion, our GBS collection contained a high number of strains with serotypes VI and VII, in contrast to findings in other countries [
7,
24,
43]. Such differences in the relative regional incidence of serotypes could compromise the efficacy of vaccine. Additionally, the present study clearly shows the equivalence of MS and CS and indicates the broader coverage of MS. Data presented here confirms previous findings on the relationship between different virulence genes and serotypes but fails to confirm the virulence contribution of the panel of potential virulence genes we screened for.