The molecular basis of glycopeptide Resistence in two Clinical Isolates: Bacillus Lentus RSA1208 and Paenibacillus Thiaminolyticus RSA 1221

9902930F Masters Disssertation Faculty of Health SciencesThe molecular mechanisms of glycopeptide resistance in two Gram-positive clinical isolates, Bacillus lentus RSA1208 and Paenibacillus thiaminolyticus RSA1221 were investigated. The glycopeptide resistance genotypes were determined by PCR. If van genes were detected, recombinant DNA techniques and sequencing were used to determine the gene sequence. The location of the resistance determinant was investigated using Southern hybridization techniques. To determine the 5’ and 3’ ends flanking the resistance operon, sub-genomic libraries were constructed. Transmission electron microscopy was used to assess possible structural changes of the B. lentus RSA1208 cell wall. B. lentus RSA1208 exhibits inducible, high-level resistance to both glycopeptides, but does not possess any known van resistance genes. Electron micrographs showed a visible increase in cell wall thickness in B. lentus RSA1208 grown in vancomycin compared to the isolate grown in vancomycin-free media. However, it remains to be confirmed as to whether this resistance is solely responsible for the high-level resistance phenotype. P. thiaminolyticus RSA1221 exhibits constitutive, high-level resistance to vancomycin only. It was found to possess a chromosomally-borne, vanA gene cassette. The vanA gene showed the highest amino acid identity to the vanA-like D-ala: D-lac gene found in P. thiaminolyticus PT-2B1 and Enterococcus faecium BM4147. All five genes of the vanA gene cluster (vanR, vanS, vanH, vanX, vanY) were amplified and sequenced. No vanZ gene was detected. The vanA operon in P. thiaminolyticus RSA1221 was found not to be associated with any known mobile elements. The observed constitutive expression of resistance maybe due to a two amino acid insertion in the VanSBpt1221 protein.

Treatment of a lower urinary tract infection in a cat caused by a multi-drug methicillin-resistant Staphylococcus pseudintermedius and Enterococcus faecalis

Staphylococci and enterococci are common causes of urinary tract infections in cats. However, both species are rarely implicated together as causes of lower urinary tract infections associated with urethral obstruction. This report describes the first case of a multi-drug methicillin-resistant Staphylococcus pseudintermedius belonging to spa type t06 and Enterococcus faecalis urinary infection in a cat with pre-existing and recurrent urethral obstruction. Both species were isolated at >105CFU/ml from a cystocentesis urine specimen. Clinical and ultrasound features, results from urinalysis, urine culture, molecular typing and susceptibility testing by minimal inhibitory concentrations determination are described. Oral treatment with nitrofurantoin, the only antimicrobial agent that constituted a viable therapeutic option, had a positive outcome

Systematic Review on Global Epidemiology of Methicillin-Resistant Staphylococcus pseudintermedius: Inference of Population Structure from Multilocus Sequence Typing Data

Background and rationale: Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of infections in dogs, also posing a zoonotic risk to humans. This systematic review aimed to determine the global epidemiology of MRSP and provide new insights into the population structure of this important veterinary pathogen.Methodology: Web of Science was searched systematically for articles reporting data on multilocus sequence typing (MLST) of S. pseudintermedius isolates from dogs or other animal or human patients and carriers. Data from the eligible studies were then integrated with data from the MLST database for this species. Analysis of MLST data was performed with eBURST and ClonalFrame, and the proportion of MRSP isolates resistant to selected antimicrobial drugs was determined for the most predominant clonal complexes.Results: Fifty-eight studies published over the last 10 years were included in the review. MRSP represented 76% of the 1428 isolates characterized by the current MLST scheme. The population of S. pseudintermedius was highly diverse and included five major MRSP clonal complexes (CCs). CC71, previously described as the epidemic European clone, is now widespread worldwide. In Europe, CC258, which is more frequently susceptible to enrofloxacin and aminoglycosides, and more frequently resistant to sulphonamides/trimethoprim than CC71, is increasingly reported in various countries. CC68, previously described as the epidemic North American clone, is frequently reported in this region but also in Europe, while CC45 (associated with chloramphenicol resistance) and CC112 are prevalent in Asia. It was estimated that clonal diversification in this species is primarily driven by homologous recombination (r/m=7.52).Conclusion: This study provides evidence that S. pseudintermedius has an epidemic population structure, in which five successful MRSP clones with specific traits regarding antimicrobial resistance, genetic diversity and geographical distribution have emerged upon a weakly clonal background through acquisition of SCCmec and other mobile genetic elements