The Gram-positive bacterium Staphylococcus intermedius is regarded as the major cause of canine pyoderma, a common skin infection of dogs. However, despite its clinical importance, the population genetic structure of S. intermedius is poorly understood. The current study examined the population genetic structure of S. intermedius using a multilocus DNA sequencing approach. A collection of 99 isolates phenotypically identified as S. intermedius and originating from a broad array of animal hosts in several different countries was investigated. Phylogenetic analysis indicated that the isolates belonged to three distinct species including S. intermedius, staphylococcus pseudintermedius, and Staphylococcus delphini, together referred to as the S. intermedius group (SIG). Importantly, it was discovered that all canine isolates investigated belonged to the S. pseudintermedius phylotype and it was concluded that S. pseudintermedius, not S. intermedius, is the common cause of canine pyoderma. Further, it was revealed that S. delphini is more clinically important than was previously thought. The allelic variation of agrD, which encodes the autoinducing peptide (AIP) of the agr quorum sensing system in staphylococci, was determined for all isolates. Four AIP variants were identified, including three which were present in all three species, suggesting that a common quorum sensing capacity has been conserved despite species differentiation in very different niches. Considerable clonal diversity was revealed within the S. pseudintermedius species, including several methicillin-resistant clones which have evolved by recent acquisition of the mecA gene. Using the sequence diversity identified, a simple diagnostic test was developed based on a PCR-RFLP approach to discriminate S. pseudintermedius from S. intermedius and S. delphini. Having established that S. pseudintermedius is the common canine pyoderma pathogen, this study aimed to investugate key host-pathogen interactions involved in colonisation of its canine host. Bioinformatic analysis of the whole genome sequence of a clinical isolate of S. pseudintermedius (strain ED99) revealed 17 genes encoding predicted LPXTG-containing cell wall-anchored (CWA) surface proteins. A diverse collection of S. pseudintermedius isolates and closely related staphylococcal species was screened for the presence of the genes encodng the novel CWA proteins. The majority of genes were widely distributed among the isolates examined, with nine genes being exclusive to S. pseudintermedius and eight being also present in other members of the SIG. In Gram-positive bacteria, a family of CWA proteins called microbial surgace components recognising adhesive matrix molecules (MSCRAMMs)mediates bacterial adherence to extracellular matrix proteins of the host. Three of the 17 predicted novel CWA proteins, designated SpsD, SpsL and SpsO, were selected for further characterisation of their role in host-pathogen interactions and were cloned and expressed on the surface of the surrogate host Lactococcus lactis. Solid phase adherence assays employing host extracellular matrix proteins and canine corneocytes were performed to identify host extracellular matrix proteins and canine corneocytes were performed to identify host receptors for the putative MSCRAMMs. L. lactis expressing SpsD demonstrated binding to fibronectin, fibrinogen and cytokeratin 10, SpsL mediated binding of L. lactis to fibronectin and canine fibrrinogen, and SpsD and SpsO both mediated L. lactis adherence to canine corneocytes. Additionally, a cell culture assay using a commercially available canine epidermal cell line was developed and the adherence of S. pseudintermedius ED99 and the L. lactis constructs to the cell line was tested. S. pseudintermedius ED99, but none of the MSCRAMM-expressing L. lactis strains, adhered to the canine epidermal cells in vitro, suggesting that receptors for S. pseudintermedius adherence which are present in ex vivo corneocytes are not present in undifferentiated canine epidermal cell line preparations. Take together, the present study provides broad new insights into the classification and evolution of the SIG, and the molecular interaction of S. pseudintermedius with its canine host.

Successful treatment of canine pyoderma has become increasingly difficult due to the development of antimicrobial resistance and recurrence of infection. The development and spread of antimicrobial resistance has major implications because treatment failures have been associated with increased mortality, morbidity and costs related to disease. All canine skin samples submitted to Vetdiagnostix Veterinary Pathology Services for microbiological culture and sensitivity between January 2007 and June 2010, from which Staphylococcus intermedius was isolated, were selected for this investigation. A total of 319 samples from dogs of various ages, sexes and breeds from various locations throughout South Africa were obtained. In addition to the antimicrobial susceptibility data, data relating to dog signalment (age, gender, breed), case history and any other aspects of the history provided e.g. diagnosis, sample type and geographical location of the patient were collected. The number of skin samples yielding Staphylococcus intermedius was high in dogs up to the age of 6 years and, then decreased with only a few cases in dogs aged 11 years or older. The distribution of samples collected in 2010 was unusual in that there were two peaks, one in dogs two years old or less and one in 6 to 9-year-old dogs. With the exception of skin samples taken in 2008, a high percentage of dogs were under the age of one year old. 2008 had a lower percentage of affected dogs less than one year of age compared to the other years. Staphylococcal pyoderma is more common in dogs below the age of five years. Dogs above this age are less likely to contract this condition. The genders tended to be equally distributed throughout the years, with almost equal proportions of affected males and females present. The Bull Terrier types and Shepherd types were grouped separately as they were over-represented and are known to be prone to pyoderma. Large short haired dogs were consistently worse affected throughout the years sampled, followed by the Bull Terrier types. In 2010, small short haired breeds were worse affected compared to the numbers between 2007 and 2009. The monthly distribution, with the exception of 2009, tended to be consistent throughout the years. The unusual temporal distribution in 2009 could be associated with a general distribution in the number of samples compared to the other years sampled. Samples tended to be mainly from practices located in the KwaZulu-Natal province of South Africa. This is most likely due to the fact that the laboratory is located in this province, with a courier network that arranges collection from these practices. Practices in other regions send their samples to the laboratory via private courier companies and the South African Post Office. Antimicrobial resistance of S. intermedius was greatest to ampicillin followed by tetracycline and then potentiated sulphonamides. The results also showed that, in general, antimicrobial resistance was low. Very few methicillin resistant isolates were detected. Temporal trends were not noted, with the exception of ampicillin where isolates became more susceptible and potentiated sulphonamides (co-trimoxazole) where isolates were becoming more resistant. Staphylococcus intermedius is significantly less resistant to erythromycin, clindamycin, cephalexin, oxacillin, amoxicillin-clavulanic acid, enrofloxacin, marbofloxacin and gentamicin, with most strains being susceptible to these drugs. Resistance to penicillin and tetracycline is frequently found in Staphylococcus intermedius and is on the increase. Resistance to most other antimicrobials, particularly newer generation antimicrobial agents such as the fluoroquinolones, is still comparatively low. In general, both the Kirby-Bauer and broth dilution MIC tests yielded similar results for the antimicrobial agents tested. The main difference between the two tests was evident in the over-estimation of resistance by the Kirby-Bauer test in the cases of ampicillin, co-trimoxazole, penicillin and doxycycline. This could be related to the instability of these particular drugs in vitro. Inoculum densities may also have played a role, with denser inocula producing smaller zone sizes for the drugs tested. Using the MIC method, all of the isolates tested were found to be completely sensitive to ticarcillin, oxacillin, amoxicillinclavulanic acid, imipenem, ceftiofur, chloramphenicol, doxycycline, gentamicin, amikacin and co-trimoxazole. Of the isolates tested using the MIC method, between 2-40% showed some level of resistance to the following antimicrobials: erythromycin, penicillin, ampicillin, enrofloxacin, clindamycin and marbofloxacin. The highest level of resistance observed was shown to erythromycin. The increase in resistance to the lincosamides, lincomycin, clindamycin and erythromycin may be attributed to the increased use of these drugs in the last decade. Knowledge of trends in bacterial resistance is important for veterinarians when determining treatment for canine skin infections. The information obtained from the analysis of the antimicrobial susceptibility profiles of Staphylococcus intermedius isolated from canine pyoderma cases will provide veterinarians with valuable information on choosing the most appropriate drug to treat S. intermedius skin infections as well as re-enforcing the need for the prudent use of antimicrobial drugs in companion animals. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Veterinary Tropical Diseases / unrestricted