Our study is the first prevalence report of direct detection of some of the GBS virulence genes and antibiotic resistance genes in pregnant women in Zimbabwe. Odds ratios showed (Table 3) that the antibiotic resistance genes are associated with drug resistance, whilst Spearman rank correlations showed (Table 4) the different associations between the investigated genes.
The atr gene and the Mobile Genetic Element
The sensitivity of the atr gene amplification, in this study was 100% (43/43) (Table 6) which is similar to what is reported in other studies done by Gavino and Wang (22), de-Paris et al., (21) and Alfa et al. (23), where sensitivity was 95.8%, 100% and 90.5% respectively. This high sensitivity may be attributed to the use of selective and enriched media prior to performing PCR. Since this PCR test has shown to be reliable and robust, future studies can focus on rapidly testing pregnant women who present in labor with unknown GBS status. This test was used as a GBS confirmatory identification test. The Mobile Genetic Element IS1548 was present in only 9.3% (4/43) (Table 6). This is contrary to an Iranian study showed that the gene is more prevalent in human than in bovine (77% vs 5%) GBS isolates (7). The origin of the isolates could be a possible explanation for such a difference.
Virulence Genes
Nine distinct virulence gene profiles were observed but 30/43 strains (69.8%) belonged to hly-scpB-bca-rib 37.2% (16/43), hly-scpB-bca 18.6% (8/43) or hly-scpB-rib 14.0% (6/43) profiles (Table 5). The PCR assay for detection of VFs revealed that high percentage of the GBS isolates were positive for hly 97.8% (42/43); scpB 90.1% (39/43) and bca 86.0% (37/43) (Table 6). The high prevalence of these genes in GBS has been previously reported (7,24–26). Comparisons with a 2008 study in country shows the importance of investigating the CPA of GBS isolates (9). A comparison with other studies done in countries such as the USA (27), Sweden (28), Argentina (29) and Egypt (20), shows that any discrepancies are possibly a result of the geographical locations among other factors.
The results also showed that the majority of the isolates had more than three virulence genes. This high prevalence of various VFs in S. agalactiae isolates from the vaginal canal of pregnant women could lead to the development of serious maternal and neonatal infections (30). The high incidences of VFs except for bac, also suggest that GBS vaccines containing the proteins ScpB, α protein (31), Rib (9) and hyaluronate lyase (32) could potentially be effective against our population of pregnant women in Zimbabwe.
The present study observed that all the isolates 100% (n=5) carrying the bac gene also carry the unrelated bca gene, whilst 86.5% (n=32) of the bca carrying isolates did not also have the bac gene (see Additional file 2: Table S2). This finding agrees with the report that, isolates that express β tend to also express the unrelated α protein, while the α protein is often expressed on its own isolates (31). The majority of the Spearman rank analysis were not statistically significant and with some showing weak associations. However, a significant negative correlation (Table 4) was detected between hly and IS1548 as well as between bac and rib genes (P <0.01). This negative association are most likely because some of these genes are not genetically linked.
Tetracyline Resistance
The high resistance for GBS to tetracycline 97.7% (42/43) (Table 2) can be attributed to a high presence of the tetM gene 97.6% (41/42) (see Additional file 1: Fig 13). The high tetM gene presence is as a result of the ubiquitous presence of tet genes in pathogens, opportunistic pathogens and members of the normal flora (33). The high resistance to tetracycline is also because the antibiotic a relatively cheap, extensively used prophylaxis in the therapy of animal and human infections and it is known that bacterial strains tend to be resistant to frequently used antibiotics (34). A low presence rate of the tetO 2.4% (1/42) (Table 6) gene indicates that it is not common in GBS isolates that are resistant to tetracycline. These results agree with work done in Canada (35) and Nigeria (36). In Kuwait, they also reported that 89.5% tetracycline-resistant isolates contained 94.5% tetM and 3.9 % tetO (37).
Erythromycin and Clindamycin Resistance
The results showed a 30.2% (13/43) and 55.8% (24/43) resistance to erythromycin and clindamycin respectively (Table 2). Such resistance decreases the options of prophylaxis in penicillin allergic women (38). We recommend that antibiotic susceptibility testing should be performed if clindamycin or erythromycin therapy is needed in the prevention of neonatal GBS infection. PCR detected 34.5% (10/29) ermB, 10.3% (3/29) ermTR and 3.4% (1/29) mefA from the intermediate and resistant erythromycin GBS (Table 6). The prevalence of the ermB determinant shows that GBS commonly use target methylation as the mechanism of macrolide resistance.
Studies done in Italy (39), South Africa (12), USA (14), Iran (13) and France (16) reported similar findings, with most of the studies also observing that the ermB gene is more prevalent in distribution than the ermTR gene among GBS strains. This study also confirmed findings by Poyart et al., (2003) that the mefA gene is rare among GBS isolates, thus efflux pumps mediated by this gene are not a common mechanism of macrolide resistance (16). Contrary to Bolukaoto et al., (2015) this current study did not find any linB genes 0% (0/35) in any of the clindamycin resistant and intermediate strains (12). In such cases, where multiple independent resistance genes can cause resistance, the observed phenotypic resistance can be attributed to any of the other known genes or genes that are yet to be discovered. This limits the usefulness of such diagnostic tests.
It was interesting to note that 63.6% (7/11) GBS which were resistant to both erythromycin and clindamycin had the ermB gene (see Additional file 2: Table S2 and S3) and that one GBS strain which was resistant to erythromycin and susceptible to clindamycin carried the ermTR gene. Such observations are similar to other studies (14,40,41).
Genetic Linkage in Macrolide/Tetracycline Resistance Determinant.
It was also observed that 100% (n=10) isolates that carried the ermB gene also carried the tetM gene. The association of erythromycin and tetracycline resistance may be due to the conjugative transposon Tn1545, which encodes erythromycin resistance via the ermB gene and tetracycline resistance via the tetM gene (42–44). This association was supported by our analysis which showed a positive correlation (Table 4) between the two genes, but this lacked statistical significance according to the Spearman Rank test. Contrary to an Egyptian study (45), we report that there is no evidence suggesting genetic linkage of tetO with ermB, ermTR or mefA in our GBS population.
Unexpressed Resistant Genotypes.
An unexpected observation in our study was found in the following three (7.0%) isolates. Isolate 127 which was tetracycline sensitive, and yet the tetM gene was detected. Isolates 243 and 322, were both erythromycin sensitive however, at least one gene ermTR or mefA was found, respectively (see Additional file 2: Table S2 and S3). Similar findings were reported in GBS and other streptococcal species (44,46–48). A confirmatory test to check Minimum Inhibitory Concentration (MIC) levels of these three isolates is recommended. However, based on the disk diffusion test, this observation suggests that the Kirby Bauer disk diffusion method is inadequate for detection of resistance. Although the reason(s) behind this lack of gene expression still has to be determined, some possible explanations include gene mutations, low expression levels of the gene and the possibility of a weak, distant or absent promoter (44). This study therefore supports the idea that the resistance genotype does not always accurately predict phenotypic resistance.