Molecular Epidemiology and Genetic Analysis of Staphylococcus species in Companion Animal Medicine
Type of Degreedissertation
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In human medicine, methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of bacterial infections in the United States (191). In veterinary medicine, an increased number of multidrug-resistant Staphylococcus spp. in dogs, horses, and swine have been reported (36, 220, 295). Methicillin-resistance in S. aureus is determined by the addition of the penicillin-binding protein 2a (PBP2a) encoded by the mecA gene (216, 217). The gene is carried on a large heterologous mobile element called the staphylococcal cassette chromosome mec (SCCmec) (114). To date, eleven SCCmec types have been identified in human MRSA clones, and several in staphylococci from animal origin (266). In horses and dogs, MRSA is frequently reported. Additionally, methicillin-resistant strains of Staphylococcus pseudintermedius (MRSP) are frequently diagnosed in dogs (220, 271, 295). The epidemiology of methicillin-resistant staphylococci from animal origin requires more investigation because of its zoonotic potential and to optimize preventative measures (14, 66, 87, 151). Using traditional microbiological methods and molecular techniques, an investigation of methicillin resistant Staphylococci sp isolated from equine and canine species was undertaken. Special emphasis was given to determining the most sensitive diagnostic method for recognition of methicillin-resistance (MR) in canine isolates due to the differing response of methicillin-resistant strains of S. schleiferi subspecies coagulans (MRSC) and S. pseudintermedius (MRSP) to cefoxitin (FOX) as compared to MRSA. From this investigation, it was hypothesized that the differences in the reaction of MRSP and MRSC to cefoxitin, when compared with MRSA, were based on variations in their penicillin-binding proteins. Thus, an analysis of the PBPs from S. pseudintermedius and S. schleiferi subsp coagulans was performed. Additionally, molecular epidemiology typing methods were used and the presence of three genes associated with virulence factors (S. intermedius exfoliative toxin [SIET], LUK-I and panton-valentine leukocidin [PVL]) were investigated in canine Staphylococcus sp). Results from the pulse field gel electrophoresis of the equine MRSA indicated a highly clonal population, with a predominance of the clone USA500, followed by USA100. Typing using multiplex PCR identified a preponderance of SCCmec type-IV, though the SCCmec type-II was also detected. In comparison, the type-V cassette was present in 66% (113/171) of the canine isolates, followed by type IV at 13% (22/171). Interestingly, 11% (18/171) of the MRSC were SCCmec type-V and all the MRSA isolates were type II. While comparing diagnostic methods, the sensitivity and specificity of oxacillin for the detection of MR strains was 83% and 93%, respectively. However, using FOX, the sensitivity was 20% and the susceptibility was 100%. By the means of PCR, the genes that encode PVL were detected in 4% of the canine MRSA, but were not detected in S. pseudintermedius or S. schleiferi subsp coagulans. In contrast, the SIET and LUK-I genes were detected in the three staphylococci, S. aureus, S. pseudintermedius and S. schleiferi subsp coagulans. Using sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), a different pattern of PBPs was found in S. pseudintermedius (5 proteins) and S. schleiferi subspecies coagulans (3 - 4 proteins), when compared with S. aureus (4 – 5 proteins). A competitive binding assay demonstrated a decreased affinity of cefoxitin towards the PBPs of MRSP as compared to MRSA. From the results of these summarized studies, it is clear that more investigation directed towards understanding the different mechanisms of resistance against antimicrobial drugs by staphylococci from animal origin are required to address the epidemiology of Staphylococcus sp in veterinary medicine.