Mupirocin is a bacteriostatic antibiotic that is used to decolonize people who carry methicillin-resistant staphylococci, primarily methicillin-resistant Staphylococcus aureus (MRSA). Mupirocin reversibly binds to bacterial isoleucyl tRNA synthetase to disrupt protein synthesis. Resistance to mupirocin is due either to a point mutation to the ileS gene that encodes the isoleucyl tRNA synthetase, classified as low-level mupirocin resistance; or, bacteria may obtain a plasmid that carries the ileS2 gene encoding an alternate isoleucyl tRNA synthetase, conferring high-level resistance. Mupirocin resistance plasmids contain insertion sequence (IS) 257 repeats, into which the ileS2 gene is inserted. Such plasmids have been characterized by their IS257-ileS2 junctions in both S. aureus and, recently, in Staphylococcus pseudintermedius in a dog from Croatia. The primary goals of this study were to determine the prevalence of mupirocin resistance in isolates of S. pseudintermedius in Texas, to determine whether resistance was due to point mutations in the native ileS or due to carriage of mupirocin resistance plasmids, and to characterize the structure of the mupirocin resistance genes carried on plasmids. In this study, 572 S. pseudintermedius isolates, collected from veterinary patients from across Texas were screened for their susceptibility to low levels of mupirocin. Of these isolates, only one out of 572 (0.17%) tested positive for mupirocin resistance and was found by polymerase chain reaction (PCR), using previously published primers mupA and mupB, to have a 458 bp fragment and, with primers M1 and M2 to have a 237 bp fragment, indicating the presence of the high-level mupirocin resistance gene, ileS2. The arrangement of the IS257-ileS2 junctions was then analyzed by PCR and the products, bands at 1816 bp for primers ileS2-5’ and IS257R and at 1127 bp for primers ileS2-3’ and IS257F, which are consistent with the amplification pattern for an S2 plasmid, were cloned into a plasmid, pT7Blue, and sequenced for comparison to published sequences in GenBank. BLAST analyses in NCBI, comparing the isolate to recently published sequences for mupirocin-resistant S. pseudintermedius isolated from a dog with pyoderma in Croatia, indicate a 100% similarity to the upstream junction, JX186508, and 97% to the downstream junction, JX186509.

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.

Made available in DSpace on 2015-03-26T13:47:04Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1115803 bytes, checksum: ef0f3b1a323ecbe3d163791baa7accb5 (MD5) Previous issue date: 2012-02-15 / Pyoderma is one of the most common skin disorders in dogs and Staphylococcus pseudintermedius is the bacterium that is most often isolated from lesions. During recent years a major concern in veterinary dermatology has been the increasing number of methicillin resistant Staphylococcus spp. isolates, that are resistant to several antimicrobials used in dermatological clinic. Infections caused by these resistant bacteria are a major cause of morbidity in pets and can be a source of transmission to humans. The objectives of this work are to review the literature on Staphylococcus pseudintermedius and the resistance in these bacteria. It is also to isolate and identify through biochemical and molecular biology tests, bacteria that causes canine pyoderma, determining their resistance profile to antimicrobials commonly used for this condition by antimicrobial susceptibility test and by the presence of the resistance gene, mecA. Seventy five bacterial isolates were obtained from the 25 animals selected to this study. A total of 97.3% of the isolates submitted to conventional phenotypic tests and API Staph Kit and 96% of those submitted to PCR were identified as Staphylococcus pseudintermedius. Of the 72 isolates submitted to PCR for the identification of the mecA gene, only four did not have this resistance gene. This study alerts to the high number of resistant Staphylococcus pseudintermedius in Brazil, and to the need of performing antimicrobial susceptibility tests to determine the best therapeutic approach, preventing the emergence of multi-resistant bacteria. / As piodermites são uma das afecções dermatológicas mais comumente encontradas nos cães sendo que o Staphylococcus pseudintermedius é a bactéria mais frequentemente isolada das lesões. Durante os últimos anos uma das maiores preocupações na dermatologia veterinária tem sido o crescente número de isolados de Staphylococcus spp. resistentes a meticilina ou seja, resistentes a vários antimicrobianos utilizados na clínica dermatológica. As infecções por estas bactérias resistentes são uma causa importante de morbidade em animais de companhia e podem ser fonte de transmissão para humanos. Objetivou-se com este trabalho rever a literatura acerca de Staphylococcus pseudintermedius e sobre a resistência nestas bactérias. Também foram objetivos isolar e identificar através de testes bioquímicos e de biologia molecular as bactérias causadoras da piodermite canina, determinando o perfil de resistência aos antimicrobianos comumente utilizados para esta afecção através de antibiograma e da presença do gene de resistência, mecA. Dos 25 animais selecionados para o estudo foram obtidos 75 isolados bacterianos. Ao todo, 97,3% dos isolados avaliados pelos testes fenotípicos convencionais e pelo Kit API Staph e 96% dos avaliados pelo PCR Foram identificados como Staphylococcus pseudintermedius. Dos 72 isolados submetidos ao PCR para a identificação do gene mecA apenas quatro não apresentaram o gene de resistência. O presente estudo alerta para o alto número de Staphylococcus pseudintermedius resistentes no Brasil e para a necessidade de se realizar antibiogramas a fim de determinar a melhor abordagem terapêutica, evitando o aparecimento de bactérias multi resistentes.

Staphylococcus pseudintermedius is the major cause of the common canine skin disease, pyoderma, and is a zoonotic pathogen of humans. Multidrug resistant strains of S. pseudintermedius have emerged and are spreading globally leading to decreased therapeutic success. The development of novel therapeutics is hindered by the lack of understanding of critical host-pathogen interactions mediating S. pseudintermedius colonization and pathogenesis. For the major human pathogen Staphylococcus aureus, interactions with host fibrinogen play a fundamental role in pathogenesis. The aim of the current study was to genetically and functionally characterise 2 cell wall-associated proteins of S. pseudintermedius, SpsD and SpsL, which mediate binding to multiple host extracellular matrix proteins including fibrinogen and fibronectin. DNA sequencing of the A- (ligand binding) domains of spsD and spsL genes for 37 phylogenetically diverse isolates revealed a highly conserved sequence for SpsL (97.1% derived amino acid identity), in contrast to more extensive variation for SpsD (76.7% derived amino acid identity). Further, recombination events were identified to have contributed to the evolution of spsD diversity. Functional analysis with gene deletion mutants of S. pseudintermedius strain ED99, constructed in the current study, demonstrated that SpsL is a major cell wall-associated fibrinogen-binding protein with enhanced affinity for canine fibrinogen. Using recombinant chains of fibrinogen it was determined that SpsL binds to the α-chain of fibrinogen similar to clumping factor B (ClfB) of S. aureus. However, ELISA and surface plasmon resonance analyses of recombinant truncated derivatives of SpsL indicated that the predicted ligand-binding N2N3 subdomains of the A-domain of SpsL are not sufficient for high-affinity fibrinogen-binding suggesting that either additional domains or post-translational modifications are required for fibrinogen-binding. Furthermore, development of a murine skin infection model allowed an investigation of the contribution of SpsD and SpsL to pathogenesis revealing a role for SpsL in focal abscess pathology. Overall these studies have provided new insights into the diversity, function and therapeutic potential of S. pseudintermedius fibrinogen-binding proteins.

Darbo tikslas: nustatyti koaguliazei teigiamų stafilokokų paplitimą tarp gyvūnų augintinių. Darbo uždaviniai: 1. išskirti koaguliazei teigiamus stafilokokus iš gyvūnų augintinių nosies ertmės; 2. išskirti koaguliazei teigiamus stafilokokus iš gyvūnų augintinių tiesiosios žarnos; 3. identifikuoti išskirtas stafilokokų padermes; 4. įvertinti įvairių veiksnių įtaką stafilokokų paplitimui; 5. nustatyti išskirtų stafilokokų atsparumą antimikrobinėms medžiagoms. Darbo apimtis – 50 puslapių. Šiame darbe yra 6 lentelės bei 14 paveikslų. Magistro darbą sudaro 4 dalys. Pirmojoje dalyje apžvelgiami literatūros šaltiniai susiję su analizuojama tema, išskiriant koaguliazei teigiamų stafilokokų virulentiškumo veiksnius, atsparumą antimikrobinėms medžiagoms, sukeliamas ligas ir šių ligų gydymą. Aptariamas Staphylococcus aureus bei Staphylococcus pseudintermedius paplitimas ir paplitimą įtakojantys veiksniai. Antrojoje dalyje nurodyti tyrimo metodai, kuriais remiantis gauti duomenys tyrimų analizei. Trečiojoje dalyje analizuojami gauti tyrimo rezultatai pagal iškeltus uždavinius. Rezultatai pateikiami atsižvelgiant į statistinių duomenų patikimumą. Ketvirtoji dalis skirta literatūros apžvalgos ir tyrimo rezultatų skirtumų ir panašumų palyginimui. Tyrimo metu iš gyvūnų augintinių nosies ertmės ir tiesiosios žarnos išskirti Staphylococcus aureus bei Staphylococcus pseudintermedius. Nustatyta, kad koaguliazei teigiamų stafilokokų paplitimas gyvūnų augintinių tarpe priklauso nuo gyvūnų... [toliau žr. visą tekstą] / The The goal of the study: to determine prevalence of coagulase positive staphylococci in companion animals. The aim of the study: 1. to isolate coagulase positive staphylococci in nasal cavity of companion animals; 2. to isolate coagulase positive staphylococci in rectum of companion animals; 3. to identificate the isolated strains of staphylococci; 4. to evaluate risk factors for prevalence of staphylococci; 5. to determine antibiotic resistance in isolated staphylococci. The master study consists of 50 pages. It includes 6 tables and 14 pictures. The master study consist of 4 major chapters. The first chapter is dedicated to review of literature that is related with analized topic. This part includes coagulase positive staphylococci virulence factors, antibiotic resistance, diseases caused by staphylococci and treatment use. Furthermore chapter contains review of the prevalence and risk factors influenced the prevalence of Staphylococcus aureus and Staphylococcus pseudintermedius. The second chapter introduce with materials and methods, that were used in the research at this master study. In the third chapter the results of the research are presented. The results are presented according to the statistical reliability. The fourth chapter is the resemblance and similarity comparision of the literature review and master study research. In this master study Staphylococcus aureus and Staphylococcus pseudintermedius were isolated from nasal cavity and rectum of companion... [to full text]

Coagulase positive staphylococci, Staphylococcus aureus and Staphylococcus pseudintermedius, are important causes of infection in human beings and dogs respectively. The rapid increase in the incidence of methicillin resistant S. aureus (MRSA) in people and its emergence in dogs has raised the profile of this organism in the veterinary community. Similarly, human S. pseudintermedius infections have also been recognized as the awareness of bidirectional human-dog transmission increases. Antimicrobial resistance has been complicating the treatment of S. aureus infections since the first penicillin resistance was observed in the 1940s. Methicillin resistance (resistance to the majority of â-lactams), is particularly troublesome as the â-lactams are a safe and effective class of antimicrobials for treating susceptible staphylococcal infections in both human beings and dogs. Additionally, resistance to other antimicrobial classes such as the macrolides, tetracyclines, sulfonamides and chloramphenicol, further complicates the treatment of staphylococcal infections. Particularly in small animal private practice, infections are often treated empirically, requiring knowledge of locally prevalent susceptibility patterns. The emergence of resistance to commonly used drugs necessitates surveillance to monitor the dissemination of resistance, and to guide antimicrobial therapy. In the last decade there have been many studies attempting to address gaps in our knowledge of the ecology of S. aureus and S. pseudintermedius in dogs. In particular, the prevalence of colonization with methicillin resistant staphylococci has been documented in different dog populations. However, failing to sample all relevant sites of colonization, may have decreased the sensitivity of these studies. The sites where coagulase positive staphylococci colonize dogs have not been systematically evaluated. The clinical and infection control implications of S. aureus infections, or colonization in the case of MRSA, requires timely laboratory identification. The tube coagulase test is arguably the most important tool used for identifying of staphylococcal species. Studies dating from the 1970s and 1980s suggested that the use of rabbit plasma, which is the current standard, may not be the ideal media for all situations and that different plasmas may need to be considered in different diagnostic situations. In this thesis, the ecology of coagulase positive staphylococci in dogs was studied from start to finish including sample collection, bacterial identification, antimicrobial susceptibility testing and molecular epidemiological investigations. This thesis will serve as a template to be used for follow up studies or by investigators setting up a surveillance program in their region. We found that multiple sites of colonization (nares, pharynx and rectum), are involved in both S. aureus and S. pseudintermedius carriage in dogs. Single site colonized dogs were identified, suggesting that maximal screening sensitivity requires sampling multiple body sites. When canine and rabbit plasma were compared, the time until clot formation was found to be significantly shorter with canine plasma. Although, the availability of canine plasma may limit its use in the diagnostic laboratory, investigators should be aware that rabbit plasma may not be ideal for all applications of the tube coagulase test. Antimicrobial susceptibility testing of canine S. aureus and S. pseudintermedius and human S. aureus isolates was done. Consistent with previous reports from Saskatoon, the S. pseudintermedius isolates were found to be overwhelmingly susceptible: pan-susceptibility was the most common phenotype identified. Antimicrobial resistance was more common among S. aureus than S. pseudintermedius including resistance to drugs which all S. pseudintermedius were susceptible to. No resistance to vancomycin, linezolid, daptomycin or quinupristin/dalfopristin was found. All isolates remained susceptible to at least one of tetracycline, clindamycin, chloramphenicol or trimethoprim/sulfamethoxazole which are often used for treating infections caused by multidrug resistant staphylococci. Finally, DNA fingerprinting revealed that the canine and human S. aureus isolates tested did not belong to mutually exclusive populations. Using AFLP, IS-typing and spa typing, many human and canine isolates were indistinguishable suggesting a common population, supporting the hypothesis that interspecies transmission occurs. The complex and under-characterized ecology of S. aureus and S. pseudintermedius requires more study so that risk factors for infection can be defined and effective infection control measures implemented. Because multiple species are involved, collaboration between veterinarians and human health professionals is imperative, and will no doubt yield the most success in our efforts to understand these potential pathogens.

Coagulase positive staphylococci, Staphylococcus aureus and Staphylococcus pseudintermedius, are important causes of infection in human beings and dogs respectively. The rapid increase in the incidence of methicillin resistant S. aureus (MRSA) in people and its emergence in dogs has raised the profile of this organism in the veterinary community. Similarly, human S. pseudintermedius infections have also been recognized as the awareness of bidirectional human-dog transmission increases. Antimicrobial resistance has been complicating the treatment of S. aureus infections since the first penicillin resistance was observed in the 1940s. Methicillin resistance (resistance to the majority of â-lactams), is particularly troublesome as the â-lactams are a safe and effective class of antimicrobials for treating susceptible staphylococcal infections in both human beings and dogs. Additionally, resistance to other antimicrobial classes such as the macrolides, tetracyclines, sulfonamides and chloramphenicol, further complicates the treatment of staphylococcal infections. Particularly in small animal private practice, infections are often treated empirically, requiring knowledge of locally prevalent susceptibility patterns. The emergence of resistance to commonly used drugs necessitates surveillance to monitor the dissemination of resistance, and to guide antimicrobial therapy. In the last decade there have been many studies attempting to address gaps in our knowledge of the ecology of S. aureus and S. pseudintermedius in dogs. In particular, the prevalence of colonization with methicillin resistant staphylococci has been documented in different dog populations. However, failing to sample all relevant sites of colonization, may have decreased the sensitivity of these studies. The sites where coagulase positive staphylococci colonize dogs have not been systematically evaluated. The clinical and infection control implications of S. aureus infections, or colonization in the case of MRSA, requires timely laboratory identification. The tube coagulase test is arguably the most important tool used for identifying of staphylococcal species. Studies dating from the 1970s and 1980s suggested that the use of rabbit plasma, which is the current standard, may not be the ideal media for all situations and that different plasmas may need to be considered in different diagnostic situations. In this thesis, the ecology of coagulase positive staphylococci in dogs was studied from start to finish including sample collection, bacterial identification, antimicrobial susceptibility testing and molecular epidemiological investigations. This thesis will serve as a template to be used for follow up studies or by investigators setting up a surveillance program in their region. We found that multiple sites of colonization (nares, pharynx and rectum), are involved in both S. aureus and S. pseudintermedius carriage in dogs. Single site colonized dogs were identified, suggesting that maximal screening sensitivity requires sampling multiple body sites. When canine and rabbit plasma were compared, the time until clot formation was found to be significantly shorter with canine plasma. Although, the availability of canine plasma may limit its use in the diagnostic laboratory, investigators should be aware that rabbit plasma may not be ideal for all applications of the tube coagulase test. Antimicrobial susceptibility testing of canine S. aureus and S. pseudintermedius and human S. aureus isolates was done. Consistent with previous reports from Saskatoon, the S. pseudintermedius isolates were found to be overwhelmingly susceptible: pan-susceptibility was the most common phenotype identified. Antimicrobial resistance was more common among S. aureus than S. pseudintermedius including resistance to drugs which all S. pseudintermedius were susceptible to. No resistance to vancomycin, linezolid, daptomycin or quinupristin/dalfopristin was found. All isolates remained susceptible to at least one of tetracycline, clindamycin, chloramphenicol or trimethoprim/sulfamethoxazole which are often used for treating infections caused by multidrug resistant staphylococci. Finally, DNA fingerprinting revealed that the canine and human S. aureus isolates tested did not belong to mutually exclusive populations. Using AFLP, IS-typing and spa typing, many human and canine isolates were indistinguishable suggesting a common population, supporting the hypothesis that interspecies transmission occurs. The complex and under-characterized ecology of S. aureus and S. pseudintermedius requires more study so that risk factors for infection can be defined and effective infection control measures implemented. Because multiple species are involved, collaboration between veterinarians and human health professionals is imperative, and will no doubt yield the most success in our efforts to understand these potential pathogens.

This thesis is an investigation of methods to remediate methicillin-resistant Staphylococcus pseudintermedius (MRSP) biofilms through conventional and microfluidic-based in vitro assays. MRSP biofilm related infections are a major concern for veterinary clinicians as they may complicate remediation by the immune system or antimicrobials. Novel antimicrobials that have been found to reduce biofilm growth in other staphylococci were assessed in both mono- and combination therapy against MRSP biofilm. Quantitative assay results (p < 0.05) suggest fosfomycin alone and in combination with clarithromycin can significantly reduce biofilm formation. Morphological examination using scanning electron microscopy and atomic force microscopy further demonstrated the effectiveness of fosfomycin alone on biofilm formation on orthopaedic screws and mica sheets. Fabricated microfluidic assays were utilized to assess multiple concentrations of antimicrobial therapy against pre-formed biofilm under physiologically relevant conditions in a quick and repeatable manner. Results demonstrated the usefulness of microfluidic platforms in determining minimum biofilm eradication concentrations.

No description available.

No description available.