Phenotypic And Molecular Characterization Of Methicillin Resistant Staphylococcus Aureus From Surgical Patients And Normal Dogs

Abstract

Staphylococcus spp. are globally recognized as colonisers of the skin and important causes of infection in the skin of animals and humans. The increasing prevalence of antimicrobial resistance, and in particular multi-drug resistant methicillin resistant Staphylococcus aureus (MRSA) and the emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) in dogs has made treatment more challenging. The objectives of this study were to determine bacterial ecology and their antimicrobial susceptibilities from wound and ear swabs with emphasis on Staphylococcus aureus and to determine the prevalence of MRSA/MRSP in normal dogs and surgical patients using phenotypic and genotypic assays. The study also undertook Basic Local Alignment Search Tool (BLAST) analysis of sequenced polymerase chain reaction (PCR) amplicons of the resistance determinant. The study was divided into two parts, retrospective and prospective components. The retrospective component of the study was designed to determine the bacterial ecology and antimicrobial susceptibility from samples taken from surgical patients. Records were retrieved from clinical laboratory of 291 bacteriological samples collected from 200 dogs submitted to the University of Nairobi Small Animal Clinic over a 10 year period between January 2004 and December 2013. Information collected included the location from where the sample was collected (wound or ear swab) as well as age, sex of the animal, microbial isolates and antimicrobial susceptibility profile. In addition, for samples obtained from wounds, records were further reviewed to determine the type, nature, location and causes of the wounds. In the prospective component of the study, investigations were done on 191 samples obtained from dogs presented at the University of Nairobi Small Animal Clinic and a community veterinary clinic. Identification of coagulase positive Staphylococcus spp. (COPS) was undertaken using mannitol salt agar as a selective medium and coagulase testing using xii reconstituted rabbit plasma. Final confirmation of COPS was done by PCR using primers specific to the 16S rRNA gene of Staphylococcus Genus and primers specific to the nuc (thermonuclease) gene of Staphylococcus aureus and Staphylococcus pseudintermedius spp. Antimicrobial susceptibility testing (AMST) was performed using Kirby-Bauer disc diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Staphylococcus aureus ATCC 25923 was used as the reference organism. Oxacillin was used as a surrogate for methicillin. For each isolate, susceptibility testing was done twice and the mean zone diameter of inhibition calculated. The mean diameter was then compared to the CLSI interpretive standard break points for Staphylococcus aureus and Staphylococcus pseudintermedius for oxacillin and the number of resistant isolates noted. DNA of the phenotypically resistant COPS isolates were extracted and thereafter specific PCR assays were used to detect the resistance determinant among the resistant isolates. The PCR amplicons were electrophoresed on 1.5 % agarose gel in Tris-acetate-EDTA buffer supplemented with 0.5μg/ml of ethidium bromide and calibrated using 100 bp DNA ladder. The gels were visually inspected by Ultra Violet (UV)-transilluminator. The amplicons obtained were purified and sequenced using the ABI PRISM 3770 genetic analyzer. BLAST analysis was done to confirm the identities of the sequenced amplicons, their location on chromosomal DNA, the geographical distribution and diversity of hosts from which genes’ homologues had previously been isolated. The sequenced resistance gene was submitted to the National Center for Biotechnology Information genetic sequence database (NCBI GenBank) for assignment of accession numbers. xiii The retrospective study findings revealed that the most prevalent microbial isolates recovered from dogs diagnosed with wounds, surgical site infections and otitis externa, were Staphylococcus aureus 50 % (133/267) and Proteus spp. 14 % (38/267) respectively. Other frequently recovered isolates included Pseudomonas spp. 10 % (28/267), other Staphylococcus spp. 8.2 % (22/267), Streptococcus spp. 6.7 % (18/267) and E. coli 5.6 % (15/267) respectively. Resistance to antimicrobial drugs was observed in the majority of the isolates in the retrospective study, with 97% (262/267) of the isolates demonstrating antimicrobial resistance to at least one drug. Resistance to sulphonamides (96%), potentiated sulphonamides (89%), ampicillin (68%), amoxicillin (62%) and tetracycline (56%) was relatively high for all bacterial species examined. Staphylococcus aureus isolates showed 95% resistance to sulfamethoxazole, 55% to ampicillin, 52% to tetracycline and 52% to amoxicillin/clavulanic acid respectively. Pseudomonas spp. showed the highest multidrug resistance with all (100%) isolates showing resistance to amoxicillin, amoxicillin/clavulanic acid and sulfamethoxazole, the isolates also showed high resistance to cotrimoxazole (93%), ampicillin (93%) and tetracyclines (80%) respectively. Low resistance to gentamicin (9%), norfloxacin (24%) and chloramphenicol (33%) was observed in all bacterial isolates. The cause of 33% (18/54) wounds was not specified in the records. Common source of wound swabs included, surgical site infections (SSI) 23.9% (11/46) followed by bite wounds 21.7% (10/46), and traumatic injuries 15.2% (7/46). Majority of the wounds 67% (31/46) were recorded on the limbs of affected animals with hindlimbs 32.6% (15/46) being more affected than hindlimbs 28.3% (13/46). xiv Data from the prospective study revealed that presumptive Staphylococcus spp. were isolated from 34% (65/191) of the samples. Coagulase positive Staphylococcus spp. (COPS) accounted for 43% (28/65) of the Staphylococcus spp. isolated. Phenotypic resistance to oxacillin was detected in 53.6% (15/28) of COPS. The PCR assay detected mecA gene as a 286 bp gene fragment amplicon in 2 of the 15 (7%) oxacillin resistant phenotypes. BLAST analysis of the sequenced PCR products revealed that one of the resistance genes had 99 % nucleotide identity to sequences in the NCBI GenBank database, while the other sample had a 95 - 97% identity. Further analysis of the resistant determinants by BLAST revealed that all the resistant Staphylococcus strains were Staphylococcus aureus strains. This study confirms Staphylococcus aureus as the most prevalent bacterial isolate from wounds, surgical site infections and otitis externa. Proteus spp., Pseudomonas spp., other Staphylococcus spp., Streptococcus spp. and Escherichia coli in descending order, were also frequently isolated. Gentamicin, norfloxacin and chloramphenicol in that order were the most effective antimicrobial agents in management of wounds, surgical site infections and otitis externa in the retrospective study. The study reports the first case of MRSA strains in dogs in Kenya which were associated with mobile genetic elements (SCCmec) and have the potential to be transferred from dogs to humans. The MRSA resistant determinants observed are similar to some human like isolates reported in several countries