Dissertations / Theses on the topic 'Staphylococcus pseudintermediu'

Staphylococcus pseudintermedius (S. pseudintermedius) is one of the most important pathogens in small animal veterinary medicine. In the dog, it is a commensal bacterium able to cause severe diseases (mainly dermatological), which rapidly evolve to persistent and chronic infections. This pathogen is also considered a public health problem due to the rapid evolution and spread of antibiotic-resistant clones, in particular, the worldwide dissemination of Multi-Drug Resistant (MDR) strains and methicillin-resistant S. pseudintermedius (MRSP). Nowadays, its zoonotic potential is under constant investigations. The first chapter of the thesis centers the attention on the typing techniques used to study a population of S. pseudintermedius isolated from the skin of dogs and raw milk of Holstein-Fresian dairy cattle. The aims of this study were the investigation of the genetic relatedness of different S. pseudintermedius strains using three molecular typing techniques; the Ribosomal Spacers Amplification PCR (RSA-PCR), the Random Amplification of Polymorphic DNA PCR (RAPD-PCR) and the Restriction Fragment Length Polymorphism PCR (RAPD-PCR). Moreover, the dissemination of species-specific genes coding for exfoliative toxins (siet, expA, and expB), enterotoxin (seCcanine) and a bicomponent leukocidin (lukS-F) was studied to understand the virulence of the strains. The study was carried out on a final population of 40 S. pseudintermedius strains isolated from 42 clinical cases of deep canine pyoderma and from 5 samples of raw milk from Holstein-Fresian dairy cattle with intramammary infection. After the microbiological examination and phenotypic typing, 47 S. pseudintermedius strains were collected and genetically analyzed. RSA-PCR revealed the presence of a unique biotype. The dendrogram obtained from RAPD-PCR disclosed two important results: i) the staphylococcal strains isolated from dogs and cows are genetically similar underlying the ability of this pathogen to colonize also dairy cattle; ii) the presence of a single cluster grouping all the S. pseudintermedius strains. Based on the presence of a specific restriction site in the phosphotransacetylase (pta) gene, RFLP-PCR confirmed the presence of 40 S. pseudintermedius and 7 non-S. pseudintermedius strains. The sequencing unveiled the effective presence of one strain of S. aureus and one of E. faecalis, 5 strains of S. schleiferi and 40 of S. pseudintermedius. All the S. pseudintermedius isolates were positive for siet gene, while expA and expB were amplified by, respectively, 10% and 17,5%. Specific enterotoxin seCcanine and leukotoxin luk-S-F were found with a prevalence of 35% and 92,5%, respectively. Therefore, the combined use of molecular typing techniques and virulence factors screening allows to better characterize S. pseudintermedius and to understand its zoonotic potential. The second chapter points out the attention to the problem of antibiotic resistance. Frequently, in Veterinary and Human medicine too, wide-spectrum antibiotics are prescribed to face with bacteria-based diseases without the microbiological examination and relative antibiotic-resistance screening. This could lead to the selection and spread of antibiotic-resistant strains. The aim of this work was to study the antibiotic-resistance profile of a large population of S. pseudintermedius strains collected in two Italian veterinary teaching hospitals, the first in Milan (Università degli Studi di Milano) and the second in Naples (Università degli Studi di Napoli “Federico II”) One-hundred sixteen and one-hundred twenty-six strains were collected in Milan and Naples, respectively. A panel of 10 different antibiotic molecules was tested with the Kirby-Bauer assay, qualitative PCR was used to describe the dissemination of mecA and tetM/K genes. The results showed a high rate of resistance for amoxicillin+clavulanate, clindamycin, tobramycin and tetracycline also confirmed by statistical significance. The prevalence of methicillin-resistant strains (MRSP) was 30% in Milan and18% in Naples also confirmed by the amplification of mecA gene. The cohort of methicillin-sensible strains in Naples (N= 103) was significantly more resistant compared to that from Milan (N= 81). In relation to the MDR population, all the MRSP in Milan and 91% in Naples are considered resistant to three or more pharmaceutical categories. These results totally agree with the recent report about the “Surveillance of antimicrobial resistance in Europe, 2017” and could be a starting point to set out a clearer use and prescription of antibiotics. The third chapter describes quorum-sensing and biofilm. It is known that bacteria are able to communicate via small peptides (Gram-positive) or lipids (Gram-negative) using a system called quorum-sensing. This complex behavior acts in response to external stimuli (e.g. cell density) and controls gene expression. Biofilm is one of the products of this communication, defined as a complex ecosystem of bacteria (sometimes also fungi and algae) enclosed in a self-produced matrix and adherent to a biotic or abiotic surface. This structure is frequently resistant to commonly used antibiotics and causes severe and more complex re-infections. The aim of the work was to describe the biofilm-forming ability of MDR and non-MDR S. pseudintermedius strains investigating the dissemination of virulence factors and antibiotic-resistance genes. A panel of 22 antibiotics was used to screen the antibiotic-resistance profiles of 73 S. pseudintermedius strains isolated from deep canine pyoderma. The biofilm-forming ability was investigated using a microtiter assay. The results clearly highlight a correlation between antibiotic-resistance and the ability to produce biofilm. All the MDR strains (N=42) are biofilm producers while 27 out of 31 non-MDR strains were able to produce biofilm. The last chapter of the thesis focused on an alternative strategy for the treatment of bacterial infections caused by Gram-positive and Gram-negative bacteria. For centuries, silver was used for its antimicrobial properties. Recently, the nanobiotechnology research area aimed to synthesize small particles (less than 100 nm) of silver metal to use as an alternative to traditional antibiotic-based therapies. The aim of this work was to synthesize silver nanoparticles using i) a Curcuma longa infusion and ii) the supernatant of E. coli. Furthermore, the antibacterial properties of synthesized nanoparticles were determined against 10 S. pseudintermedius strains and 10 Pseudomonas aeruginosa isolates by Kirby-Bauer disk diffusion assay and via Minimum Inhibitory Concentration (MIC). The anti-biofilm capacities were also investigated using an in vitro model of biofilm growth. Results showed that silver nanoparticles exert strong antibacterial activity compared to antibiotic alone and these properties are magnified when antibiotic was adjuvated with silver. Nanoparticles are also able to disrupt mature biofilm by directly linking to the cell wall of bacteria causing bacterial lysis. The use of silver nanoparticles obtained with green technology (e.g plant or organism derived molecules) is an eco-friendly alternative to classical antibiotic-based therapies and can be used to fight against both Gram-positive and Gram-negative infections.

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]

Tese especialmente elaborada para a obtenção do grau de Doutor em Ciências Veterinárias, especialidade de Clínica
Staphylococci are a group of bacteria with clinical, agricultural, and economic importance because of their wide range of virulence factors and ability to become resistant to antimicrobials. This thesis has pursued three main objectives: I. Determine the frequency of methicillin-resistant S. aureus (MRSA) strains in several animal species, identify the characteristics of strains present in animals and comparison with human strains MRSA nasal screening was performed in 71 horses and 307 calves, and the observed frequencies were 3% and 2%, respectively. Seventy-four MRSA isolated from 2001 to 2014 were characterized: fourteen spa types, three SCCmec types and three clonal complexes (CC) 5, CC22 and CC398, were found. Most isolates were multidrug-resistant. Fourteen MRSA CC398 strains had qac genes (13 qacG and 1 qacJ), while 4 isolates (three CC5 and one CC22) had insertions in the norA promoter gene. MRSA linages from pets (CC5 and CC22) harboured specific sets of virulence genes and a lower number of resistance genes than CC398 from livestock-animals. II. Reveal antimicrobial/biocide susceptibility patterns/trends and resistance genes in methicillin-resistant staphylococci (MRS) Several antimicrobial resistance patterns and genes were found in MRS from horses. Minimum bactericidal concentrations of biocides chlorhexidine acetate, benzalkonium chloride, triclosan and glutaraldehyde were lower than the recommended in-use concentrations for veterinary medicine, although two MRS carried plasmid-borne qacA and sh-fabI or qacB and qacH-like genes. An investigation on the evolution of resistance to 38 antimicrobials, corresponding mechanisms and molecular characteristics of 644 clinical Staphylococcus spp. isolates obtained from companion animals between 1999-2014 revealed resistance to the majority of antimicrobials and the number of mecA-positive strains increased significantly over time. Considering S. pseudintermedius, the methicillin-susceptible (MSSP) were genetically more diverse than methicillin-resistant (MRSP). All MRSP and two MSSP strains were multidrug- resistant, with several antimicrobial resistance genes identified. One MSSP isolate harbored a qacA and another a qacB gene. Three biocide products had high bactericidal activity (Otodine®, Clorexyderm Spot Gel®, Dermocanis Piocure-M®), while Skingel® failed to achieve a five log reduction in the bacterial counting. III. Study of the pathogenesis of S. pseudintermedius in dogs The agr type III predominated in MRSP. Five virulence genes were found in all strains and only spsO gene was significantly associated with MSSP. MSSP produced more biofilm on BHIB and BHIB+1% glucose than MRSP isolates. Several virulence genes encoding surface proteins and toxins were highly expressed in the MRSP strain (compared to MSSP). By whole proteome characterization of S. pseudintermedius through 2DE MALDI-TOF/TOF MS approach we were able to identify 367 unique proteins, of which 39 were surface proteins. By subsequent use of the serological proteome analysis (SERPA) approach we identified 4 antigenic proteins with promising features for vaccine development. These results indicate that MRS were widely disseminated in the studied animal population, the environment and people in contact with these animals. The resistant trends and mechanisms detected in MRS strains are worrying and make animals a reservoir of important MRS clones and genes. Biocides are still a good therapeutic choice, even in the presence of efflux genes. Higher expression of virulence genes may play a role in the rapid and widespread of MRSP clones. Dogs are able to mount an IgG-response against S. pseudintermedius and the proteins identified by the immune system can in the future be used as vaccine candidates.
RESUMO - Estudo da dinâmica de estafilococos meticilina-resistente em animais – um foco no Staphylococcus pseudintermedius em cães - Os estafilococos são um grupo de bactérias com importância clínica, agrícola e económica devido à ampla gama de fatores de virulência e pela sua capacidade de se tornarem resistentes aos antimicrobianos. Esta tese debruçou-se sobre três objetivos principais: I. Determinar a frequência de estirpes S. aureus meticilina-resistente (MRSA) em diversas espécies animais, identificar as características das estirpes presentes em animais e comparar com estirpes humanas Colhemos zaragatoas de 71 cavalos e 307 vitelos para pesquisa de MRSA, e observaramse frequências de 3% e 2%, respetivamente. Foram caracterizadas setenta e quatro estirpes MRSA isoladas entre 2001-2014: catorze tipos de spa, três tipos de SCCmec e três complexos clonais (CC) 5, CC22 e CC398, foram encontrados. A maioria das estirpes (74%) eram multirresistentes. Catorze estirpes de MRSA CC398 tinha genes qac (13 qacG e 1 qacJ), enquanto 4 (três CC5 e um CC22) tinham inserções no gene promotor norA. As linhagens de MRSA de animais de estimação (CC5 e CC22) tinham conjuntos específicos de genes de virulência e um menor número de genes de resistência do que as linhagens associadas aos animais de produção (CC398). II. Revelar padrões/ tendências de suscetibilidade antimicrobiana/biocida e genes de resistência em estafilococos meticilina-resistente (MRS) Foram encontrados vários padrões e genes de resistência antimicrobiana em MRS de cavalos. As concentrações bactericidas mínimas dos biocidas acetato de clorhexidina, cloreto de benzalcónio, triclosan e glutaraldeído foram menores do que as recomendadas em medicina veterinária, embora dois MRS tivessem os genes plasmídicos qacA e sh-fabI ou qacB e um qacH-semelhante. Uma investigação sobre a evolução da resistência a 38 antimicrobianos, mecanismos correspondentes e características moleculares de 644 Staphylococcus spp. clínicos obtidos de animais de companhia entre 1999-2014 revelou resistência à maioria dos antimicrobianos. O número de estirpes mecA-positivo aumentou significativamente ao longo do tempo. Quanto aos S. pseudintermedius, os meticilina-suscetível (MSSP) eram geneticamente mais diversos do que os meticilina-resistente (MRSP). Todos os MRSP e 2 MSSP eram multirresistentes, com vários genes de resistência identificados. Um MSSP tinha um gene qacA e outro um qacB. Três produtos biocidas tinham elevada atividade bactericida (Otodine®, Clorexyderm Spot Gel®, Dermocanis Piocure-M®), enquanto Skingel® não conseguiu atingir uma redução de 5 log na contagem bacteriana. III. Estudo da patogenicidade de S. pseudintermedius em cães O tipo III agr predominou nos MRSP. Cinco genes de virulência foram encontrados em todas as estirpes e só o gene spsO foi significativamente associado com MSSP. MSSP produziu mais biofilme em BHIB e BHIB + 1% glucose que as estirpes de MRSP. Vários genes de virulência que codificam proteínas e toxinas de superfície foram altamente expressos na estirpe MRSP (em comparação com MSSP). Através da caracterização do proteoma total de S. pseudintermedius pela abordagem 2DE MALDI-TOF/TOF MS fomos capazes de identificar 367 proteínas únicas, das quais 39 eram proteínas de superfície. Posteriormente utilizámos a análise do proteoma serológico (SERPA) que identificou quatro proteínas antigénicas com características promissoras para o desenvolvimento de vacinas. Estes resultados indicam que MRS estavam amplamente disseminados na população animal estudada, no ambiente e nas pessoas em contato com esses animais. As tendências de resistência e os mecanismos detetados em estirpes MRS são preocupantes tornando os animais um reservatório de clones MRS e genes. Os biocidas ainda são uma boa opção terapêutica, mesmo na presença de bombas de efluxo. Uma maior expressão de genes de virulência pode desempenhar um papel na rápida expansão de clones de MRSP. Os cães foram capazes de montar uma resposta IgG contra S. pseudintermedius e as proteínas identificadas pelo sistema imunológico podem, no futuro, ser utilizadas como candidatos vacinais.

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.

Šio darbo tikslas - Staphylococcus aureus ir Staphylococcus pseudintermedius išskyrimas iš gyvūnų augintinių, jų savininkų ir antimikrobinių medžiagų atsparumo nustatymas. Darbo uždaviniai: 1. Išskirti stafilokokus iš gyvūnų augintinių. 2. Išskirti stafilokokus iš gyvūnų augintinių savininkų. 3. Identifikuoti koaguliazei teigiamus stafilokokus. 4. Įvertinti įvairių veiksnių įtaka šunų nosies ertmės ir tiesiosios žarnos mikroflorai. 5. Įvertinti įvairių veiksnių įtaka gyvūnų augintinių savininkų nosies ertmės mikroflorai. 6. Nustatyti atsparumą antimikrobinėms medžiagoms. Iš viso paimti 123 mėginiai, iš jų 82 mėginiai paimti iš šunų laikomų namuose ir lauke, ir 41 mėginys iš jų savininkų. Mėginiai mikrobiologiniam tyrimui buvo imami į transportines terpes TRANSWAB® (naudojama pernešimo terpė aerobams ir anaerobams išskirti (Amies, Liofilchem, Italija)). Mikroorganizmų išskirta iš 37 (30,1 proc.) mėginių. Po vieną mikroorganizmų rūšį nustatyta 33 (89,1 proc.) mėginiuose. Po dvi mikroorganizmų rūšis (mišri infekcija), nustatyta 4 (10,9 proc.) mėginiuose. Iš šunų nosies ertmės ir tiesiosios žarnos paimto 41 mėginio mikroorganizmai buvo išskirti iš 27 (59,3 proc.). Staphylococcus pseudintermedius sudarė 32,4 proc. išskirtų mikroorganizmų, Staphylococcus aureus – 12,2 proc., Bacillus spp. – 14,7 proc. Iš šunų savinikų nosies ertmės paimto 41 mėginio mikroorganizmai buvo išskirti iš 16 (55,9 proc.) mėginių. Staphylococcus aureus sudarė 36,5 proc. išskirtų mikroorganizmų... [toliau žr. visą tekstą]
The objective of the research: Staphylococcus aureus and Staphylococcus pseudintermedius isolation from a pet and their owners, and determine the resistance of isolates to antimicrobial agent. Tasks of the research: 1. To isolate staphylococci from pet. 2. To isolate staphylococci from pet owners. 3. To identify coagulase-positive staphylococci. 4. Evaluate the influence of canine nasal and rectal flora. 5. Evaluate the influence of the pet owners nasal cavity flora. 6. To determine the resistance of isolates to antimicrobial agent. 41 samples of dogs and 41 from humans were investigated. Total take 123 samples, including 82 samples were taken from dogs kept in the home and outdoors, and 41 samples of their owners. Samples for microbiological analysis were collected in transport medium TRANSWAB ® (used to transport medium for aerobes and anaerobes release (Amies, Liofilchem, Italy)). Microorganisms were identified in 37 (30.1 percent.) samples. Single type of microorganisms were identified in 33 (89.1 percent) samples. Two types of microorganisms (mixed infection) were identified in 4 (10.9 percent) samples. In dogs, nasal and rectal were taken 41 samples. Microorganisms were identified in 27 (59.3 percent) samples. Staphylococcus pseudintermedius accounted 32.4 percent of identified microorganisms, Staphylococcus aureus – 12.2 percent, Bacillus spp. – 14.7 percent. Dogs owners nasal were taken 41 samples. Microorganisms were identified in 16 (55.9 percent) samples... [to full text]

A nivel mundial la piodermia es una de las enfermedades de la piel más diagnosticada en caninos. Entre los agentes involucrados se encontraba Staphylococcus intermedius, el que se creía el principal agente de las piodermias caninas; sin embargo, en el año 2005 fue reclasificado en 3 especies: S. intermedius, S. pseudintermedius y S. delphini. Posteriormente, estudios en diferentes países reportaron que el Staphylococcus pseudintermedius es en realidad el agente bacteriano más frecuentemente aislado en piodermias, seguidamente surgieron los primeros reportes de aislados resistentes a meticilina. Por otro lado, Staphylococcus aureus, patógeno importante en medicina humana, se aísla con más frecuencia en muestras de piodermias caninas, siendo estos potenciales reservorios para reinfecciones en humanos de cepas resistentes a antibióticos. Por ello, este estudio buscó determinar la presencia S. pseudintermedius y S. aureus en 141 aislados de Staphylococcus sp. provenientes de casos de piodermia canina del periodo 2016 - 2018. El ADN de los 141 aislados fue extraído y analizado mediante PCR-RFLP, el cual consistió en la amplificación del gen pta y la digestión con la enzima MboI. Obteniendo que los aislados de Staphylococcus sp. fueron identificados como Staphylococcus pseudintermedius en un 87.9%, 12.1% como otros Staphylococcus sp. y no se detectó Staphylococcus aureus. Concluyéndose que el Staphylococcus más frecuentemente involucrado en piodermias caninas es el Staphylococcus pseudintermedius; sin embargo, no se debe omitir el rol potencial que puede cumplir Staphylococcus aureus como patógeno en caninos.
Universidad Nacional Mayor de San Marcos (Lima). Vicerrectorado de Investigación y Posgrado
Tesis

Dissertação de Mestrado Integrado em Medicina Veterinária
Methicillin-resistant coagulase-positive staphylococci (MRCPS) colonization in companion animals is an emerging and significant problem in public and animal health. During one year, nasal swabs were obtained from 40 cats and 146 dogs admitted to the Teaching Hospital of the Faculty of Veterinary Medicine - Technical University of Lisbon. MRCPS colonization was screened by plating enrichment cultures on a selective medium, Chrom MRSA ID. Bacterial species and mecA were confirmed by PCR. Clonality of the isolates was assessed by pulsed-field gel electrophoresis (PFGE). All isolates were subjected to spa and SCCmec typing. They were also tested by PCR for the lukF/lukS genes encoding Panton-Valentine leukocidin (PVL) in Staphylococcus aureus and Luk-I and Staphylococcus intermedius exfoliative toxin (SIET) in Staphylococcus pseudintermedius. Methicillin-resistant S. aureus (MRSA) was found in two cats (5 %) and one dog (0.6 %). Isolates were spa type t032, SCCmec IV and shared identical PFGE profiles. These were similar to the EMRSA-15 human clone. Strains were PVL-negative. Nine dogs carried methicillin-resistant S. pseudintermedius (MRSP) (6 %), whereas none of the cats was positive. The PFGE type A strain (n=1) showed identical characteristics as the American MRSP clone strains (CC68-MRSP-V), while PFGE cluster B grouped European MRSP isolates (CC71-MRSP-III) (n=8). All isolates were SIETnegative. The 8 European MRSP isolates were positive for the lukF/lukS genes and the American MRSP isolate was negative for both genes. Strains were multidrug-resistant, which represents a major challenge for veterinarians in terms of antibiotic therapy.
RESUMO - Frequência de colonização por staphylococci coagulase-positivo meticilinaresistente em cães e gatos internados no hospital escolar da Faculdade de Medicina Veterinária – Universidade Técnica de Lisboa, Portugal - A colonização por staphylococci coagulase-positivo meticilina-resistente (MRCPS) é um problema emergente e de grande importância em termos de saúde animal e pública. Durante um ano, zaragatoas nasais de 146 cães e 40 de gatos foram obtidas de animais internados no Hospital Escolar da Faculdade de Medicina Veterinária - Universidade Técnica de Lisboa. A colonização por MRCPSfoi pesquisada por inoculação de culturas de enriquecimento num meio selectivo, Chrom MRSA ID. As espécies de MRCPS e a amplificação do gene mecA por feita por PCR. A clonalidade dos isolados foi confirmada por PFGE. Todos os isolados foram sujeitos a tipagem spa e SCCmec. Os isolados de S. aureus meticilina-resistente (MRSA) e de S. pseudintermedius meticilina-resistente (MRSP) foram testados por PCR para a presença dos genes lukF/lukS que codificam, respectivamente, a leucocidina Panton-Valentine (PVL) e a leucocidina-I (Luk-I). Os isolados de MRSP foram ainda testados para a presença da toxina exfoliativa do S. intermedius (SIET). Nesta amostra, 0,6 % (n=1) dos cães testados e 5 % (n=2) dos gatos apresentaram MRSA. Os isolados de MRSA eram spa tipo t032, SCCmec IV e partilhavam padrões idênticos de PFGE. As estirpes eram idênticas ao clone humano EMRSA-15. Os 3 isolados eram PVL negativos. Nove cães apresentaram MRSP (6 %), enquanto nenhum dos gatos foi positivo. PFGE tipo A mostrou características idênticas ao clone americano de MRSP (CC68-MRSP-V) e PFGE tipo B agrupou os isolados europeus de MRSP (CC71-MRSP-III) (n=8). Os isolados de MRSP PFGE tipo B eram Luk-I positivos mas SIET negativos. Todas as estirpes de MRSP eram multirresistentes a várias classes de antibióticos, o que representa um desafio para os médicos veterinários em termos de estratégias de antibioterapia.

The extensive use of antibiotics has led to the emergence of methicillin-resistant Staphylococcus pseudintermedius, a bacterium causing difficult-to-treat canine skin infection (pyoderma). The administration of bacteriophages (phage therapy) can be an alternative to antibiotic therapy. Lytic phages, which lyse their host, are considered the only appropriate type of phages for phage therapy as opposed to temperate phages, which can survive within their host (lysogeny). However, it is possible to mutate temperate phages so that they cannot establish lysogeny anymore. Phage λ virulent (Vir) mutants have lost the operator to which the CI repressor binds to inhibit the expression of lytic genes. As a result, Vir mutants are strictly lytic. The work presented in this thesis was undertaken to isolate S. pseudintermedius phages and gain knowledge about their biology with the aim to develop phage therapy to treat pyoderma. The work was novel; very few data were available on S. pseudintermedius phages and no data have been published on phage therapy to treat canine skin infection. Four temperate phage candidates were selected after phenotypic and genotypic characterisation. No lytic phages were found. Random mutagenesis approaches were unsuccessful for the isolation of Vir mutants. An operator and three point mutations leading to the absence of CI repressor binding to this operator were identified through gel shift assay. These mutations should lead to a virulent phenotype if introduced in the relevant phage genome through site-directed mutagenesis. A PCR-based assay was performed to explore how widespread lysogeny was in S. pseudintermedius: 11 out of 45 tested strains were positive for the presence of prophage genes. Bioinformatic analyses revealed some of the genetic characteristics of S. pseudintermedius phages: genomic circular permutation and the presence of a genetic switch similar to that of phage λ. The work reported in this thesis represents a first step towards understanding the biology of S. pseudintermedius phages and developing phage therapy.

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.

Memoria para optar al Título Profesional de Médico Veterinario
El pioderma canino corresponde a la contaminación bacteriana de la piel, siendo una de las patologías dermatológicas infecciosas más comunes en la clínica veterinaria (Gortel, 2013). El Staphylococcus pseudintermedius es el agente etiológico más común de esta enfermedad, siendo un patógeno oportunista zoonótico (Weese y van Duijkeren, 2010). El pioderma canino generalmente se produce como consecuencia de causas subyacentes como heridas, ectoparásitos, enfermedades metabólicas y/o inmunológicas (Muller, et al., 2001); por lo que tradicionalmente su manejo se ha basado en la identificación y tratamiento de éstas apoyado por una terapia antimicrobiana, ya sea sistémica y/o tópica. Durante las últimas décadas los staphylococcus han mostrado una mayor resistencia a los antimicrobianos, especialmente a la meticilina, tanto en medicina humana como veterinaria (Cain, 2013). Actualmente, el tratamiento de las infecciones de la piel producidas por agentes resistentes a antimicrobianos constituye un verdadero desafío, limitando las opciones terapéuticas y obligando a los médicos veterinarios a incorporar nuevos enfoques de tratamiento contra el pioderma canino. En esta revisión se analizarán las consecuencias del incremento en la prevalencia de Staphylococcus pseudintermedius meticilino resistente, las indicaciones para la prevención del desarrollo de la resistencia microbiana y el tratamiento del pioderma canino.

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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.

Dissertação de Mestrado Integrado em Medicina Veterinária
Nos cães com dermatite atópica, as infeções cutâneas recorrentes causadas pelo Staphylococcus pseudintermedius são extremamente comuns. Estudos prévios demonstraram que cães atópicos com piodermite recorrente têm títulos mais elevados de imunoglobulina E anti-Staphylococcus no soro. Os principais objetivos deste estudo foram definir qual a concentração ideal de extrato de alergénios de S. pseudintermedius a utilizar nos testes intradérmicos de cães atópicos e posteriormente determinar a frequência de hipersensibilidade cutânea ao S. pseudintermedius em cães atópicos com história de infeção cutânea recorrente. Para isso, realizaram-se testes intradérmicos, onde se testaram diferentes concentrações de extrato de um S. pseudintermedius resistente à meticilina (MRSP), estirpe 5819/10. Esta estirpe foi isolada de um cão com piodermite profunda e apresentava os seguintes genes de virulência: luk-I, se-int, siet, speta, ebpS e spsL. As concentrações utilizadas foram 2 μg/ml, 20 μg/ml e 200 μg/ml, assim como um controlo positivo (fosfato de histamina 0.001%) e um controlo negativo (PBS 0.9%). Foram incluídos neste estudo, vinte e três cães saudáveis e vinte e quatro cães atópicos com infeção cutânea recorrente por S. pseudintermedius, diagnosticados por citologia cutânea e/ou cultura bacteriana. Dos 23 cães saudáveis presentes neste estudo, 14 (61%) apresentaram reação positiva na concentração de 200 μg/ml. Assim sendo, é bastante provável que a concentração de 200 μg/ml seja irritante e por isso resulte em reações falso-positivas. A concentração de 20 μg/ml foi a concentração mais indicada para ser usada no diagnóstico de hipersensibilidade ao S. pseudintermedius em cães atópicos. Posteriormente, foram realizados testes intradérmicos em 24 canídeos atópicos com piodermite e/ou otite bacteriana recorrente com a concentração de 20 μg/ml; quatro destes animais apresentaram reações positivas nesta concentração, o que parecer indicar a existência de reações de hipersensibilidade imediata a componentes do S. pseudintermedius. No entanto, limitações que envolviam o extrato impediram a obtenção de resultados reprodutíveis e uniformes. Acreditamos que a imunoterapia específica para esta bactéria pode ser benéfica para o tratamento destes pacientes, o que torna fundamental o diagnóstico de hipersensibilidade ao S. pseudintermedius. Nesse sentido, deverão ser realizados mais estudos, de modo a obter um extrato fidedigno que permite alcançar consistência e reprodutibilidade em ambiente clínico e assim melhorar a qualidade dos resultados obtidos nos testes intradérmicos.
ABSTRACT - THE USE OF STAPHYLOCOCCUS PSEUDINTERMEDIUS EXTRACT IN SKIN ALLERGY TESTS ON THE DOG – A PRELIMINARY STUDY - In dogs with atopic dermatitis, recurrent skin infections caused by Staphylococcus pseudintermedius are extremely common. Previous studies demonstrated that atopic dogs with recurrent pyoderma have higher titters of anti-Staphylococcus immunoglobulin E. The main objectives of this study were to define which is the optimal concentration of allergen extract of S. pseudintermedius for intradermal tests use in atopic dogs and subsequently determine the frequence of skin hypersensitivity to S. pseudintermedius in atopic dogs with a history of recurrent skin infection. For this purpose, intradermal tests were carried out with different concentrations of the extract from a methicillin-resistant S. pseudintermedius (MRSP), strain 5819/10. This strain was isolated from a dog with deep pyoderma and presented the following virulence genes: luk-I, se-int, siet, speta, ebpS and spsL. The concentrations used were 2 μg/ml, 20 μg/ml and 200 μg/ml, a positive control (0.001 % histamine phosphate) and a negative control (PBS 0.9 %). Twenty-three healthy dogs and twenty-four atopic dogs with recurrent pyoderma by S. pseudintermedius, diagnosed by skin cytology and/or bacterial culture were included in this study. Of the 23 healthy dogs, 14 (61 %) showed a positive reaction in the concentration of 200 μg/ml. Therefore, it is quite likely that the concentration of 200 μg/ml is irritant and so resulted in false-positive reactions. A concentration of 20 μg/ml appeared to be more suitable for use in the diagnosis of S. pseudintermedius hypersensitivity in atopic dogs. Subsequently, intradermal tests in 24 atopic dogs with recurrent pyoderma with the concentration of 20 μg/ml were performed. Four of these animals showed a positive reaction in this concentration, which seems to indicate the existence of an immediate hypersensitivity reaction to components of S. pseudintermedius. However, limitations involving the extract prevented obtaining reproducible and uniform results. We believe that specific immunotherapy for these bacteria can be beneficial for the treatment of atopic patients, which makes the diagnosis of S. pseudintermedius hypersensitivity fundamental. However, more studies should be conducted in order to obtain a reliable extract that can achieve consistency and reproducibility in a clinical environment and thus improve the quality of results obtained in intradermal tests.

Las piodermas y otitis externas en perros son enfermedades multifactoriales que afectan con mayor frecuencia a esta especie, y representan la gran mayoría de los problemas dermatológicos en la práctica clínica veterinaria. Staphylococcus pseudintermedius es el agente más importante asociado a la pioderma canina. Dicho agente bacteriano produce toxinas estafilocócicas que se clasifican en tres grupos principales: superantígenos de toxina pirogénica (PTSAgs), que comprenden la toxina del síndrome de shock tóxico (TSST) codificada por el gen tst, las enterotoxinas estafilocócicas (SE); toxinas exfoliativas codificadas por el gen siet; y el tercer grupo compuesto por las citotoxinas tales como leucocidinas codificadas por el gen luk-I (lukS, lukF) y hemolisinas, responsables de lesiones cutáneas graves y lisis celular de los polimorfonucleares. Además, existe evidencia de la transmisión de S. pseudintermedius entre humanos y perros. Sin embargo, no se conocen las características virulentas de los aislados circulantes en nuestro país. Por ello, el objetivo del presente estudio buscó determinar el perfil patogénico genético asociado a toxinas de S. pseudintermedius. Así, se evaluaron 141 aislados identificados genéticamente como S. pseudintermedius provenientes del cepario del Laboratorio de Microbiología y Parasitología Veterinaria de la FMV-UNMSM, durante el 2016-2018, de aislados obtenidos de casos clínicos de caninos con diagnóstico de piodermas y otitis externas. Se realizó en primer lugar la reactivación de las cepas, seguido de la extracción del ADN, y la posterior detección de los genes de virulencia asociados a toxinas tst, siet, lukS, lukF mediante PCR-Múltiple. Obteniendo que el 98.58% de los aislados de muestras clínicas de piodermas y otitis externas contienen tres genes de virulencia asociados a toxinas siet, lukS y lukF. Consecuentemente existe un potencial patogénico genético asociado a toxinas de estas cepas de S. pseudintermedius, que podrían agravar la infección y resolución de estos cuadros dermatológicos en caninos y además de impacto en la salud pública.
Perú. Universidad Nacional Mayor de San Marcos. Vicerrectorado de Investigación y Posgrado. Programa de Promoción de Tesis de Pregrado. A19080891-PTPGRADO
Tesis

Dissertação de Mestrado Integrado em Medicina Veterinária
At a time when antimicrobial resistance is rising steadily and the involved microorganisms are demonstrating zoonotic potential, honey and its derived products may prove useful in this ongoing battle. Otitis externa in dogs is considered to be one of the most prominent causes for presentation at veterinary practice. Some of the regularly administered agents in otitis treatments are no longer effective, as resistance has increased, perhaps due to the often long-term periods which are necessary for resolution and the accompanying tendency towards chronicity. In order to address the need for efficient alternative treatments, LMesitran ® Soft, a medical grade honey gel was used to treat 15 dogs with otitis externa of bacterial and/or fungal involvement. Success was based on clinical score decrease, cytology and owner input over time and with basis on culture results. 70% of enrolled dogs achieved clinical cure between days 7 to 14 and over 90% on day 21, the maximum established time limit, with a confidence interval of 95%. Furthermore, by day 7, 20% of dogs had obtained both clinical and cytological cures. This study was successfully able to demonstrate that the use of L-Mesitran® was effective in managing otitis externa in dogs, including cases in which highly resistant pathogens were present, such as methicillinresistant Staphylococcus pseudintermedius (MRSP), thus paving the way to future studies.
ABSTRACT - L-MESITRAN® NO MANEIO DE OTITE EXTERNA CANINA – UM ESTUDO PILOTO - Numa altura em que nos deparamos com um aumento de bactérias com resistências aos antibióticos e em que os organismos envolvidos apresentam por vezes inclusivamente potencial zoonótico, o recurso ao mel e seus derivados pode ser uma inestimável ferramenta no decurso desta batalha. A otite externa em cães é um estímulo iatrotrópico frequente e das principais causas de idas ao médico veterinário. Alguns dos tratamentos habitualmente utilizados deixaram de ser eficazes à medida que as resistências surgiram, talvez consequência de terapêuticas prolongadas e recorrentes. Neste estudo avaliou-se uma potencial alternativa à terapêutica, recorrendo-se ao L-Mesitran® Soft, uma pomada contendo mel de grau clínico, para tratar 15 cães com otite externa de envolvimento bacteriano e/ou fúngico. A resposta foi considerada positiva de acordo com a diminuição da pontuação clínica, citologia e opinião dos donos, no decorrer do tempo. Estabelecido o limite de 21 dias, 70% dos cães tratados obteve cura clínica entre os dias 7 e 14 e mais de 90% no dia 21, com um intervalo de confiança de 95%. Ainda, até ao dia 7, 20% dos cães havia obtido cura clínica e citológica. Este estudo demonstrou que o L-Mesitran® foi eficaz no maneio dos casos de otite externa, incluindo aqueles em que estavam presentes bactérias com várias resistências aos antibióticos, como é o caso do Staphylococcus pseudintermedius com resistência à meticilina (SPRM), abrindo assim caminho para futuros estudos.

OBJECTIVE – The aim of the study was to evaluate the phenotypic and genotypic characteristics of methicillin-resistant Staphylococcus pseudintermedius in samples from dogs and cats processed in the laboratory of a veterinarian teaching hospital. We also performed tests for vancomycinresistance on the same isolates. MATERIALS AND METHODS – A total of 36 strains of Staphylococcus were isolated from 33 dogs and 3 cats (from skin, eyes, ears, urine, surgical wounds and others). The strains were identified by cultivation on selective medium (MSA), by biochemical system (API-Staph®) and by molecular method (HRM-PCR). The evaluation of the resistance pattern of the strains was performed using Kirby-Bauer method, testing different antimicrobial classes in addition to methicillin and vancomycin. We also performed MIC for methicillin and vancomycin. The presence of mecA gene was evaluated by PCR. RESULTS – 31 strain of S. pseudintermedius, 1 strain of S. aureus and 4 strains of other Staphylococcus species were identified by HRM-PCR method. Among the 36 strains of S. pseudintermedius isolated, 19 were phenotypically resistant to methicillin and 16 of these MRSP carried the mecA gene. The 50% of the 36 isolates were found to be multiresistant and 16 of these strains were also methicillin-resistant. No vancomycin-resistant strains were found. CONCLUSIONS – This study confirmed the increasing prevalence of MRSP and the absence so far of vancomycin-resistance in strains of Staphylococcus isolated from small domestic animals. The most reliable methods for the identification of species were the molecular ones, instead of the biochemical, less effective to have conclusive results.

Staphylococcus pseudintermedius is the most commonly isolated bacteria in dogs and is often associated with pyoderma and otitis in these animals. In the past years, methicillin resistant Staphylococcus pseudintermedius (MRSP) was described. These microorganisms have in there genome the mecA gene, responsible for the transcription of the PBP2a protein that reduces the susceptibility of these bacteria to all beta - lactamic antibiotics. The aim of this study was to evaluate the frequency of MRSP in dogs with superficial pyoderma presented at the Veterinary Hospital of UFMG. Forty-three dogs with superficial pyoderma were selected for the study diagnosed by physical and dermatological examination from march to July of 2013. From the selected animals, 75% had a history of prior antibiotic therapy. The samples were collected in sterile swab secretion of skin lesions and right nostril and were streaked on sheep blood agar and subsequently evaluated phenotypically and genotypically. At the phenotypic identification, 88 % of the isolates were classified as SIG (Staphylococcus pseudintermedius Group). At the genotypic identification for classification as Staphylococcus pseudintermedius, this number grows to 91 %, showing a significant correlation with the result of biochemical identification. A significant correlation between the species identified in skin lesions and nostrils. In the antibiogram, prevalence of resistance was amikacin 7% to 23% amoxicillin + clavulanate , 24% cephalexin , chloramphenicol 18% , 38% enrofloxacin 40% streptomycin , gentamicin 30% , 34% oxacillin, 77% penicillin , polymyxin B 6% , 62% and 77% tetracycline trimethoprim sulfa . Thirty-five percent of the samples had the mecA gene, demonstrating a significant correlation with the prevalence of oxacillin resistance. We also observed a significant correlation between the frequency of mecA in the lesions and nostrils. There was no correlation between previous antibiotic therapy and frequency of mecA gene in samples. In conclusion, S. pseudintermedius is the most common bacteria in canine pyoderma and the mecA gene is present in significant frequency in the lesions and nostrils of the tested animals.
O Staphylococcus pseudintermedius é a bactéria mais comumente isolada de cães e está frequentemente associada à piodermites e otites nesses animais. Atualmente, tem sido descrito o Staphylococcus pseudintermedius resistente à meticilina (MRSP). Estes microorganismos são portadores do gene mecA, responsável pela transcrição da proteína PBP2a que reduz a susceptibilidade dessas bactérias a todos os antibióticos betalactâmicos. O objetivo deste estudo foi avaliar a prevalência de MRSP em cães com piodermite superficial atendidos no Hospital Veterinário da UFMG. Foram selecionados para o estudo 43 cães com piodermite superficial diagnosticados por exame físico e dermatológico entre março e julho de 2013. Dos animais selecionados, 75% tinham histórico de antibioticoterapia prévia. Foram coletadas amostras em haste de algodão estéril de secreção de lesões de pele e narina direita. As amostras foram cultivadas em ágar sangue de carneiro e, posteriormente, avaliadas fenotipicamente e genotipicamente. Na identificação fenotípica, 88% dos isolados foram classificados como bactéria do SIG (Staphylococcus pseudintermedius Group). Na identificação genotípica para classificação como Staphylococcus pseudintermedius, este número cresceu para 91%, demonstrando correlação significativa com o resultado da identificação bioquímica. Observou-se correlação significativa entre a espécie identificada nas lesões de pele e narinas. No antibiograma a prevalência de resistência foi de 7% a amicacina, 23% a amoxacilina + clavulanato, 24% a cefalexina, 18% ao cloranfenicol, 38% a enrofloxacina, 40% a estreptomicina, 30% a gentamicina, 34% a oxacilina, 77% a penicilina, 6% a polimixina B, 62% a tetraciclina e 77% a sulfa + trimetoprim. Trinta e cinco porcento das amostras apresentava o gene mecA, demonstrando correlação significativa com a frequência de resistência à oxacilina. Observou-se também correlação significativa entre a prevalência de mecA nas narinas e lesões de pele. Não houve correlação entre a antibioticoterapia prévia e a frequência de gene mecA nas amostras. Desta forma, conclui-se que o S. pseudintermedius é a bactéria mais prevalente em piodermite canina e o gene mecA está significativamente presente nas lesões e narinas dos animais testados.

Dissertação de Mestrado Integrado em Medicina Veterinária
ABSTRACT - Objective: This study aims to characterize the dynamics of antibiotic resistance gene transmission in dogs with skin and soft tissue infections (SSTI) and human beings co habiting with them. We also aim to evaluate the gut colonization of these individuals for the presence of extended spectrum beta-lactamases (ESBL’s) in Enterobacteriaceae and to assess the effect of antibiotherapy on the selection of MDR bacteria from human and canine gut microbiota. Methods: Two types of biological samples were gathered in a teaching veterinary hospital in Portugal, at the dermatology department, from twelve dogs diagnosed with SSTI and their household members. Collections included a swab from the infection site (ISS) and a faecal sample (FS). Gathering of samples was performed at two different times. The ISS were cultured and an AST was performed. The FS was also cultured, and the bacteria isolated subjected to molecular analysis. Antibiotic resistance patterns were obtained by disk diffusion antimicrobial susceptibility’s testing and Enterobactereaceae ESBL’s production was confirmed by amplification of the specific gene by PCR and sequencing. Results: High levels of Methicillin resistant Staphylococcus pseudintermedius (MRSP) were isolated, and high levels of other multidrug resistant bacteria (MDR) as well. One family was suspected of carrying the same E. coli clone, shared by two humans and one dog of the same household, with a blaCTX-M-15 gene. All of the isolated Enterobactereaceae displayed susceptibility to carbapenems. The most common ESBL genes found were from the blaCTX-M group, followed by blaOXA-1 and then blaTEM, no gene from the blaSHV gene was found. Conclusions: There is a high prevalence of ESBL-producing E. coli. Interspecies transmission of antimicrobial resistance is real. This issue should be addressed with introduction of antimicrobial stewardship strategies on a wider scale and better use of antimicrobials like chlorohexidine, especially in SSTI.
RESUMO - Esta dissertação enquadra-se nos objetivos de um projeto muito maior e ainda mais ambicioso, chamado PetRisk. Pretende-se analisar o impacto e as interações dos genes de resistência aos antibióticos, percebendo de que forma as barreiras interespécie podem ser ultrapassadas. Assim sendo, o estudante integrou o núcleo desse mesmo empreendimento em Portugal, o Laboratório de Resistência aos Antibióticos da FMV-UL, liderado pela Professora Doutora Constança Pomba. Objetivo: Este estudo teve como objetivo principal compreender a dinâmica de transmissão de genes de resistência aos antibióticos, entre cães com infeção de pele e tecidos moles e as pessoas com quem vivem em regime de co-habitação. Pretendeu-se avaliar a colonização do tubo digestivo desses mesmos indivíduos quanto à presença de Enterobactereacea produtoras de beta-lactamases de largo expectro e verificar o efeito da antibioterapia na seleção de estirpes multiresistentes da microbiota intestinal canina e humana. Outro obejtivo da realização deste trabalho é o de aumentar os recursos biológicos – com isto subentende-se, bactérias de diferentes agregados familiares e o seu perfil de resistência aos antimicrobianos – para o projeto mãe, o PetRisk. Métodos: Para tal, recolheram-se dois tipos de amostras biológicas no departamento de dermatologia do hospital veterinário escolar da FMV-ULisboa. A amostra inclui doze cães com infeções de pele e tecidos moles, assim como os restantes membros do seu agregado familiar. Os materiais recolhidos foram zaragatoas do local de infeção (ZLI) e amostras fecais (AF). As colheitas decorreram em dois tempos diferentes. Às ZLI após cultura microbiológica realizaram-se testes de suscetibilidade aos antibióticos de rotina. Os isolados provenientes das AF foram sujeitos a cultura microbiológica e análise molecular. Os padrões de resistência aos antibióticos foram obtidos pelo método de difusão de discos e a confirmação da produção das beta-lactamases de largo expectro pelas Enterobactereaceae por amplificação do respetivo gene por PCR e sequenciação. Resultados: Foram encontrados elevados níveis de Staphylococcus resistentes à meticilina, assim como a resistência a multiplos antibióticos. Também as nas Escherichia coli provenientes das AF foram encontrados elevados nívreis de resistência a múltiplos antibióticos. Uma familia foi suspeita de partilhar o mesmo clone de E.coli (duas pessoas e um cão) com o mesmo filogrupo e o gene blaCTX-M-15. Todas as Enterobactereaceae isoladas demonstraram suscetibilidade aos carbapenemos. Os genes de beta-lactamases de largo expectro detetados foram (da maior para a menor frequência): blaCTX-M , blaOXA-1 e blaTEM, com nenhum registo de blaSHV. Conclusões: Existe uma elevada prevalência de E. Coli produtoras de beta-lactamases de largo expetro. A transmissão de informação genética inter-espécies é uma realidade. A prática clínica beneficiaria de uma administração mais prudente e integrada de antibióticos, com uma equipa a trabalhar apenas para a gestão destes recursos num hospital, assim como maior atenção ao uso de antisséticos como a clorohexidina, especialmente em infeções de pele e tecidos moles onde os produtos disponivéis são de fácil aplicação.
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During my PhD, I mainly worked on two different fields of research: 1. Analysis of microRNAs in canine hematopoietic malignancies for diagnostic purposes. MicroRNAs (miRNAs) are a new class of small non-coding RNAs involved in the negative regulation of gene expression. In humans, it has been shown that miRNAs play a key role in the modulation of the innate immune response and in the regulation of normal and neoplastic hematopoiesis. The expression profile of these molecules varies in the different stages of differentiation of the hematopoietic system cellular components and any alteration of these processes is directly associated with dysregulation of one or more miRNAs. The frequency and nature of some of these tumors are similar in dogs and humans. In dog, the studies of hemato-oncology performed to date have shown that the currently used methodologies are not always able to obtain comprehensive information about the staging, grading, immunophenotyping and prognosis of the diseases. This makes miRNAs very attractive as new potential diagnostic and prognostic markers allowing better subclassification of these tumors in dogs. The purpose of the study undertaken in this project was to examine, using stem-loop TaqMan real time RT-PCR, the expression profile of a panel of miRNAs in some hematological malignancies of the dog, such as chronic lymphocytic leukemia (CLL), lymph node lymphoma and splenic lymphoma, in relation to their non cancer tissues. Panels of miRNAs, already known in humans because of their importance in hematopoietic malignancies, have been selected for investigation, according to the information available in the literature and databases, and to their level of conservation in the canine genome. As a preliminary step, for each studied canine neoplasia, an accurate validation of suitable candidate endogenous control (EC) genes for normalization of miRNA expression levels in normal and neoplastic lymphoid tissues was made. This was performed by means of real time RT-PCR followed by statistical analysis using the algorithms NormFinder and geNorm. In all studied lymphoid malignancies, and for each miRNA investigated, a relative quantification (by calculating the delta-delta Ct to the endogenous control gene selected) and an absolute quantification (interpolating its Ct value on standard curves obtained from serial dilutions of an equimolar pool of synthetic miRNAs) was performed after selecting the best housekeeping genes. The results of this study confirm that in dogs, as already shown in humans, there is differential expression of some miRNAs in various hematologic malignancies, suggesting the potential utility of miRNA monitoring as a new diagnostic strategy in canine hematopoietic malignancies. In particular, in lymph node lymphoma samples miR-181a was upregulated in canine T-cell lymphoma and miR-17-5p was upregulated in canine B-cell lymphoma. Moreover, the molar ratio between miR-181a and miR-17-5p clearly distinguished between T-cell and B-cell lymphoma samples. In CLL samples, the molar ratio miR-150/miR-125b was correlated with the immunophenotype. Finally, molar ratio miR-17-5p/miR-155 was correlated with grading in canine splenic lymphoma. Furthermore, in dogs is often difficult to obtain a sufficient number of fresh clinical samples to perform large diagnostic studies. From here the need arises to carry out studies of retrospective nature based on appropriately validated methods of analysis that make possible to analyze archival samples. Therefore, one of the goals of this study was to develop a methodology for extraction and analysis of miRNAs from smears of aspirated lymph node (for the study of lymph node lymphoma) and peripheral blood (for the study of CLL) and from tissue sections fixed in formalin and included in paraffin (for the study of splenic lymphoma). The results obtained during this study confirm the possibility to analyze miRNAs for retrospective studies starting from such archival samples, as the data obtained from these samples are comparable to those obtained from fresh samples. 2. High Resolution Melting Analysis (HRMA) of genic polymorphisms for diagnostic purposes. High Resolution Melting (HRM) analysis is a new, post-PCR analysis method used for identifying genetic variation in nucleic acid sequences. Simple and fast, this method is based on DNA melting (dissociation) curve techniques and is enabled by the recent availability of improved double-stranded DNA (dsDNA)–binding dyes along with next-generation real-time PCR instrumentation and analysis software. HRM analysis can discriminate DNA sequences based on their composition, length, GC content, or strand complementarity. Accordingly, HRMA allows the detection of multiple types of variations in DNA sequences such as single base changes, insertions, deletions and duplications. In this work this new methodology was utilized as a successful rapid diagnostic technique for the evaluation of the resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples, and for the detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood.

Staphylococcus pseudintermedius affecting dogs is analogous to S. aureus on humans, acting as both normal flora and opportunistic pathogen. Methicillin resistance in S. pseudintermedius is recent, with the first documented occurrence of an isolate bearing the methicillin resistance gene, mecA, in 1999. This gene encodes penicillin binding protein 2a, which renders all beta-lactam drugs ineffective and functions as a “gateway” antibiotic resistance determinant. In the presence of ineffective antibiotics, opportunities for mutational events and acquisition of mobile genetic elements increase as microbial densities increase, often leading to multi-drug resistance. Methicillin-resistant S. pseudintermedius (MRSP) infections have become increasingly common. For example, approximately 30% of the S. pseudintermedius isolates tested by the University of Tennessee College of Veterinary Medicine Clinical Bacteriology Laboratory are resistant to methicillin. An increasing number of MRSP isolates are also resistant to most clinically useful antibiotics available to veterinarians except for chloramphenicol, and resistance to this antibiotic is common among European MRSP isolates. Chloramphenicol resistance has begun to appear in the US and if this trend continues there may soon be few viable antibiotic treatment options. Compared with the arrival of methicillin-resistant S. aureus in the 1960s, the opportunity currently exists to apply advanced molecular methods early in this recognized emergence of MRSP. To that end I have pursued projects utilizing multilocus sequence typing, pulsed-field electrophoresis, and SCCmec characterization of both susceptible and resistant S. pseudintermedius. The initial result was the detection of a clonal population of MRSP in the southeastern United States. Further characterization of this and other clonal lineages using genomic sequencing and real-time RT-PCR expression analysis of antibiotic resistance and quorum sensing genes revealed a marked difference in the regulation of antibiotic resistance between regional clones. These discoveries have interesting epidemiological implications and provide a foundation for the development of novel therapeutics to circumvent the expanding antibiotic resistance repertoire of MRSP. Potential targets identified by this work include membrane-bound beta-lactamase receptors responsible for the regulation of mecA, non-cognate auto-inducing peptides, and synthetic antisense oligonucleotides.

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.

A descoberta dos antibióticos representou um importante avanço no tratamento de infeções bacterianas, tanto na medicina humana como na medicina veterinária, constituindo a base da medicina moderna. Os antibióticos são amplamente utilizados na rotina de clínicas e hospitais veterinários e o seu uso excessivo e indevido tem conduzido a fenómenos de resistências sendo atualmente uma preocupação mundial na saúde animal e com impacto em saúde pública. Este estudo teve como objetivo avaliar e comparar o perfil de resistência a antibióticos de bactérias isoladas em amostras de animais atendidos no Hospital Veterinário Universitário de Coimbra, entre março de 2016 e dezembro de 2018. O estudo foi dividido em duas partes, numa primeira parte foi realizada a caracterização das 206 amostras correspondentes a antibiogramas de bactérias isoladas em canídeos e felídeos. Seguidamente após a aplicação dos critérios de inclusão foram estudadas apenas as infeções simples, tendo sido avaliado o seu perfil de resistência nos três anos em estudo. Em relação à avaliação do perfil de resistência ao longo dos três anos, para as amostras de urina (n=54), verificou-se um aumento da resistência à amicacina dos agentes bacterianos isolados, no ano de 2018 relativamente aos dois anos anteriores (p=0,02). No que respeita aos agentes etiológicos isolados em amostra de zaragatoa cutânea (n=13), verificou-se que ao longo dos três anos, um aumento da resistência destes agentes ao trimetoprim/sulfametoxazole (p=0,04). Os agentes etiológicos identificados em maior percentagem foram Escherichia coli (61%) e Staphylococcus pseudintermedius (27%), sendo que para E.coli foi possível encontrar diferença significativa no perfil de resistência para a tetraciclina (p=0,002) ao longo dos três anos. Verificou-se uma tendência (p=0.06) para o aumento no perfil de resistência nos antibióticos cloranfenicol e amicacina. Este estudo permitiu verificar uma alteração no perfil de resistências em animais de companhia, nomeadamente em infeções provocadas por E.coli, tal como se tem verificado em estudos nacionais e internacionais publicados recentemente. Considera-se assim crucial a consciencialização dos profissionais de saúde e população em geral em relação à utilização e prescrição prudentes dos antibióticos.
The antibiotics discovery was a big advance in the treatment of bacterial infections, in human and in veterinary medicine, being the basis of modern medicine. Antibiotics are widely used in veterinary clinics and hospitals in its daily routine and their excessive or inadequate use in animals has led to the appearance of resistant bacteria. This phenomenon is nowadays a worldwide concern due to its impact in animal health as well as in human health. The main objective of this study was to analyse and compare antibiotics resistance profiles in bacteria isolated from animal samples attended at the Hospital Veterinário Universitário de Coimbra, from March 2016 to December 2018. This study was conducted in two steps. Firstly, a descripitive characterization of the 206 samples corresponding to the results obtained from antimicrobial susceptibility tests in bacteria isolated from dogs and cats, was performed. Secondly, and after considering the defined inclusion criteria, an analisys was developed using only cases of single bacterial infections by the evaluation of its resistance profile among the three years of study So, during the study period, the resistance profiles in isolates obtained in urine samples (n=54) showed an increase in amikacin resistance in 2018, when compared to the previous two years (p=0,02). Bacterial agents isolated from cutaneous swab (n=13), evidenced an increase in the resistance to trimethoprim/sulfamethoxazole (p=0,04) during the three years of study. A higher percentage of isolates belonged to Escherichia coli (61%) and Staphylococcus pseudintermedius (27%) species, and considering E. coli a significant difference in the resistance profile for tetracycline (p=0.002) was observed from 2016 to 2018. Also, regarding the same bacterium, it was observed a tendency (p=0,06) for an increase of the resistance profile in amikacin and cloranfenicol. This study demonstrated an increase in the resistance profiles in isolates obtained from companion animals such as cats and dogs, especially in infections caused by E.coli. These data is in accordance to other national and international studies recently published. Thus, an awareness of healthcare professionals and of the general population is crucial for the conscient and prudent prescription and use of antibiotics.

Staphylococcus pseudintermedius é uma bactéria colonizadora da pele e mucosas do cão, podendo ser também encontrada em gatos. Esta bactéria é o principal agente etiológico do pioderma superficial bacteriano canino e uma das principais causas para tratamento sistémico com recurso a antibióticos em animais de companhia. Este trabalho teve como principal objetivo estudar a atividade de efluxo em S. pseudintermedius e a sua relevância na resistência a compostos antimicrobianos efluxáveis como as fluoroquinolonas, utilizando o brometo de etídeo (EtBr) como marcador indireto da atividade de efluxo. Estudou-se um conjunto de 52 S. pseudintermedius, associados a infeções da pele e dos tecidos moles (SSTIs) em cães e gatos, isolados em dois laboratórios de diagnóstico veterinário do distrito de Lisboa, Portugal. Numa primeira abordagem, foi determinada a concentração mínima inibitória (CMI) para o EtBr e a distribuição deste valor utilizada para definir o valor de cut-off (COWT) possibilitando a separação da população wild-type (WT) e non wild-type (NWT) para este composto. Estabeleceu-se um COWT de 8 mg/L e a aplicação deste valor à coleção em estudo indicou a inexistência de uma população NWT para o EtBr. Posteriormente, a CMIEtBr foi determinada na presença dos inibidores de efluxo (IEs) verapamil e tioridazina. Estes IEs promoveram uma redução da CMIEtBr, sugerindo a presença de atividade de efluxo nestes isolados. A pesquisa dos genes plasmídicos qacA/B e smr, que codificam para bombas de efluxo MDR com capacidade para extrusar compostos como o EtBr, foi realizada no grupo de 52 isolados tendo-se posteriormente estendido a toda a coleção de 157 S. pseudintermedius disponível, não se tendo encontrado nenhum dos genes. Numa segunda abordagem, e porque o efluxo tem sido implicado na resistência a fluoroquinolonas (FQ) noutras espécies de estafilococos, determinou-se a CMI da ciprofloxacina (CMICIP) na presença e ausência dos mesmos compostos IEs. Os valores obtidos para a CMICIP variaram entre 0,06-64 mg/L. Na presença dos dois IEs apenas se verificou diminuição significativa da CMICIP em três isolados, todos eles suscetíveis à CIP e à meticilina. Esta aparente ausência de efluxo pode estar encoberta pela presença de mutações nas zonas quinolone-resistance determining region (QRDR) dos genes alvo grlA e gyrA. Estas mutações foram pesquisadas para 42 isolados dos 52 em estudo, revelando que 27 apresentam pelo menos uma mutação QRDR. Todas estas mutações já tinham sido previamente descritas, com exceção da mutação Asp83Asn de GyrA, identificada pela primeira vez neste trabalho para S. pseudintermedius. Assim, verificou-se que a resistência a FQ nesta batéria ocorre maioritariamente por mutações nos genes alvo. Verificou-se ainda a presença de atividade de efluxo basal, revelando o potencial de emergência de resistências pela exposição a estes antibióticos através da ativação de sistemas de efluxo. Os resultados obtidos nesta Dissertação refletem a necessidade de uma abordagem Uma Só Saúde, que reconhece a importância de uma visão global da saúde humana, animal e do meio ambiente para uma resposta adequada e eficaz aos principais desafios de saúde na atualidade, como é a resistência aos antimicrobianos.
Staphylococcus pseudintermedius is a bacterium that colonizes the skin and mucous membranes of dogs and can also be found in cats. This bacterium is the main etiological agent of superficial bacterial canine pyoderma and one of the main causes for systemic treatment using antibiotics in companion animals. The main objective of this work was to study the efflux activity in S. pseudintermedius and its relevance in the resistance to effluxable antimicrobial compounds such as fluoroquinolones, using ethidium bromide (EtBr) as an indirect marker of efflux activity. A set of 52 S. pseudintermedius, associated with skin and soft tissue infections (SSTIs) in dogs and cats, isolated at two veterinary diagnostic laboratories in the district of Lisbon, Portugal, was studied. In a first approach, the minimum inhibitory concentration (MIC) for EtBr was determined and the distribution of this value used to define the cut-off value (COWT) enabling the separation of the wild-type (WT) and non wild-type (NWT) population for this compound. A COWT value of 8 mg/L was established and the application of this value to the collection under study indicated the inexistence of an NWT population for EtBr. Subsequently, CMIEtBr was determined in the presence of the compounds verapamil and thioridazine, known as efflux inhibitors (EIs). These EIs promoted a reduction in CMIEtBr, suggesting the presence of efflux activity in these isolates. The search for plasmid genes qacA/B and smr, encoding MDR efflux pumps with the capacity to extrude compounds such as EtBr, was carried out in a group of 52 isolates and was later extended to the entire collection of 157 isolates of S. pseudintermedius available at the host laboratory. None of the genes were found. In a second approach, and because efflux has been implicated in fluoroquinolone (FQ) resistance in other staphylococcal species, ciprofloxacin MIC (CMICIP) was determined in the presence and absence of the same EIs. The values obtained for CMICIP ranged between 0.06-64 mg/L. In the presence of the two EIs, there was only a significant decrease in CMICIP in three isolates, all of them susceptible to CIP and methicillin (MSSP). This apparent absence of efflux may be overshadowed by the presence of mutations in the quinolone-resistance determining region (QRDR) of the grlA and gyrA target genes. These mutations were searched for 42 isolates of the 52 S. pseudintermedius, revealing that 27 have at least one QRDR mutation. All these mutations have been previously described except the GyrA mutation Asp83Asn, identified for the first time in this work for S. pseudintermedius. Thus, it was found that FQ resistance in this bacterium occurs mainly through mutations in target genes. It was also verified the presence of basal efflux activity, revealing the potential for emergence of resistance by exposure to these antibiotics through the activation of efflux systems. The results obtained in this Dissertation support the need for a One Health approach, which recognizes the relevance of a global perspective of the human, animal and environmental health for an adequate and effective response to the main health challenges, such as antimicrobial resistance

Orientação : Margarida Alves ; co-orientação : Ana Oliveira
Staphylococcus pseudintermedius é uma bactéria comensal da pele e mucosa do cão, sendo o principal agente etiológico de piodermite bacteriana superficial e uma das principais causas para prescrição antibiótica em clínica de pequenos animais. O presente estudo teve como objetivo a determinação do perfil de resistência aos antibióticos recomendados pelas linhas de orientação da International Society for Companion Animal Infectious Disease (ISCAID), num grupo de isolados de S. pseudintermedius suscetíveis e resistentes à meticilina, provenientes de cães com piodermite superficial. Avaliou-se, igualmente, a incidência de multirresistência e a aplicabilidade das linhas de orientação da ISCAID neste grupo de isolados. Este constitui um estudo prospetivo, tendo sido utilizados 55 isolados de S. pseudintermedius, testados para a suscetibilidade à oxacilina pela técnica de Kirby-Bauer e, posteriormente, divididos em dois grupos: S. pseudintermedius resistente à meticilina (MRSP, 25/55, 45%) e S. pseudintermedius suscetível à meticilina (MSSP, 30/55, 55%). No grupo MRSP, foi observado 96% de resistência a ambas clindamicina e eritromicina; 92% à amoxicilina-ácido clavulânico, trimetoprim-sulfametoxazol e pradofloxacina; 88% à cefalotina, enrofloxacina e gentamicina; 80% à tetraciclina; 40% à minociclina; 12% ao cloranfenicol e rifampicina; e 8% à amicacina. No grupo MSSP, verificou-se 70% de resistência à tetraciclina; 67% à minociclina; 47% à eritromicina; 43% à clindamicina; 27% ao trimetoprim-sulfametoxazol; 23% ao cloranfenicol; 10% à gentamicina; 7% a ambas enrofloxacina e pradofloxacina; 3% à rifampicina e amicacina; e 0% de resistência à amoxicilina-ácido clavulânico e cefalotina. Foi possível, ainda, verificar que 66% dos isolados demonstraram-se multirresistentes, sendo que 67% eram MRSP e 33% MSSP; 33% demonstraram-se não multirresistentes, dos quais 95% eram MSSP e apenas 5% MRSP. Verifica-se, assim, a existência de níveis elevados de resistência a antibióticos de primeira escolha, muitas vezes utilizados de forma empírica e sem realização de antibiograma prévio. Este facto limita a sua utilização por parte do Médico Veterinário em isolados MRSP, apesar de no grupo MSSP ainda se detetar evidentes suscetibilidades. O número de isolados multirresistentes é, também, alarmante, diminuindo o número de alternativas antibióticas disponíveis em casos cujas opções antibióticas são limitadas, o que ocorre frequentemente em clínica de referência.
Staphylococcus pseudintermedius is part of the normal microbiota of canine skin and mucosa. It is also the main etiological pathogen of canine superficial pyoderma, in addition to being a major cause for antimicrobial prescription in small animal practice. This study aimed to determine the resistance of the antibiotics recommended by the guidelines of the International Society for Companion Animal Infectious Disease (ISCAID) guidelines, in a group of S. pseudintermedius isolates susceptible and resistant to methicillin, from dogs with superficial pyoderma. The incidence of multidrug resistance and the applicability of the ISCAID guidelines in this group of isolates was also evaluated. This was a prospective study, where 55 isolates of S. pseudintermedius were tested for oxacillin susceptibility by the Kirby-Bauer technique and, then, divided into two groups: methicillin resistant S. pseudintermedius (MRSP, 25/55, 45%) and methicillin susceptible S. pseudintermedius (MSSP, 30/55, 55%). In the MRSP group, 96% of the isolates evidenced resistance to both clindamycin and erythromycin; 92% to amoxicillin-clavulanic acid, trimethoprim-sulfamethoxazole and pradofloxacin; 88% to cephalothin, enrofloxacin and gentamicin; 80% to tetracycline; 40% to minocicline; 12% to chloramphenicol and rifampicin; and 8% to amikacin. In the MSSP group, 70% of the isolates showed resistance to tetracycline; 67% to minocicline; 47% to erythromycin; 43% to clindamycin; 27% to trimethoprim-sulfamethoxazole; 23% to chloramphenicol; 10% to gentamicina; 7% to both enrofloxacine and pradofloxacin; 3% to rifampicin and 3% to amikacin; and 0% to amoxicillin-clavulanic acid and cephalothin. It was also determined that 66% of the isolates were multiresistant, in which 67% were MRSP and 33% MSSP; 33% were non multiresistant, with 95% of the isolates being MSSP and only 5% MRSP. The results reveal the existence of high levels of resistance to first tier antibiotics, often used empirically and without previous susceptibility testing. This limits their use by the Veterinarian in MRSP isolates, although there are still obvious susceptibilities in the MSSP group. The number of multiresistant isolates is also alarming, decreasing the number of antibiotic alternatives available in cases whose options are limited, which often occurs in reference practice.

碩士
國立中興大學
獸醫學系暨研究所
106
The skin microbiota of dogs includes several species of bacteria, which contributes to the skin defense mechanism. Staphylococcus pseudintermedius plays the role of an opportunistic pathogen, which is cultured at high frequency from the skin of dogs with superficial pyoderma, and effective treatment against infections caused by this pathogen is important in veterinary medicine. Although antibiotic is the standard medication when dealing with bacterial infection, multiple antibiotic-resistant mechanisms gradually developed by bacteria make treatment more challenging nowadays. If S. pseudintermedius was transferred from dogs to humans, it may not only cause direct infection but also exchange drug-resistant gene with other staphylococci on humans. The objective of this survey has three parts. First, we investigate the bacterial species that cause canine superficial pyoderma, the antibiotic-resistant characteristics, drug-resistant genes of isolated S. pseudintermedius, the possible risk factors lead to MRSP infection, and we also compare previous findings with outcomes this time. Second, we investigate the detection rate of S. pseudintermedius from the dog owners and evaluate possible risk factors causing S. pseudintermedius colonizing owners by information from the questionnaire. Third, we find out prevalent dru types of MRSP in Taiwan. Samples were obtained at the National Chung Hsing University from Jun. 2017 to Jan. 2018, and 60 pairs of dogs and owners were included. Sixty-five bacteria were isolated from the dogs, which included 47 S. pseudintermedius (72.3%), 12 other staphylococci (18.5%), 4 other Gram-positive bacteria (6.2%) and 2 Gram-negative bacteria (3.1%). The positive rates of possessing mecA and blaZ of S. pseudintermedius were 34.04% and 80.85%, respectively. More mecA- or blaZ-positive isolates showed resistance against most tested antibiotics and had multiple-drug resistance compared with mecA- or blaZ-negative ones. Dogs which received antimicrobial treatment within a recent month were at significantly higher risk of MRSP infections compared with those which didn’t (p ＜0.05). The detection rate of S. pseudintermedius from 60 samples of owners was 8.33%, and there were no significant risk factors that associated with S. pseudintermedius colonizing owners could be found in this survey. Each MRSP isolate belonged to one of the following three dru types: dt11y (29.41%), dt11a (47.06%) and dt10cp (23.53%). In conclusion, the major pathogen of canine superficial pyoderma is found to be S. pseudintermedius in Taiwan, and isolates which are mecA- or blaZ-positive are generally more resistant to commonly used antibiotics. Compared with the outcomes of previous investigation, we found that S. pseudintermedius isolated from samples of canine superficial pyoderma are apparently more resistant to doxycycline, augmentin, cephalexin, and cephazolin after few years. Although S. pseudintermedius isolated from the owners might be transferred from their dogs, definite risk factors should be further examined in the future study.

Doctoral Thesis in Veterinary Sciences - Clinic
O mel de grau médico é considerado uma opção eficaz e económica no tratamento de feridas infetadas. O mel manuka (MH) e um gel à base de mel (HBO) encontram-se disponíveis no mercado permitindo a prescrição de produtos seguros, sem antibióticos, eficazes e padronizados. Neste trabalho, o primeiro objetivo consistiu em determinar a metodologia de diagnóstico e tratamento das doenças dermatológicas causadas por Staphylococcus pseudintermedius e Malassezia pachydermatis em clínica de pequenos animais em Portugal. Inicialmente, demonstrámos que ambos os microrganismos causam frequentemente várias doenças, nomeadamente, a foliculite bacteriana superficial (SBF), a dermatite das pregas de pele (FD), a dermatite generalizada por Malassezia spp. (MD) e otite externa (OE). Os antibióticos orais foram largamente prescritos para o tratamento de SBF, particularmente amoxicilina-ácido clavulânico (100%), cefalexina (94%), enrofloxacina (67%) e marbofloxacina (60%). Para o tratamento de FD e OE, os antibióticos orais foram administrados em 88% e 82% dos casos, respetivamente. Os antifúngicos orais foram prescritos para o tratamento da MD (85%), FD (70%) e OE (59%). Todas as doenças foram tratadas topicamente com antibióticos, antifúngicos e glucocorticóides. Alternativas, como produtos à base de mel, não foram frequentemente prescritas pelos clínicos portugueses. Casos clíncos causados por S. pseudintermedius multirresistentes (MDR) são frequentemente observados em clínica o que reduz dramaticamente as opções de antibioterapia. O segundo trabalho avaliou a incidência de multirresistência em S. pseudintermedius sensíveis (MSSP) e resistentes à meticilina (MRSP), previamente recolhidos de cães com SBF. Observou-se que todos os MRSP eram resistentes à amoxicilinaácido clavulânico, clindamicina e eritromicina. Foram observados elevados níveis de resistência ao trimetoprim-sulfametoxazol (97%), tetraciclina e gentamicina (87%), cefalotina (83%), enrofloxacina (83%), pradofloxacina (80%) e minociclina (50%). Observou-se um baixo nível de resistência ao cloranfenicol (17%), amicacina (7%) e rifampicina (7%). A maioria dos isolados eram MDR (38/60). Todos os isolados não-MDR eram MSSP. A resistência à meticilina foi associada à multirresistência a outras classes de antibióticos. O terceiro trabalho apresentava como objetivos determinar a eficácia in vitro do MH contra S. pseudintermedius e M. pachydermatis e o tempo necessário para obter o efeito bactericida ou fungicida. Sessenta isolados de S. pseudintermedius recolhidos de cães com SBF, foram divididos em grupos MRSP/MSSP e MDR/não-MDR. Testaram-se também vinte isolados de M. pachydermatis recolhidos de cães com OE. O MH foi testado não diluído e diluído a 80%, 40%, 30%, 20%, 15%, 10%, 7,5%, 5%, 3,7% e 2,5% p/v. A concentração mínima bactericida (MBC) de S. pseudintermedius foi de 20% p/v, sem diferença entre MSSP/MRSP ou MDR/não-MDR. Para M. pachydermatis a concentração mínima fungicida (MFC) foi de 40% p/v. Realizou-se um ensaio de “time-kill” em períodos distintos. O MH diluído eliminou ambos os microrganismos após 4 horas. O MH diluído a 40% p/v manteve atividade contra S. pseudintermedius mas demorou mais tempo a eliminar os isolados de M. pachydermatis. Finalmente, determinou-se a eficácia in vitro do HBO contra os isolados de S. pseudintermedius e M. pachydermatis. Avaliou-se a eficácia da componente de mel (HO) do produto. Os isolados de S. pseudintermedius e M. pachydermatis foram testados contra diluições seriadas de HBO (40%, 20%, 10%, 5% e 2,5% p/v). O HBO contém HO na concentração de 40%. O HO foi testado puro e em diluições seriadas (40%, 20%, 10%, 5% e 2,5% p/v). Aplicou-se o mesmo protocolo após exposição à catalase para avaliar a presença de peróxido de hidrogénio. Realizou-se um ensaio de “time-kill” para determinar tempo de exposição para obter efeito bactericida/fungicida. O MBC para S. pseudintermedius foi de 20% p/v (5-20% p/v) para HBO e HO. O HBO demonstrou valores de MBC inferiores em comparação com o HO (P=0,003). Não houve diferença entre MSSP/MRSP (HBO P=0,757; HO P=0,743). Apenas o HO foi afetado pela catalase (P=0,015). O MFC para o HBO foi de 10% p/v (5-10% p/v) e 40% p/v para o HO (20-≥40% p/v). Todos os isolados foram eliminados após 4 horas de exposição ao HBO. Resumindo, este trabalho contribui para melhorar o nosso conhecimento sobre o efeito do mel sobre o S. pseudintermedius e a M. pachydermatis. Demonstra especificamente que ambos microrganismos são sensíveis ao MH, HBO e ao mel que compõem este produto. Tratamentos à base de mel não são, no entanto, prescritos frequentemente pelos clínicos portugueses e, os antibióticos orais, são frequentemente utilizados na prática clínica, apesar da propagação do S. pseudintermedius resistente à meticilina. Finalmente, este trabalho acrescenta evidências de que S. pseudintermedius resistente à meticilina pode ser multirresistente, reforçando a necessidade de opções não-antibióticas. Os resultados poderão ser usados futuramente em ensaios clínicos que tenham como objetivo tratar infecções cutâneas e de canal auditivo causados por S. pseudintermedius e/ou M. pachydermatis.
Medical honey is considered an effective economic option for the treatment for infected wounds. Medical grade Manuka honey (MH) and a honey-based gel (HBO) are now available, allowing for the prescrition of safe, non-antibiotic, effective and standardised products. In the present doctoral thesis, we aimed to determine how clinicians diagnosed and treated dermatological conditions caused by Staphylococcus pseudintermedius and Malassezia pachydermatis in Portugal. We demonstrated to both pathogens often cause superficial bacterial folliculitis (SBF), fold dermatitis (FD), Malassezia dermatitis (MD) and otitis externa (OE). Oral antibiotics were widely prescribed for treatment of SBF, particularly amoxicillin-clavulanic acid (100%), cephalexin (94%), enrofloxacin (67%), or marbofloxacin (60%). Fold dermatitis and OE, were also treated with oral antibiotics in 88% and 82% of cases, respectively. Oral antifungals were often prescribed for MD (85%), FD (70%), and OE (59%). All the diseases were frequently treated topically with antibacterials, antifungals, and glucocorticoids. Alternative options such as honey-based products were not frequently used by clinicians. Multidrug resistant (MDR) S. pseudintermedius isolates are frequently observed, a fact that dramatically decreases antimicrobial treatment options. The second part of this work evaluated the incidence of multidrug resistance in methicillin-susceptible and resistant S. pseudintermedius isolates (MSSP/MRSP) previously collected from dogs with SBF. All MRSP exhibited resistance to amoxicillin-clavulanic acid, clindamycin and erythromycin. High resistance levels were observed to trimethoprimsulfamethoxazole (97%), tetracycline and gentamicin (87%), cefalothin (83%), enrofloxacin (83%), pradofloxacin (80%) and minocycline (50%). Low resistance level was observed for chloramphenicol (17%), amikacin (7%) and rifampicin (7%). Most isolates were multidrug resistant (MDR, 38/60). All nonMDR isolates were MSSP. Methicillin resistance was associated with MDR to other classes of antibiotics. The third part of this work, aimed to determine the in vitro efficacy of MH against S. pseudintermedius and M. pachydermatis and necessary exposure time for a bactericidal or fungicidal effect. Sixty S. pseudintermedius, previously isolated from dogs with canine SBF, were divided into MRSP/MSSP and MDR/non-MDR groups. Twenty M. pachydermatis isolates, also previously isolated from dogs with OE were tested. Manuka honey was tested undiluted and diluted at 80%, 40%, 30%, 20%, 15%, 10%, 7.5%, 5%, 3.7% and 2.5% w/v. For S. pseudintermedius the minimal bactericidal concentration (MBC) was 20% w/v with no difference between MSSP/ MRSP or MDR/non-MDR. For M. pachydermatis the minimal fungicidal concentration (MFC) was 40% w/v. A time-kill assay was performed at different times. Undiluted MH killed both microorganisms after 4 hours of exposure. Diluted MH at 40% w/v maintained activity against S. pseudintermedius but took longer time to kill M. pachydermatis isolates. Finally, we determined the in vitro efficacy of HBO, against isolates of S. pseudintermedius and M. pachydermatis. The efficacy of the product’s honey component (HO) was also evaluated. All isolates were tested against serial dilutions of HBO (40%, 20%, 10%, 5% and 2.5% w/v). HBO contains HO at 40%. HO was tested pure and after serial dilutions (40%, 20%, 10%, 5% and 2.5% w/v). The same protocol was applied after exposure to catalase to determine the influence of hydrogen peroxide. A time-kill assay was performed determine the period of time necessary for a bactericidal/fungicidal effect. MBC for S. pseudintermedius was 20% w/v (5-20% w/v) for HBO and HO. HBO had lower MBC values when compared to HO (P=0.003). No difference was observed between MSSP/MRSP isolates (HBO P=0.757, HO P=0.743). Only HO was affected by catalase (P=0.015). MFC for HBO was 10% w/v (5-10% w/v) and 40% w/v for HO (20-≥40% w/v). All isolates were killed after 4 h of exposure to HBO. In summary, our work indicates that honey was effective against S. pseudintermedius and M. pachydermatis. We could demonstrate that both microorganisms are susceptible to MH, HBO and the honey that composes this product. Nevertheless, honey treatments are not frequently prescribed by Portuguese clinicians and oral antibiotics are overused in small practice, despite the spread of antibiotic resistant S. pseudintermedius. Finally, the work adds further evidence that methicillin resistant S. pseudintermedius may be multidrug resistant which reinforces the need for non-antibiotic options. The results obtained can be used in future clinical trials aiming to treat skin and ears infections caused by S. pseudintermedius and M. pachydermatis.

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.

碩士
國立臺灣大學
獸醫學研究所
107
Staphylococcus pseudintermedius is an opportunistic pathogen for causing pyoderma in dogs. The number of human cases with S. pseudintermedius infection, mainly acquired from dogs, is also increasing during recent years. S. pseudintermedius-induced soft tissue infection (STI) is a typical biofilm-related infectious disease, which is refractory to routine antibiotic treatment. In addition, methicillin resistance S. pseudintermedius-induced STIs in dogs and human are also rapidly emerging recently. Therefore, to understand the pathogenesis of S. pseudintermedius infection for developing the effective strategies for the prevention and treatment of S. pseudintermedius-induced infections is imperative. The biochemical properties of S. pseudintermedius are similar to those of Staphylococcus aureus, but the current understanding of the pathogenesis of S. pseudintermedius is still limited. In S. aureus, accessory gene regulator (agr) system, which is a quorum sensing and a two-component system, mediate S. aureus virulence for causing diseases, but the role of agr in the pathogenesis of S. pseudintermedius is still unknown. Therefore, in this study, the virulence of different agr types of S. pseudintermedius for causing STI was characterized. To investigate the pathogenesis of S. pseudintermedius-induced skin infection, the mouse pyoderma model was established. Fourty-seven clinical MRSP isolates were classified into four types by agr classification, namely type I (52%), type II (2%), type III (35%) and type IV (10%). The agr type III and type IV of MRSP isolates showed stronger ability to cause skin infection in vivo. The abilities of MRSP to resist neutrophil killing and to form the biofilm are not correlated to the virulence of MRSP for causing skin abscesses. The results of cytotoxicity assay, cell adhesion and cell invasion tests showed that agr type III and type IV isolates perform significantly better than other agr types of MRSP for causing skin infection. Taken together, agr type III and type IV of S. pseudintermedius isolates show greater abilities to adhere, invade and damage host cells, which contribute to bacterial virulence for causing pyoderma.