In the past decade, Staphylococcus aureus has developed two distinct vancomycin resistance mechanisms. First, the bacterium is capable of generating a thickened, poorly crosslinked cell wall that creates false targets. These targets cause vancomycin to bind at the periphery of the thickened peptidoglycan, allowing normal cell wall formation to continue at the cell membrane. Second, S. aureus has acquired genes from Enterococcus that encode an alternative stem peptide. The genes, known as van genes, alter the target of vancomycin, rendering vancomycin treatment ineffectual.In this work, we attempted to further characterize both mechanisms of vancomycin resistance. First, a potential link between up-regulated purine biosynthesis and increased vancomycin resistance due to a thickened cell wall was examined. Despite exploration of multiple mechanisms to increase purine levels within the cell, increased purine synthesis did not provide S. aureus with any advantage in the presence of vancomycin. However, during the investigation, purine biosynthesis in S. aureus was further characterized by confirming purr as the repressor of the purine pathway and demonstrating its sensitivity to mutation.Next, the relationship between homotypic oxacillin resistance and increased vancomycin resistance in the absence of the van genes was investigated. Vancomycin passage of two heterotypic methicillin resistant S. aureus (MRSA) caused these strains to convert to homotypic oxacillin resistance in the absence of oxacillin exposure. Additionally, conversion of heterotypic oxacillin resistant strains to homotypy by oxacillin passage increased strain survival in vancomycin. The SOS response was examined as the possible link between conversion to homotypic oxacillin resistance and increasing vancomycin resistance due to a thickened cell wall. The current study, however, detected no induction of the SOS response during vancomycin exposure.Lastly, the relationship between oxacillin resistance and vancomycin resistance due to the acquisition of the van genes was examined. In vitro and in vivo methods were utilized to determine the effectiveness of a combination of β-lactam antibiotics and vancomycin to treat vancomycin resistant S. aureus (VRSA) infections. Combination therapy provided a significant advantage over untreated control or either antibiotic alone in the rabbit model of endocarditis.

The Problem: Novel community-acquired, non-multiresistant strains of oxacillin- (methicillin-) resistant Staphylococcus aureus (ORSA) have emerged in many parts of the globe. Little is known of the clinical features, the epidemiology, and the antibiotic treatment of these strains. Materials and Methods: A retrospective chart review was performed on patients presenting to Emergency Departments or Dermatology Clinics with staphylococcal infections. Patients were stratified into three groups, non-multiresistant ORSA (NORSA), multiresistant ORSA (MORSA) and oxacillin-susceptible S. aureus, and clinical comparisons made. Strains of NORSA and MORSA were typed using antibiograms, phage typing and pulsed-field gel electrophoresis. Antimicrobial studies were performed to compare different methods of detecting resistance to oxacillin and to non-beta-lactams. Time-kill studies were performed with one drug to explore killing kinetics. The interaction between drug combinations was examined using disk approximation and time-kill methodologies. A single point pharmacodynamic analysis was performed. Results: There was an increase in infections with NORSA, MORSA and OSSA. NORSA strains appeared to be more virulent than OSSA and MORSA strains. NORSA was strongly associated with skin and soft tissue infections and with Polynesians. Most of the NORSA strains were related to New Zealand ????Western Samoan Phage Pattern???? (WSPP) isolates, and unrelated to community-acquired, non-multiresistant MRSA strains from Western Australia. Two patients were found to have British EMRSA-15 strains. NORSA strains were unrelated to MORSA strains. Resistance to rifampicin, fusidic acid, ciprofloxacin and trimethoprim emerged in the time-kill assays. Combinations of antibiotics, particularly with ciprofloxacin, often showed antagonism. Gentamicin, fusidic acid, clindamycin, teicoplanin, vancomycin, and linezolid were predicted to perform well. Ciprofloxacin, erythromycin, doxycycline, flucloxacillin and quinupristin/dalfopristin were predicted to fail. Conclusions: WSPP strains of New Zealand and EMRSA-15 strains from Britain exist in South Western Sydney. These organisms are virulent, and increasing in incidence in several areas of Australia. Antimicrobial treatment of infections with these strains is problematic and requires further study.

The Staphylococci are gram-positive bacteria that cause infections in humans and can produce severe morbidity and mortality. Methicillin resistant S. aureus (MRSA) isolates are resistant to all beta-lactam antibiotics, such as methicillin, and cephalosporins making treatment of these infections more difficult. MRSA has become prevalent throughout the United States, spreading in the health care setting and the community.The purpose of this study is to examine methicillin resistance among S. aureus isolates in an outpatient population in the state of Florida and asses possible associations between methicillin resistance and age group, gender, and geographic area. It is important to define methicillin resistance in a population so that clinical practice can adjust to the prevalence of resistance.The dataset used for this analysis is a record of all the S. aureus isolates tested by a large lab company in the state of Florida from January 1, 2003 to December 31, 2005. This is the first study to asses methicillin resistance with a population based dataset and not patients from hospitals. The percent of isolates that were methicillin resistant increased as year increased. This increase in the number of methicillin resistant isolates was significant for both the crude and adjusted analysis. When treated as a continuous variable and adjusted for age category, gender, and county of residence the odds ratio for year is 1.446, 95% CI: 1.410- 1.484. In 2005, 49.7% of the isolates were methicillin resistant. Methicillin resistance also varied by age category, gender, county, and region. For age group and gender the differences were not large and may not be clinically significant. However, there was substantial variation in methicillin resistance by region and county of residence.With nearly half of the S. aureus isolates being methicillin resistant, the beta-lactam antibiotics may no longer be an ideal choice for treating S. aureus infections in Florida. The percentage of MRSA isolates that were resistant to trimethoprim-sulfamethoxazole, tetracycline, gentamycin, and rifampin was low. These antibiotics may be feasible alternatives to treat outpatient S. aureus infections in Florida.

The Problem: Novel community-acquired, non-multiresistant strains of oxacillin- (methicillin-) resistant Staphylococcus aureus (ORSA) have emerged in many parts of the globe. Little is known of the clinical features, the epidemiology, and the antibiotic treatment of these strains. Materials and Methods: A retrospective chart review was performed on patients presenting to Emergency Departments or Dermatology Clinics with staphylococcal infections. Patients were stratified into three groups, non-multiresistant ORSA (NORSA), multiresistant ORSA (MORSA) and oxacillin-susceptible S. aureus, and clinical comparisons made. Strains of NORSA and MORSA were typed using antibiograms, phage typing and pulsed-field gel electrophoresis. Antimicrobial studies were performed to compare different methods of detecting resistance to oxacillin and to non-beta-lactams. Time-kill studies were performed with one drug to explore killing kinetics. The interaction between drug combinations was examined using disk approximation and time-kill methodologies. A single point pharmacodynamic analysis was performed. Results: There was an increase in infections with NORSA, MORSA and OSSA. NORSA strains appeared to be more virulent than OSSA and MORSA strains. NORSA was strongly associated with skin and soft tissue infections and with Polynesians. Most of the NORSA strains were related to New Zealand ????Western Samoan Phage Pattern???? (WSPP) isolates, and unrelated to community-acquired, non-multiresistant MRSA strains from Western Australia. Two patients were found to have British EMRSA-15 strains. NORSA strains were unrelated to MORSA strains. Resistance to rifampicin, fusidic acid, ciprofloxacin and trimethoprim emerged in the time-kill assays. Combinations of antibiotics, particularly with ciprofloxacin, often showed antagonism. Gentamicin, fusidic acid, clindamycin, teicoplanin, vancomycin, and linezolid were predicted to perform well. Ciprofloxacin, erythromycin, doxycycline, flucloxacillin and quinupristin/dalfopristin were predicted to fail. Conclusions: WSPP strains of New Zealand and EMRSA-15 strains from Britain exist in South Western Sydney. These organisms are virulent, and increasing in incidence in several areas of Australia. Antimicrobial treatment of infections with these strains is problematic and requires further study.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / The large use of oxacillin in Brazilian hospitals and the ausence of scientific data confirming its safety, justify the necessity of carrying out well âstructured studies, identifying the existence of possible triggering factors of the reactions. AIM: To study the use of oxacillin and the occurrence of adverse reactions in two different groups, identifying risk factors associated. METHODS: Children using oxacillin were monitorized from October 2000 to July 2001 in the Universitary Hospital Walter CantÃdio (HUWC), as well as ones under treatment with oxacillin of block D from Childrenâs Hospital Albert Sabin in the period of July 2001 and March 2002 (FIRST STUDY). Also, patients under treatment with oxacillin from HUWC, during the period of July until October 2003, were followed up (SECOND STUDY). The follow-up was done through daily visits in the infirmary and analysis of medical records and prescriptions, being observed the clinical history, prescribed drugs, laboratorial exams, oxacillin use, adverse reaction reports related to oxacillin (AROx) and procedures done due to these AROx. The reactions were reported and classified according to causality and gravity (both studies) and hypersensitivity type (in the SECOND STUDY). Statistical tests were used promptly. RESULTS: FIRST STUDY: In 130 children monitorized were observed both a large oxacillin exposure in the masculine sex, being used the oxacillin average dose of 216,3mg/kg/day. The accumulative incidence (AI) of AROx was equal to 20,8%, of the total of 3352 patients-day monitorized (incidence density/ ID = 0,8). Of the 43 reactions reports, the most frequent reactions were fever (50%) and cutaneous rash (35,7%). The procedure more used in the case of occurrence of AROx was the suspension of oxacillin use (51,9%). The most of reactions presented causality and gravity as Probable (55,6%) and Moderate (92,6%), respectively. In addition, the average of exposure time to oxacillin and hospitalization time were significantly different between the groups of patients that presented AROx or no. The relative risk (RR) of oxacillin exposure by more than 14 days was 5,46 to AROx occurrence. SECOND STUDY: Of the 76 patients, 36,8% ones related to allergic antecedents. Of these, 72,5% were associated to use of drugs. Oxacillin was considered as first choice drug for treatment of 75% of patients. The association oxacillin plus ceftazidima was the most used (22%) during treatments. The average dose of oxacillin prescribed was 206mg/kg/day. The average time of treatment with oxacillin was 15 days. The ineffective therapeutic (21,1%) and occurrence of adverse reaction (15,8%) caused the suspension of this antibiotic. The permanence time in the hospital of patients was 32,4% (patients-day total: 2463; ID = 0,97). The most frequent reactions were the increase of transaminases (22,1%), fever (17%) and cutaneous rash (13,6%). The majority of reactions showed causality, gravity and hypersensitivity as Probable (44,1%), Moderate (66,1%) and type B (86,4%). The presence of AROx was frequent between patients with age equal or under 14 years old (P = 0,0159)/ RR = 2,22). The incidence of adverse reactions to other medicaments was higher in the AROx patient group (P = 0,0036)/ RR = 2,66). 34% of followed patients presented at least one drug related problem involving oxacillin (DRPOx). A total of 71 DRPOx was identified. Of these DRPOx, the detection of adverse reactions was predominant (33,8%). CONCLUSION: The careful use of oxacillin is recommended in paediatrics, with duration of treatment established promptly. The empirical and prolonged exposure of this drug should be avoided. / O elevado grau de utilizaÃÃo da oxacilina nos hospitais do Brasil e a inexistÃncia de dados cientÃficos que comprovem sua seguranÃa justificam a necessidade da realizaÃÃo de estudos bem planejados que visem testar a existÃncia de fatores desencadeantes das reaÃÃes. OBJETIVOS: Estudar a utilizaÃÃo da oxacilina e a ocorrÃncia de reaÃÃes adversas em duas populaÃÃes distintas, apontando fatores de risco associados. METODOLOGIA: Foram acompanhadas crianÃas expostas à oxacilina no Hospital UniversitÃrio Walter CantÃdio (HUWC), entre outubro/2000 e julho/2001, e no bloco D do Hospital Infantil Albert Sabin, entre julho/2001 e marÃo/2002 (PRIMEIRO ESTUDO); e tambÃm, os pacientes adultos e as crianÃas expostos à oxacilina no HUWC, no perÃodo de julho a outubro de 2003 (SEGUNDO ESTUDO). O seguimento de pacientes foi feito atravÃs de visitas diÃrias Ãs enfermarias, anÃlise de prontuÃrios e prescriÃÃes, sendo observada a histÃria clinica, medicamentos prescritos, resultados de exames laboratoriais, perfil de utilizaÃÃo da oxacilina, ocorrÃncia de reaÃÃo adversa à oxacilina (RAOx) e procedimentos adotados devido à RAOx. Os casos foram notificados e classificados quanto à causalidade e gravidade (nos dois estudos) e quanto ao tipo de hipersensibilidade (no segundo estudo), sendo realizados testes estatÃsticos pertinentes. RESULTADOS: PRIMEIRO ESTUDO: A amostra foi composta por 130 crianÃas, sendo observada uma maior exposiÃÃo do sexo masculino (56,9%) e uma dose mÃdia de oxacilina utilizada, equivalente a 216,3 mg/kg/dia. A incidÃncia acumulada (IA) de RAOx foi igual a 20,8%, para um total de 3352 pacientes/dia acompanhados (densidade de incidÃncia/ DI = 0,8). De 43 reaÃÃes relatadas, as mais freqÃentes foram febre (50%) e rash cutÃneo (35,7%), A conduta mais utilizada na ocorrÃncia de RAOx foi a suspensÃo do uso da oxacilina (51,9%). A maioria das reaÃÃes tiveram causalidade ProvÃvel (55,6%) e gravidade Moderada (92,6%). As mÃdias do tempo de exposiÃÃo à oxacilina e do tempo de internamento, diferiram significantemente entre os grupos de pacientes com e sem RAOx. O risco relativo (RR) da exposiÃÃo à oxacilina por mais de 14 dias foi de 5,46 para a ocorrÃncia de RAOx. SEGUNDO ESTUDO: Dos 76 pacientes monitorizados, 36,8% referiram antecedentes alÃrgicos, dos quais 72,5% foram atribuÃdos ao uso de medicamentos. A oxacilina foi primeira escolha para o tratamento de 75% dos pacientes monitorizados e a ceftazidima, a associaÃÃo mais utilizada (22%). A dose mÃdia prescrita de oxacilina foi de 206mg/kg/dia e o tempo mÃdio de exposiÃÃo, igual a 15 dias. A ineficÃcia terapÃutica e a ocorrÃncia de reaÃÃo adversa, foram os causadores da suspensÃo do tratamento com oxacilina em 21,1% e 15,8% dos casos, respectivamente. O tempo de internaÃÃo dos pacientes foi em mÃdia 32,4 dias. Observou-se uma IA de RAOx igual a 31,6%, para um total de 2463 pacientes-dia monitorizados (DI = 0,97). As reaÃÃes mais freqÃentes foram: aumento de transaminases (22,1%), febre (17,0%) e rash cutÃneo (13,6%). A maioria das reaÃÃes foi considerada de causalidade ProvÃvel (44,1%), gravidade Moderada (66,1%) e hipersensibilidade do tipo B (86,4%). A ocorrÃncia de RAOx foi mais freqÃente entre os pacientes com idade igual ou menor que 14 anos (P = 0,0159/ RR = 2,22) e a incidÃncia de reaÃÃo adversa a outros medicamentos, foi maior no grupo de pacientes com RAOx (P = 0,0036/ RR = 2,66). Cerca de 34% dos pacientes monitorizados apresentaram pelo menos 01 problema relacionado com medicamento envolvendo a oxacilina (PRMOx), sendo identificado um total de 71 PRMOx, dos quais a ocorrÃncia de reaÃÃo adversa foi o mais freqÃente (33,8%). CONCLUSÃO: Recomenda-se a administraÃÃo cautelosa de oxacilina em crianÃas, com duraÃÃo do tratamento estabelecida, evitando-se tratamento empÃrico e uso prolongado.

A capacidade do Staphylococcus aureus escapar do sistema imune do hospedeiro é atribuída à existência de algumas exotoxinas, as quais são codificadas por genes acessórios. A expressão de genes codificadores de exotoxinas é controlada por reguladores, tais como o agr (acessory gene regulator), sendo identificados 4 tipos polimórficos deste regulador. Neste estudo, foram utilizadas 37 isolados de Staphylococcus aureus, sensíveis à oxacilina, originados de laboratórios e hospitais públicos e privados brasileiros. Utilizou-se a técnica de PCR para a amplificação dos genes codificadores dos fatores de virulência estudados e do tipo de agr presente em cada cepa. A técnica da eletroforese em campo pulsado (PFGE) foi utilizada para se verificar a existência clonal entre os isolados. Observou-se que o gene codificador de enterotoxina mais freqüentemente isolado foi o gene sea, todos relacionados com o tipo III de agr. Observou-se, também, alguns pontos divergentes com publicações anteriores como a presença dos genes seb e tst na mesma cepa (33%), cuja relação julgava-se não ser muito freqüente, o mesmo acontecendo com os genes tst e lukElukD. Não se encontrou isolado portador dos genes codificadores das toxinas esfoliatinas. Segundo a técnica de PFGE, não houve relação clonal entre os isolados. Conclui-se, portanto, que as bases moleculares da patogenicidade do S. aureus são multi-fatoriais, dependendo não somente da presença como também da expressão de alguns genes acessórios como também do tipo de agr presente. / The capacity of the Staphylococcus aureus to escape from the immune system of the host is conferred to the existence of some exotoxins, which are codified by accessory genes. The expression of the genes of exotoxins is controlled by regulators, such as agr (acessory gene regulator), being identified four different types of this regulator. In this study, it had been used 37 strains of Staphylococcus aureus, sensible to the oxacillin, proceeding from public and private Brazilians laboratories. It had been used the PCR technique for detention of the genes of the virulence factors and the agr type present in each strain. The PFGE had been used for detectation of clonal relationship between the strains. Thus, it was observed that the most frequently determined gene coder of enterotoxin was sea (100% were related as type of agr III). it was observed divergent points to the studies carried through in other countries, like strains with coders genes of seb and tst (33%).This correlation, the literature judged not to be frequent, the same happening for the simultaneous presence of tst and lukE-lukD. It was not found in this study strains carrying of exfoliatin toxins. According to PFGE, there wasn\' t evidence of clonal relationship between the strains. It is concluded, therefore, that the molecular bases of the pathogenicity of the S. aureus are multifactorial, depending not only on the presence, as also of the expression of some accessory genes and the agr type present.

A capacidade do Staphylococcus aureus escapar do sistema imune do hospedeiro é atribuída à existência de algumas exotoxinas, as quais são codificadas por genes acessórios. A expressão de genes codificadores de exotoxinas é controlada por reguladores, tais como o agr (acessory gene regulator), sendo identificados 4 tipos polimórficos deste regulador. Neste estudo, foram utilizadas 37 isolados de Staphylococcus aureus, sensíveis à oxacilina, originados de laboratórios e hospitais públicos e privados brasileiros. Utilizou-se a técnica de PCR para a amplificação dos genes codificadores dos fatores de virulência estudados e do tipo de agr presente em cada cepa. A técnica da eletroforese em campo pulsado (PFGE) foi utilizada para se verificar a existência clonal entre os isolados. Observou-se que o gene codificador de enterotoxina mais freqüentemente isolado foi o gene sea, todos relacionados com o tipo III de agr. Observou-se, também, alguns pontos divergentes com publicações anteriores como a presença dos genes seb e tst na mesma cepa (33%), cuja relação julgava-se não ser muito freqüente, o mesmo acontecendo com os genes tst e lukElukD. Não se encontrou isolado portador dos genes codificadores das toxinas esfoliatinas. Segundo a técnica de PFGE, não houve relação clonal entre os isolados. Conclui-se, portanto, que as bases moleculares da patogenicidade do S. aureus são multi-fatoriais, dependendo não somente da presença como também da expressão de alguns genes acessórios como também do tipo de agr presente. / The capacity of the Staphylococcus aureus to escape from the immune system of the host is conferred to the existence of some exotoxins, which are codified by accessory genes. The expression of the genes of exotoxins is controlled by regulators, such as agr (acessory gene regulator), being identified four different types of this regulator. In this study, it had been used 37 strains of Staphylococcus aureus, sensible to the oxacillin, proceeding from public and private Brazilians laboratories. It had been used the PCR technique for detention of the genes of the virulence factors and the agr type present in each strain. The PFGE had been used for detectation of clonal relationship between the strains. Thus, it was observed that the most frequently determined gene coder of enterotoxin was sea (100% were related as type of agr III). it was observed divergent points to the studies carried through in other countries, like strains with coders genes of seb and tst (33%).This correlation, the literature judged not to be frequent, the same happening for the simultaneous presence of tst and lukE-lukD. It was not found in this study strains carrying of exfoliatin toxins. According to PFGE, there wasn\' t evidence of clonal relationship between the strains. It is concluded, therefore, that the molecular bases of the pathogenicity of the S. aureus are multifactorial, depending not only on the presence, as also of the expression of some accessory genes and the agr type present.

Made available in DSpace on 2016-04-28T20:16:28Z (GMT). No. of bitstreams: 1 2008 - Shana de Mattos de Oliveira Coelho.pdf: 1309260 bytes, checksum: d56e11e43855d92c6e9b5e2b5f5f4ab0 (MD5) Previous issue date: 2008-12-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The present study was done to characterize phenotypic and genotypically the virulence factors and resistance profile to antibiotics, specifically to the oxacillin, of species of Staphylococcus spp. coagulase-positives isolated from milk samples of cows with mastitis. Of 150 isolated, the species S.aureus, S.intermedius and S.hyicus were identified. The antimicrobial susceptibility test yielded high resistance to penicillin and ampicillin and distinct antibiotyps had been identified, being of sensitivity to all the antibiotics prevalent. A total of 94,6% and 70,7% was positive for the production of betalactamases and presence of the mecA gene, respectively. The femA gene was detected in 80,9% of isolated mecA positive. The isolated presented distinct standards of growth in the tests of susceptibility to oxacillin. The tests of agar screen and the evaluation of the microdilution in plate, presented considerable values of sensitivity and specificity when the isolated ones were considered as an only group (Staphylococcus spp coagulase-positives) or when the species had been discriminated, respectively. The addition of 4% of NaCl in the agar plate can have effect in the increase of the sensitivity of the test of diffusion using the oxacillin and the test of diffusion presented the better capacity in the detention of the resistance isolates with cefoxitin disk. A total of 80% of the S.aureus was classified as MRSA and had presented resistance higher than 15% to the ampicillin and sulbactam association, vancomicin and gentamicin. About the virulence factors, the technique of the microplate disclosed 82.8% of isolated producers of slime and the intensity of the production was not associated with the presence of the icaA and icaD genes. In addition, the agar with the congo red presented effectiveness in the detention of isolated the not producing ones of slime . In all hemolitics isolates were detected the beta-hemolisin and 11.3% of the non-hemolitics had produced hemolisins in the presence of beta-hemolitic strain, indicating the presence of the delta hemolisin. spaA, coa and agr genes had been carried through the amplifications of the genes the S.aureus. The spaA gene, that it codifies protein A region X, was positive in all isolated ones, disclosed only amplicon for each isolated one and presented polimorfism being the prevalent size of 315pb. The amplification of the coa gene presented three distinct polimorfics types, with only amplicon for each isolated and the prevalent profile was of 900pb. The amplification of the agr gene resulted in amplicons with 350 and 550 pb in 74% of the isolated. The test of biotyping disclosed the bovine ecovar as the prevalent in the S.aureus. Seven S.aureus that had presented the same profiles of the coa, spaA, agr genes, antibiotyps and ecovars was detected being 4 pertaining the same properties and 3 isolated from distinct properties. / O presente estudo foi conduzido para caracterizar feno-genotipicamente os fatores de virul?ncia e perfil de resist?ncia aos antibi?ticos, especificamente ? oxacilina, de esp?cies de Staphylococcus spp. coagulase-positivos isolados de amostras de leite de vacas com mastite. Do total de 150 isolados, foram identificadas as esp?cies S.aureus, S.intermedius e S.hyicus. O teste de suscetibilidade antimicrobiana revelou elevada resist?ncia ? penicilina e ampicilina e distintos antibiotipos foram identificados, sendo o de sensibilidade a todos os antibi?ticos o prevalente. Um total de 94,6% e 70,7% foi positivo para a produ??o de betalactamases e presen?a do gene mecA, respectivamente. O gene femA foi detectado em 80,9% dos isolados mecA positivos. Os isolados apresentaram distintos padr?es de crescimento nos testes de suscetibilidade ? oxacilina. Os testes de ?gar screen e a avalia??o da microdilui??o em placa, apresentaram valores consider?veis de sensibilidade e especificidade quando os isolados foram considerados como um ?nico grupo (Staphylococcus spp coagulase-positivos) ou quando as esp?cies foram discriminadas, respectivamente. A adi??o de 4% de NaCl no meio de cultura pode ter efeito no aumento da sensibilidade do teste de difus?o em disco utilizando o disco de oxacilina e o teste de difus?o em disco apresentou melhor capacidade na detec??o dos isolados resistentes ? oxacilina com discos de cefoxitina. Um total de 80% dos S.aureus foi classificados como MRSA e apresentaram resist?ncia superior a 15% ? associa??o de ampicilina e sulbactam, vancomicina e gentamicina. Quanto aos fatores de virul?ncia, a t?cnica da microplaca revelou 82,8% de isolados produtores de slime e a intensidade da produ??o n?o foi associada ? presen?a dos genes icaA e icaD. Em adi??o, o ?gar contendo vermelho congo apresentou efic?cia na detec??o do isolados n?o produtores de slime . Em todos os isolados hemol?ticos foi detectada a beta hemolisina e 11,3% dos n?o-hemol?ticos produziram hemolisinas na presen?a da cepa beta-hemol?tica, indicando a presen?a da delta hemolisina. Foram realizadas as amplifica??es dos genes spaA, coa e agr em todos os S.aureus. O gene spaA, que codifica a regi?o X, foi positivo em todos isolados, revelou um ?nico amplicon para cada isolado e apresentou polimorfismo sendo o tamanho prevalente o de 315pb. A amplifica??o do gene coa apresentou tr?s tipos polim?rficos distintos, com ?nico amplicon para cada isolado e o perfil prevalente foi o de 900 bp. A amplifica??o do gene agr resultou em amplicons com 350 e 550 pb em 74% dos isolados. A prova de biotipifica??o revelou o ecovar bovino como o prevalente nos S.aureus. Foram identificados 7 S.aureus que apresentaram os mesmos perfis dos genes coa, spaA, agr, antibiotipos e ecovares, sendo 4 pertencentes mesma propriedades e 3 provenientes de propriedades distintas.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2017-04-05T19:32:12Z No. of bitstreams: 1 2012 - Gabriela Viana da Silva.pdf: 1385561 bytes, checksum: ebedf6cb2382daee2a3bf246edc9848f (MD5) / Made available in DSpace on 2017-04-05T19:32:12Z (GMT). No. of bitstreams: 1 2012 - Gabriela Viana da Silva.pdf: 1385561 bytes, checksum: ebedf6cb2382daee2a3bf246edc9848f (MD5) Previous issue date: 2012-03-27 / CAPES / The sheep milk production in Brazil is a relatively new activity, whose production is directed towards the manufacture of cheeses, yogurts and other products. Therewith a deficit of scientific papers related to this activity. Considered an excellent substrate, many pathogens can be transmitted to humans through consumption of milk and its derivatives, including Staphylococcus spp., one of the main agents involved in outbreaks of food poisoning. Moreover, the increasing of antimicrobial resistance is becoming a global public health problem. Within this problematic, this study aimed to identify the species of Staphylococcus and its antimicrobial susceptibility profile of current use in human and veterinary medicine, isolated from milk of sheep origin from rural properties in the Meridional Agreste of Pernambuco. We identified 13 different species, three coagulase-positive and 10 coagulase-negative and two strains identified only as SCN, especially Staphylococcus aureus (29) followed by S. chromogenes (15) and S. intermedius (9) by its frequency. The determination of antimicrobial susceptibility by disk diffusion revealed high rates of resistance to penicillin (53.2%) and ampicillin (45.6%). The susceptibility to oxacillin was evaluated by the disk diffusion methods for oxacillin and cefoxitin, Screen Agar and determination of Minimum Inhibitory Concentration through tests of broth microdilution and agar, the latter two detected resistance in 24% and 6% of isolates, respectively. All isolates were negative for the presence of blaZ and mecA genes and was not observed a correlation between phenotypic and genotypic testing for resistance to beta-lactams. The results show that this species is a source of infection, through its products, Staphylococcus potentially pathogenic to humans. / A produ??o de leite ovino no Brasil ? uma atividade relativamente nova, cuja produ??o est? direcionada para a confec??o de queijos, iogurtes e outros derivados. Com isso h? uma defict de trabalhos cient?ficos ligados a esta atividade. Considerado um excelente substrato, muitos micro-organismos patog?nicos podem ser veiculados ao homem atrav?s do consumo de leite e seus derivados, entre eles Staphylococcus spp., um dos principais agentes envolvidos em surtos de intoxica??es alimentares. Al?m disso, o crescente aumento da resist?ncia aos antimicrobianos vem se constituindo um problema de sa?de p?blica global. Dentro desta problem?tica, o presente estudo objetivou identificar as esp?cies de Staphylococcus e seu perfil de suscetibilidade aos antimicrobianos de uso corrente em medicina humana e veterin?ria, isoladas de leite de origem ovina provenientes de propriedades rurais no Agreste Meridional de Pernambuco. Foram identificadas 13 esp?cies diferentes, sendo tr?s do grupo Staphylococcus coagulase-positivo e 10 de Staphylococcus coagulase-negativo e duas cepas identificadas apenas como SCN, destacando-se por sua frequ?ncia Staphylococcus aureus (29) seguida pelo S. chromogenes (15) e S. intermedius (9). A determina??o da suscetibilidade aos antimicrobianos pelo teste disco difus?o revelou elevados percentuais de resist?ncia ? penicilina (53,2%) e ampicilina (45,6%). A suscetibilidade a oxacilina foi avaliada atrav?s dos m?todos de disco-difus?o para oxacilina e cefoxitina, Screen Agar e determina??o da Concentra??o Inibit?ria M?nima atrav?s dos testes de Microdilui??o em Caldo e em Agar, tendo estes dois ?ltimos detectado resist?ncia em 24% e 6% dos isolados, respectivamente. Todos os isolados foram negativos para a presen?a dos genes blaZ e mecA, n?o tendo sido observada uma correla??o entre os testes fenot?picos e genot?picos para a resist?ncia aos beta-lact?micos. Os resultados obtidos evidenciam que esta esp?cie animal ? mais uma fonte de infec??o e veiculadora, atrav?s de seus produtos, de Staphylococcus potencialmente patog?nicos para o homem.

Nas últimas décadas, os Staphylococcus coagulase-negativo, têm sido considerados como patógenos verdadeiros, sendo um dos principais grupos bacterianos responsáveis pelas infecções relacionadas a assistência a saúde (IRAS). O presente estudo teve como objetivo geral: avaliação da relação entre a resistência a oxacilina e a produção de biofilme de amostras Staphylococcus coagulase-negativo de origem comunitária e hospitalar. Neste sentido, foram desenvolvidos os seguintes objetivos específico: identificar ao nível de espécie os Staphylococcus coagulase-negativo; analisar por técnica fenotípica (Ágar vermelho do Congo) a produção de slime; avaliar quantitativamente, a produção de biofilme; correlacionar a produção de polissacarídeos extracelulares (slime) com a produção de biofilme; avaliar a relação da resistência a oxacilina como indicador da presença do gene mecA; avaliar a relação entre a concentração inibitória mínima e a concentração bactericida mínima para oxacilina; pesquisar a presença dos genes mecA, icaAD e atlE, pela técnica de PCR. Foi estudado um total de 150 amostras, sendo 50 isoladas de fômites, 50 isoladas de sangue e 50 isoladas de comunidade. Independente da origem, foram identificadas 14 espécies de Staphylococcus coagulase-negativo, sendo mais frequentes S. epidermidis 42,6%, S. haemolyticus 13,3% e S. cohnii cohnii 10,7%. A análise geral da expressão fenotípica de slime mostrou que 64% das amostras avaliadas eram produtoras de slime. Das 150 amostras testadas neste estudo, 95,3% foram produtoras de biofilme. Ao considerarmos a análise da quantificação do biofilme em relação às origens das amostras estudadas não encontramos diferenças significativas e a maioria das amostras foi considerada moderadamente produtora de biofilme. O gene mecA foi detectado em 6 amostras comunitárias, 34 amostras de fômites e 34 amostras de sangue. Não houve diferença significativa entre as amostras de fômites e sangue. Porém, houve diferença significativa entre as amostras de origem comunitária e as de origem hospitalar - fômites e sangue (p < 0,0001). Ao compararmos as três origens de isolamento quanto a presença do gene atlE observamos que houve diferença significativa (p = 0,0012) entre elas. Sendo, as amostras isoladas de sangue, as que apresentaram maior número de amostras que possuíam o gene atlE (n = 18). Das 150 amostras testadas observamos a presença do gene icaAD em 46% amostras comunitárias, 56% amostras de fômites e 60% amostras de sangue, não encontramos diferença significativa (p = 0,5750). Observamos uma correlação entre a resistência a oxacilina e a produção de slime, pois as amostras de origem hospitalar (fômites e sangue) apresentaram altos níveis de resistência a oxacilina e em sua grande maioria foram produtoras de slime. A espécie S. epidermidis foi a mais isolada e deve-se ressaltar que, quando comparada com as outras espécies, apresentou altos níveis de resistência a oxacilina, sendo a maioria produtora de slime e biofilme. / In recent decades, coagulase-negative Stapphylococci have been considered as true pathogen, one of the major bacterial groups responsible for hospital infection. The present study aimed to: assess the relationship between oxacillin resistance and biofilm production samples coagulase-negative Stapphylococci of community and hospital. In this sense, we have developed the following specific objectives: to identify to species level coagulase-negative Staphylococci; analyze by phenotypic test (Congo red Agar) slime production, evaluate quantitatively the biofilm production; correlate the production of extracellular polysaccharides (slime) with biofilm production; evaluate the relationship of resistance to oxacillin as an indicator of the presence of the mecA gene; evaluate the relationship between minimal inhibitory concentration and minimum bactericidal concentration for oxacillin; investigate the presence of the mecA gene, atlE and icaAD, by PCR. We studied a total of 150 samples, 50 were isolated from fomites, 50 from community and 50 isolated from blood. Regardless of origin, 14 species of coagulase-negative Stapphylococci were identified , being more frequent 42.6% S.epidermidis, 13.3% S. haemolyticus and 10.7% S. cohnii cohnii. A general analysis of the phenotypic expression of slime showed that 64% of the samples were slime producers. Of the 150 samples tested in this study, 95.3% produced biofilm. When we consider the analysis of quantification of biofilm in relation to the origins of the samples studied we did not find significant differences and most of the samples were considered moderately biofilm producers. The mecA gene was detected in 6 community samples, 34 samples of fomites and 34 blood samples. There was no significant difference between the samples of blood and fomites. However, there was significant differences between the samples from the community and nosocomial - fomites and blood (p <0.0001). Comparing the three origins insulation as the presence of the gene atlE we observed a significant difference (p = 0.0012) between them. Being the isolated blood samples which showed the highest number of samples that had the gene atlE (n = 18). From the 150 tested samples we observed the presence of the gene icaAD 46% in community samples, 56% samples from fomites and 60% of blood samples, we found no significant difference (p = 0.5750). We observed a correlation between oxacillin resistance and slime production, because the nosocomial (fomites and blood) samples showed high levels of resistance to oxacillin and mostly were slime producers. The species S. epidermidis were the most isolated and it should be noted that, compared with other species, it showed high levels of resistance to oxacillin, mostly producing slime and biofilm.