Antibiotic resistance profile of staphylococcus species isolated from human clinical samples in Mafikeng, North West Province, South Africa

Abstract

Vaginal colonisation with, coagulase-negative Staphylococci (CNS) and coagulase-positive Staphylococci (CPS) among pregnant women has resulted in greater usage of antibiotics and consequently an increase in the occurrence of multiple antibiotic resistance strains among the genus Staphylococcuus. This has, however, resulted in serious limitations in the treatment of staphylococcal infections due to high levels of resistance to a range of antibiotics. The aim of the study was to isolate, identify and characterise antibiotic resistant Staphylococci strains from pregnant women in the Mafikeng area, North-West Province, South Africa using phenotypic and genotypic assays. Vaginal samples were collected from women who were in their 35 to 37th week of pregnancy and reported for routine pre-natal check-ups at two local clinics in the Mafikeng area. The samples were placed in transport medium, labelled properly and transported on ice to the laboratory for analysis. Selective isolation of staphylococci was performed using Mannitol Salt Agar and potential colonies subjected to both preliminary (Gram staining, catalase , haemolysis , mannitol fermentation and coagulase tests) and confirmatory (Latex agglutination test, and MALDI-TOF-MS and nuc PCR analysis) identification tests. The percentage of antibiotic resistant profiles and multiple antibiotic resistant phenotypes of the isolates were determined using (which method). The presence of the Penicillin Binding Protein 2a (PBP2a) was determined using a serological assay while the virulence potential of isolates was determined using sea-to-see genes specific multiplex PCR analysis. The genetic similarities of isolates was also determined using ERIC and RAPD PCR analysis. All the sixty-eight 68 (100%) Gram positive cocci were catalase positive; a small proportion 32 (47%) produced the coagulase enzyme and 33 (49%) were positively identified based on the presence of the clumping factor. Of the 68 isolates, 36 (53%) were non-hemolytic, while 32 (47%) were beta- hemolytic. MALDI-TOF MS analysis revealed that only 33 (49%) were positively identified to the species level as belonging to staphylococci. Among these isolates, S. aureus was most frequently isolated 14 (43%)

than S. vitulinus 7 (21%), S. epidermidis 4 (12%), S cohnii 3 (9%), S. equorum, 3 (9%) and S. succinus 2 (6%). Out of these, 25 (76%) isolates were positive for the nuc gene. Antibiotic resistance data revealed that large proportions (76% - 100%) of isolates were resistant to the antibiotics tested. All the isolates were most often, resistant to penicillin and, ampicillin (100%), and variously resistant to oxacillin (97%), oxytetracycline (97%), vancomycin (97%), rifampin (85%), erythromycin (82%) and streptomycin (76%). In this study, a large proportion of these isolates were resistant to three (3) or more antibiotics and therefore, were termed multiple antibiotic resistant (MAR) strains. Despite this, isolates from different sample sites portrayed slight variations in antibiotic resistance patterns. The MARPs PG-AMP-OX-VA-GM-VA-ERY-RIP-OT, PG-AMP-OX-K-VA-ERY-RIPOT and PG-AMP-OX-S-VA-ERY-RIP-OT were predominant among S. aureus while PG-AMP-OX-GMVA- ERY-RIP-OT, PG-AMP-OX-VA-ERY-RIP-OT were common among S. vitulinus. In addition, PG-APOX- K-VA-OT and PG-AMP-OX-K-VA-ERY-RIP-OT were predominant among S. cohnii and S. epidermidis respectively. Despite the fact that only one S. succinus isolate possessed the PG-AP-OXS- K-GM-VA-ERY-RIP-OT, a cause for concern is the fact that this isolate was resistant to all the ten antimicrobial agents tested. A large proportion 25 (76%) of isolates possessed the PBP2a. These isolates are of clinical importance since they are able to destroy ~-lactam antibiotics. Cluster analysis based on antibiotic inhibition zone diameter data revealed two major clusters (1-11) and when the clusters were further analysed for patterns of associations of the different species present in a given cluster, 21 of the these isolates were present in cluster II which was the larger cluster while a small proportion 12 (36 %) was present in cluster I. Cluster I was divided into three sub-clusters designated A-C and sub-cluster IA contained 2 (16.6%) S. cohnii and only 1 (8.3%) S. aureus. On the contrary, cluster IB contained only S. aureus isolates whereas cluster IC was represented by 2 (17%) of S. aureus and S. vitulinus respectively, including (8.3%) S. equorum and S. epidermidis respectively.

Cluster II was sub-divided into 5 clusters designated A to E. Sub-clusters (cluster IIC and IIE), respectively contained the largest number of isolates. Sub-cluster IIC contained only the S. aureus. while sub-cluster IIE contained 2 (9.5%) isolates each of S. aureus and S. vitulinus. In addition, 1 isolate each of S. epidermidis and S. cohnii were present in this sub-cluster. In sub-cluster IIA, 2 S. succinus isolates clustered with 1 each of S. aureus and S. vitulinus. With the exception of sub-clusters 1B and IIC that contained only S. aureus isolates, all the other sub-clusters mixed. A total of 4 (12%) which were positive for the seb gene and all these were S. aureus. All 33 Staphylococcus isolates were subjected to non-specific PCR analysis using the primers S, AP? and ERIC2 to determine their genetic relatedness. The band patterns of the isolates ranged from O to 11 per isolate. A total of 31 isolates that produced reliable band patterns were subjected to cluster analysis in order to determine their genetic relatedness. The clustering patterns generated with the ERIC-PCR protocol when the primers were combined revealed six main clusters (I-VI). Generally, isolates belonging to different species and isolated from different locations clustered together and were considered mixed clusters. The largest cluster was cluster one (I) with seven (23%) out of 31 Staphylococcus isolates and S. aureus was the predominant species (43%) within this cluster. In addition, a large proportion of these isolates were obtained from samples collected in Stad Clinic. Clusters Ill and VI respectively contained 6 isolates each. Despite the fact that S. aureus was also the predominant species in cluster Ill, an interesting observation is that all these isolates were obtained from Montshioa clinic. In addition, one isolate each of S. cohnii, S. equorum and S. vitulinus were present in this cluster. The species composition of isolates in cluster VI was similar to that of cluster Ill since S. aureus was predominant. However, in this cluster, S. cohnii, S. epidermidis and S. vitulinus that were resistant to multiple antibiotics were also present. Overall, the remarkable similarities (65 to 95%) between the ERIC profiles of some isolates

from humans in the same clinic that also belonged to the same or different species indicated that the ERIC-PCR protocol used in this study could be very effective in differentiating between isolates.