Dissertations / Theses on the topic 'MecA gene and PVL'

Clinical S. aureus strains were gathered from four hospitals, two in Turkey (Hacettepe hospital 200 strains and Ankara Hospital 106 strains) and the other two from Libya (Aljalla Hospital 88 strains and Jamahyria Hospital 62 strains). The clinical specimens were collected form different sources including blood, urine, wound, pus, burn, sputum, semen, catheter and aspiration. Patients were aged between 0 to 84 years and from both sexes. Resistance to Methicillin was determined by measuring the Oxacillin MIC
this was done by using the oxacillin E-test, with resistance defined as an MIC of >
2 µ
g ml. In this study all isolates displayed an Oxacillin MIC of &
#8805
256µ
g/ml. The MRSA strains were (56%) in Turkish hospitals, and (59%) in Libyan hospitals. The percentage of the VRSA and VISA in Libyan hospitals was (7%) and (26%) respectively, although the percentage of VRSA in Turkish hospitals was only 2% and there were no intermediately susceptible Staphylococcus aureus (VISA). Besides the MRSA isolates, Coagulase Negative Staphylococcus showing Methicillin resistance was collected from clinical isolates in thirteen patients in Turkish hospitals. In both countries, the majority MRSA isolates were multiresistant to more than five classes of antibiotics including
Ampicillin, Amoxicillin, Tetracycline, Erythromycin and Ciprofloxacin. Most of the MRSA isolates were from blood (68%), wounds (57%) and pus (50%).The results of genetic investigations indicated that the mecA gene was present in the majority of isolates in both countries
the community acquired MRSA type (ccr-BIV) was present in three samples out of thirty in Turkish hospitals and in one case out of twenty in Libyan hospitals
There was no case out of fifty specimens that carry the hospitals acquired MRSA type (ccr-BI, II, III) in both countries. Besides the Methicillin resistance gene, the incidence of Tetracycline resistance gene was quite high (tetM and tetK 50%) in Turkish hospitals isolates, and the prevalence of Panton-Valentine Leukocidin gene was high (PVL 70%) in Libyan hospitals specimens.

Os Staphylococcus spp coagulase negativa (SCoN) são reconhecidos como agentes etiológicos importantes de infecções humanas. A maioria dos países desenvolvidos relata um aumento de infecções nosocomiais por SCoN resistentes a meticilina e outros antibióticos. A resistência a meticilina é uma característica importante destas bactérias, porém difícil de detectar pelos métodos convencionais de suscetibilidade. Esta resistência é devido à presença de um elemento genético chamado staphylococcal cassette chromosome (SCCmec), que codifica o gene mecA. A identificação dos tipos de SCCmec é relevante para epidemiologia da resistência a meticilina dos Staphylococcus spp. Este estudo prospectivo teve como objetivo determinar a prevalência do gene mecA e a distribuição dos tipos de SCCmec nos SCoN. Também foi avaliada a eficiência do teste de disco difusão com cefoxitina e oxacilina para caracterizar a resistência a meticilina mediada pelo gene mecA. Foram analisadas um total de 181 SCoN de hemoculturas. A prevalência do gene mecA entre estes isolados foi de 71,3%. A sensibilidade dos discos de cefoxitina e oxacilina para todas as espécies de SCoN foi de 100% e 98,4%, respectivamente, enquanto que a especificidade foi de 93% e 89,3%. As espécies mais prevalentes de SCoN foram S. epidermidis (64%), seguido pelo S. hominis (10%), S. haemolyticus (8,8%) e S. capitis (7,7%). A percentagem de isolados positivos para o gene mecA foi maior para S. haemolyticus (98,3%), seguido pelo S. epidermidis (75%) e S. hominis (72,2%). Entre os 129 isolados resistentes, 36 (27.9%) foram identificados com o SCCmec tipo I, 4 (3.0%) com o SCCmec tipo II, 67 (52%) com o SCCmec tipo III, 1 (0.8%) com o SCCmec tipo IV e 4 (3.0%) com os SCCmec tipos I e III. Dezessete isolados foram não tipáveis. O SCCmec tipo III foi o mais prevalente entre os isolados de S. epidermidis (52%). Entre estas cepas, 30 (23%) apresentaram um SCCmec tipo III modificado, que amplificou uma região dcs adicional. Os SCoN meticilina resistentes (MR-SCoN) mostraram um nível de resistência aos antibióticos não -lactamicos maior quando comparados aos SCoN meticilina suscetíveis (MS-SCoN). Os isolados com o SCCmec tipo III foram mais resistentes aos antibióticos não -lactamicos do que os isolados com SCCmec tipos I, II, e IV.
Coagulase negative staphylococci (CoNS) are now recognized as important etiological agents of infections in humans. Most developed countries have reported an increase in CoNS infection in hospitalized patients, which are resistant to methicillin and other antibiotics. The methicillin resistance is an important characteristic of these bacteria. Moreover, the resistance to methicillin may not be easily detected by conventional susceptibility methods. Methicillin resistance is due to the acquisition of a large DNA element termed staphylococcal cassette chromosome mec (SCCmec), which harbored the mecA gene. The SCCmec typing is essential for understanding the molecular epidemiology of methicillin resistant Staphylococcus spp. This prospective study was aimed to determine the prevalence of mecA gene and the distribution of SCCmec types in CoNS. Also was evaluated the efficiency of the cefoxitin and oxacilin disk diffusion test to characterize the meticillin resistance mediated by the gene mecA. A total of 181 CoNS from bloodstream isolate were available for the study. The prevalence to mecA gene was 71.3%. The sensivity of oxacillin and cefoxitin disks for all CoNS species were 100% and 98.4% respectively, whereas the specificity were 93% and 89,3 %. The most prevalent SCoN species were: S. epidermidis 116 (64%) followed by S. hominis 18 (10%), S. haemolyticus 16 (8.8%) and S.capitis 14 (7.7%). The percentage of mecApositive isolates was highest for S. haemolyticus (93.8%), followed by S. epidermidis (75%) and S. hominis (72.2%). Among the 129 bloodstream isolates witch mecA gene, 36 (27.9%) harbored SCCmec type I, 4 (3.0%) harbored SCCmec type II, 67 (52%) harbored SCCmec type III, 1 (0.8%) harbored SCCmec type IV and 4 (3.0%) harbored SCCmec type I and III. Seventeen isolates were not typeable. The SCCmec type III was the most prevalent among the isolates of S. epidermidis (52%). Among these strains, 30 (23%) harbored a modified SCCmec type III, which contained, in contrast to regular type III, an additional dcs region. Methicillin resistant CoNS showed higher level of resistance to all non beta lactamics antimicrobials as compared to methicillin susceptible. The isolates with SCCmec type III were more resistant to non-beta lactamics antimicrobials than the isolates harboring SCCmec type I, II and IV.

Staphylococcus aureus is an extra-ordinarily versatile pathogen causing a wide spectrum of infections. The aims of this study are to analyze 10 clinical isolates of S. aureus from the UK by Multi Locus Sequence Typing (MLST) and determining their PVL-type variants. In addition to that, to study the effect of several antibiotics at sub inhibitory concentrations on a number of virulence factors at mRNA using quantitative PCR and protein levels using proteomic methods. Western blotting was used to study differential expression of Spa at protein levels. Data showed that the 10 clinical isolates belong to seven clonal complexes (CCs), which are CC1, CC5, CC8, CC22, CC30, CC88, and CC121. Genetic variation within lukSF-PV gene showed that three of these isolates were belong to the same PVL type variant of CA-MRSA USA300 strain, R variant. From which, two isolates were found to belong to the same CC of USA300, CC8. The remaining 7 isolates were found to belong to H variant. Data presented here showed that the sub-MIC levels of both cell wall inhibitors reduced lukSF-PV and spa steady-state mRNA levels when cells were grown in the presence of these antibiotics for one hour. However, after 5 hrs post antibiotic addition of these two antibiotics, vancomycin remained depressed lukSF-PV and spa steady-state mRNA levels as well as at protein levels, but oxacillin increased spa and lukSF-PV mRNA levels, as well as Spa at protein levels. Protein synthesis inhibitors clindamycin and linezolid were both caused an increase of lukSF-PV mRNA levels, but they both decreased spa mRNA levels, when cultures grown in the presence of these antibiotics for one hour. However, when cultures grown with these antibiotics for 5 hrs, clindamycin remained to increase lukSF-PV and decrease spa mRNA levels and protein levels, but linezolid decreased both virulence factors at mRNA and protein levels. The data showed in this study confirmed that growing S. aureus in the presence of oxacillin induce toxin expression and might enhance the virulence of this bacterium, therefore using these antibiotics to treat S. aureus infections may contribute to worse outcomes. These data also confirmed that linezolid and vancomycin, are both important selections of antimicrobial agents to treat serious infections caused by the bacterium.

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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.

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A espécie bacteriana Staphylococcus aureus é considerada um dos mais importantes patógenos humanos. Uma característica notável dessa espécie é a capacidade de adquirir resistência aos antibióticos, sendo a resistência à meticilina uma das mais significativas. Estudos recentes têm mostrado a presença de Staphylococcus aureus resistente à meticilina (MRSA) em certos grupos da população, tais como os pacientes HIV positivos, nos quais foi observado maiores riscos de infecções por esta linhagem. O objetivo deste estudo é determinar a prevalência de MRSA colonizando pacientes HIV positivos atendidos em um hospital de referência em doença infecciosas, na cidade do Natal-RN, Brasil. Para tanto, todos os pacientes HIV positivos em tratamento, no hospital selecionado para o estudo, foram convidados a participar da pesquisa e solicitado à assinatura do Termo de Consentimento Livre e Esclarecido (TCLE). Realizou-se um estudo transversal e descritivo, em que as amostras biológicas dos participantes foram obtidas através de swabs nasais. Os espécimes obtidos foram semeados no meio de cultivo ágar manitol salgado para o isolamento de S. aureus. As colônias sugestivas dessa espécie foram submetidas a testes laboratoriais de identificação como coloração de Gram, susceptibilidade à bacitracina e as provas da catalase e coagulase livre. A identificação de MRSA foi realizada através da técnica de disco-difusão, utilizando como marcador, o disco de cefoxitina, conforme recomendado pelo Clinical and Laboratory Standards Institute (CLSI) 2017. A mesma técnica foi utilizada para avaliar a susceptibilidade a outros antimicrobianos. Foi realizada ainda a detecção dos genes mecA e lukF através da Reação em Cadeia da Polimerase (PCR). As informações relativas à condição clínica dos participantes foram obtidas mediante entrevista, realizada por meio de um questionário contendo 16 perguntas. Dos 400 pacientes que participaram do estudo, 129 (32,2%) estavam colonizados por S. aureus. Destes, nove (2,2%) eram MRSA, confirmados pela presença do gene mecA. Quanto ao gene lukF, apenas cinco abrigavam esse gene. A maioria dos MRSA apresentou sensibilidade à maioria dos antibióticos testados. No entanto, três amostras apresentaram resistência a mais de duas classes dos antimicrobianos. Não foi encontrada nenhuma associação entre a colonização por S. aureus, incluindo a linhagem MRSA e os fatores relacionados aos indivíduos estudados. Contudo, a presença da linhagem MRSA, reconhecida pela sua virulência e facilidade em adquirir mecanismos de resistência aos antimicrobianos, colonizando pacientes que apresenta maior vulnerabilidade a infecções pode representar um fator de risco para esse segmento da população.
The bacterial species Staphylococcus aureus is considered one of the most important human pathogens. And, a notable feature of this species is the ability to acquire resistance to antibiotics, with methicillin resistance being one of the most significant. Recent studies have shown the presence of methicillin-resistant Staphylococcus aureus (MRSA) in certain population groups, such as HIV-positive patients, in whom increased risk of infection by this strain has been observed. The objective of the study is to determine the prevalence of MRSA colonizing HIV positive patients treated at a referral hospital in the city of Natal-RN and to relate the presence of MRSA with factors associated with the clinical condition of the individuals. To that end, all HIV-positive patients being treated at the hospital selected for the study were invited to participate in the study and asked to be enrolled in the ICF. A crosssectional and descriptive study was carried out, in which the biological samples of the participants were obtained through nasal swabs. These were seeded in the salted mannitol agar medium for the isolation of S. aureus. The colonies suggestive of this species were submitted to laboratory tests of identification as Gram staining, susceptibility to bacitracin and the tests of catalase and free coagulase. The identification of the S. aureus strain resistant to methicillin (MRSA) was performed using the disk-diffusion technique, using as a marker the cefoxitin disc as recommended by the CLSI 2017. The same technique was used to evaluate the susceptibility to other antimicrobials. Detection of mecA and lukF genes through Polymerase Chain Reaction (PCR) was also performed. The information regarding the clinical condition of the participants was obtained through an interview, consisting of 16 questions. Of the 400 patients who participated in the study, 129 (32.2%) were colonized by S. aureus. Of these, nine (2.2%) were MRSA, confirmed by the presence of the mecA gene. As for the lukF gene, only five harbored this gene. Most MRSA showed sensitivity to most of the antibiotics tested. However, three samples showed resistance to more than two classes of antimicrobials. No association was found between S. aureus colonization, including the MRSA lineage and the factors related to the individuals studied. However, the presence of MRSA lineage, recognized for its virulence and ease in acquiring mechanisms of antimicrobial resistance, colonizing patients that presents greater vulnerability to infections may represent a risk factor for this segment of the population.

For many years coagulase negative Staphylococcus (CoNS), skin commensals, were regarded as mere contaminants. However, in recent decades, they emerged as important agents of nosocomial infections, particularly in immunocompromised patients and patients with biomaterials. This is attributed to increasing antimicrobial resistance by these microorganisms, and oxacillin resistance was the main mechanism presented by CoNS. Thus, this study aimed to characterize this resistance, as well as the susceptibility of 160 isolates of CoNS obtained from patients admitted to HUSM, and also compare phenotypic tests used in laboratory detection of oxacillin resistance with genotypic detection of mecA gene, considered the gold standard. The antimicrobial susceptibility tests were performed by the qualitative technique of disk diffusion (CLSI 2009), as well as by semi-quantitative method (MicroScan ®). The phenotypic detection of resistance was performed by using cefoxitin and oxacillin disk. The genotypic identification of mecA gene was performed by PCR. Together with the molecular detection of mecA gene was performed the detection of gene icaD, responsible for biofilm formation. S. epidermidis (62%) was the main species identified among the samples selected for this study, and the prevalent clinical material was blood (38%). Due to the fact that they are skin commensals, an evaluation of clinical significance of the selected samples was performed. It presented that 48% of the isolates were considered more likely to be the etiologic agents of infections. The adult and neonatal ICUs represent prevalent clinical units in isolation of CoNS, with 33% and 29% of the isolates, respectively. Regarding the antimicrobial susceptibility, a high rate of resistance among the isolates was observed. These were grouped into seven prevalent susceptibility profiles, in which three were characterized by resistance to most of the antimicrobials tested. All strains were susceptible to linezolid, teicoplanin and tigecycline, in total 59 isolates (36.8%). All isolates were susceptible to vancomycin. Through the results of phenotypic tests, 89% and 94% of the isolates were characterized as oxacillin resistant by using cefoxitin and oxacillin disks, respectively. The molecular technique revealed that 79% of isolates carried mecA gene. A susceptibility of 96% to cefoxitin disk and 95% to oxacillin disk could be found from the statistical analysis when compared to the gold standard. Biofilm formation was observed in 45% of samples tested, in which S. epidermidis has been identified as prevalent in positive biofilm samples (74%). The phenotypic methods used in this study are equivalent for detecting oxacillin resistance when compared to the gold standard, and the use of oxacillin disk together with cefoxitin disk should be encouraged, since the oxacillin disk can identify other resistance mechanisms not mediated by mecA. In relation to susceptibility of the isolates, there was a high resistance to first-choice antimicrobial used for the treatment of CoNS.
Por muitos anos os Staphylococcus coagulase negativos (SCoN), comensais da pele, foram considerados como simples contaminantes. No entanto, nas últimas décadas, emergiram como importantes agentes de infecções nosocomiais, principalmente em imunocomprometidos e portadores de biomateriais. Isso é atribuído a crescente resistência antimicrobiana apresentada por estes microrganismos, sendo a resistência à oxacilina o principal mecanismos apresentado pelos SCoN. Desta forma, este estudo objetivou caracterizar esta resistência, bem como o perfil de sensibilidade de 160 isolados de SCoN obtidos de pacientes admitidos no HUSM. Além de comparar testes fenotípicos utilizados na detecção laboratorial da resistência à oxacilina com a detecção genotípica do gene mecA, considerado padrão ouro. Os testes de sensibilidade aos antimicrobianos foram efetuados pela técnica qualitativa de difusão do disco (CLSI 2009), bem como pelo método semi-quantitativo (MicroScan®). A detecção fenotípica da resistência foi realizada com os discos de cefoxitina e oxacilina. A identificação genotípica do gene mecA foi realizada por PCR. Juntamente a detecção molecular do gene mecA foi realizado a detecção do gene icaD, responsável pela formação de biofilme. S. epidermidis (62%) foi à principal espécie identificada entre as amostras selecionadas para este estudo e sangue, o material clínico prevalente (38%). Devido a serem comensais da pele, foi realizada uma avaliação da significância clínica das amostras selecionadas, de onde evidenciamos que 48% dos isolados foram considerados os prováveis agentes etiológicos das infecções. As UTIs adulto e neonatal representam as unidades clínicas prevalentes no isolamento de SCoN, com 33% e 29% dos isolados, respectivamente. Em relação a sensibilidade aos antimicrobianos observou-se um elevado índice de resistência entre os isolados. Estes foram agrupados em 7 perfis de sensibilidade prevalentes, sendo que 3 caracterizaram-se pela resistência a maioria dos antimicrobianos testados. Todas as cepas foram sensíveis à linezolida, teicoplamina e tigeciclina, totalizando 59 (36,8%). Todos os isolados foram sensíveis à vancomicina. Através dos resultados dos testes fenotípicos, verificou-se que 89% e 94% dos isolados foram caracterizados como resistentes à oxacilina, utilizando os discos de cefoxitina e oxacilina, respectivamente. Pela técnica molecular, 79% dos isolados carreavam o gene mecA. A partir da análise estatística evidenciou-se sensibilidade de 96% para o disco de cefoxitina e de 95% para o disco de oxacilina, quando comparados ao padrão ouro. A formação de biofilme foi evidenciada em 45% das amostras testadas, onde o S. epidermidis foi identificado como prevalente nas amostras biofilme positivas (74%). Os métodos fenotípicos utilizados neste estudo mostrarm-se equivalentes na detecção da resistência à oxacilina quando comparados ao padrão ouro, sendo que o uso do disco de oxacilina em conjunto com o de cefoxitina deve ser encorajado, uma vez que, o disco de oxacilina pode identificar outros mecanismos de resistência não mediados pelo gene mecA. Em relação ao perfil de sensibilidade dos isolados houve grande resistência aos antimicrobianos de primeira escolha usados para o tratamento de SCoN. Palavras-chave: SCoN, resistência, oxacilina, cefoxitina, gene mecA.

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Coagulase negative staphylococci (CNS) is in human and animal microbiota, but may cause with infections with significant morbidity and mortality rates. Healthy care workers may be carriers of many microorganisms and spread resistant ECN in the hospital. This study aimed to identify the CNS species isolated from healthy care workers saliva, establishe the oxacillin resistance pattern and detect the mecA gene in resistant isolates. We evaluated 100 ECN, isolated from the saliva of an institution of professional health of large Riberão Preto in Sao Paulo state. The ECN identification was based on biochemical tests, and 41 were identified as S. epidermidis, 25 S. saprophyticus, 18 S. haemolyticus, 8 S. cohnii, 4 S. lugdunenses, 3 S. capitis, and 1 S. simulans. Thirty-two percent were nonsusseptible to oxacillin, 84.4% to mupirocin, 43.7% to cefoxitin, but all were vancomycin susceptible. The oxacillin nonsusseptible ECN, detected by disk diffusion test were grown in agar screening oxacillin (6 μ g) supplemented with sodium chloride (4.0%) and submited to mecA detection by the PCR. Of the 32 nonsusseptible oxacillin CNS,, 93.7% developed in the oxacillin agar and the mecA gene was detected in 75.0%. This is the first report of mecA gene presence in CNS isolated from the saliva of healthy care workers. Attention must be given to CNS species identification, as well as the characterization of the nonsusceptible microorganisms, since healthy care workers may represent a reservoir of CNS
Estafilococos coagulase negativa (ECN) fazem parte da microbiota autóctone humana e animal, embora possam causar infecções. Profissionais da saúde podem ser portadores de inúmeros microrganismos virulentos entre eles os ECN resistentes a oxacilina que podem ser disseminados por esses profissionais. O estudo teve como objetivo identificar espécies de ECN isolados da saliva de profissionais da saúde, de uma instituição de saúde de grande porte em Riberão Preto no estado de São Paulo, determinar o perfil de resistência à oxacilina e detectar o gene mecA. Foram avaliados 100 ECN, e através de testes bioquímicos 41 estafilococos foram identificados como S. epidermidis, 25 S. saprophyticus, 18 S. haemolyticus, 8 S. cohnii, 4 S. lugdunenses, 3 S. capitis e um S. simulans. Trinta e dois por cento apresentaram resistência in vitro a oxacilina, 84,4% a mupirocina, 43,7% a cefoxitina, e todos suscetíveis a vancomicina. Os ECN resistentes a oxacilina, detectados pelo teste de disco difusão, foram cultivados no agar triagem oxacilina (6µg) suplementado com cloreto de sódio e a detecção do gene mecA foi realizada pela técnica de PCR. Dos 32 ECN resistentes a oxacilina, 93,7% desenvolveram no agar oxacilina e o gene mecA foi detectado em 75,0% das amostras analisadas. Vale ressaltar que este estudo é pioneiro em mostrar a presença do gene mecA em ECN isolados da saliva de profissionais da saúde saudáveis. Uma maior atenção deve ser dada na identificação das espécies de ECN, e na caracterização da resistência desse grupo de microrganismos, uma vez que os profissionais da saúde podem representar um reservatório de ECN resistentes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Multiple antibiotic resistant Staphylococcus aureus represent a big problem in the control of hospital infections. Resistance pattern of isolated S. aureus presented in central vascular catheter of patients interned in the Intensive Therapy Center of the School Hospital of the Universidade Federal do Triângulo Mineiro was evaluated by antimicrobial tests, in which it was possible to detect high level of resistance to penicillin (94.7%) and ampicillin (86.8%), only considering the samples that presented resistance, beyond one strain that presented resistance to vancomycin. The oxacillin resistance evaluation was confirmed by PCR with the presence of the gene mecA. The association of the results obtained in the phenotypic test with the presence of the gene mecA, considered the reference method, was confirmed through the Table of Contingency and the Test of Χ2 with Yates correction. In 49 isolates evaluated, 23 were resistant to oxacillin, being possible to detect the mecA gene in 21 samples. The test of molecular screening by RAPD allowed the separation of the phenotypic groups in two different grouping patterns, the ones that presented resistance to antimicrobials and the sensible ones, with a dissimilarity of 73,3%. There is a higher genetic similarity between groups that present the same type of resistance, thus confirming the phenotypic analyses. Molecular markers for detection of resistance to oxacillin, like the gene mecA, were more sensitive than the phenotypic markers.
Staphylococcus aureus resistentes a múltiplos antibióticos representam um grande problema no controle das infecções hospitalares. O perfil de resistência a antimicrobianos de isolados de S. aureus presentes em cateteres vasculares central de pacientes internados em leitos do centro de terapia intensiva do Hospital Escola da Universidade Federal do Triângulo Mineiro foi avaliado por meio de testes antimicrobianos, pelos quais foi possível detectar um elevado nível de resistência à penicilina (94,7%) e ampicilina (86,8%), considerando-se somente as amostras que possuíam resistência, além de uma cepa resistente à vancomicina. A avaliação da resistência à oxacilina foi confirmada por PCR através da presença do gene mecA. A associação dos resultados obtidos no teste fenotípico com a presença do gene mecA, considerado um método de referência, foi confirmada através da Tabela de Contingência e do Teste de Χ2 com correção de Yates. Em 49 amostras avaliadas, 23 apresentaram resistência à oxacilina, sendo possível detectar a presença do gene de resistência mecA em 21 amostras. O teste de tipagem molecular por RAPD permitiu a separação dos grupos fenotípicos em dois padrões diferentes de agrupamento, os que possuíam resistência e os sensíveis aos antimicrobianos, com uma dissimilaridade de 73,3%. Há maior similaridade genética entre grupos que apresentam o mesmo tipo de resistência, confirmando assim as análises fenotípicas. Marcadores moleculares para detecção de resistência à oxacilina, como o gene mecA, foram mais sensíveis que os marcadores fenotípicos.
Mestre em Genética e Bioquímica

Staphylococcus aureus belongs to the normal flora. Many healthy people are colonized by the bacterium mainly in the nose but also on the skin and on other mucous membranes without showing symptoms. After damage to the skin, the bacterium can enter the wound and cause infections. Methicillin-resistant S. aureus (MRSA) is resistant to b-lactam antibiotics such as penicillin and methicillin. The gene that gives resistance characteristic of MRSA is the mecA-gene. MRSA strains are spread in both hospitals and in the community, and it is important to identify these bacteria with rapid and sensitive methods. In this study, Taq Man RT-qPCR was compared with SYBR Green RT-qPCR (LightCycler480, Roche) to explore which method had the best sensitivity with the least working hours. In addition, Bullet for automated DNA extraction and CAS 1200 ™ for automated pipetting of the samples were evaluated. Twelve patient isolates and 232 patient samples for MRSA screening were included in the study. The results showed that the primers were of major importance for the outcome of the amplification. It was also shown that the Ct-values were clearly lower when the Bullet, CAS 1200 ™ and LightCycler480 were combined compared with manual DNA extraction, manual pipetting and the Rotor-Gene 6000. In future, the former method will be used by the laboratory when screening patient samples for MRSA.

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FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais
Acredita-se que os resíduos de serviços de saúde (RSS) possam atuar como veículos de disseminação de microrganismos potencialmente patogênicos e de marcadores de resistência a drogas antimicrobianas. Assim, considerando-se os riscos para saúde humana e ambiental associados ao fenômeno da resistência microbiana a drogas, nossos objetivos foram identificar linhagens de Staphylococcus coagulase negativo (SCN) isoladas do chorume percolado dos RSS no aterro sanitário de Juiz de Fora, MG, determinar o seu perfil de susceptibilidade a antimicrobianos de interesse clínico-microbiológico e detectar o marcado molecular mecA. Linhagens bacterianas presuntivamente caracterizadas como SCN (n=109) foram identificadas utilizando-se sistema comercial semiautomatizado e o seu perfil de susceptibilidade a antimicrobianos, determinado pela técnica de diluição em ágar, de acordo com as recomendações do Clinical and Laboratory Standards Institute (CLSI). Apenas 26,6% das amostras de SCN foram identificadas pelo sistema comercial, distribuídas em S. sciuri (31%), S. epidermidis (27,6%), S. lentus (20,7%), S. vitulinus (10,3%) S. saprophyticus (6,9%), S. haemolyticus (3,5%). As outras linhagens foram identificadas como Staphylococcus spp. (73,4%). Entre os antimicrobianos avaliados, a penicilina e a oxacilina foram as drogas menos efetivas (61,4% de resistência), seguidas pela eritromicina e azitromicina (27,3% e 22,7% de resistência respectivamente). Os antimicrobianos mais eficazes foram a gentamicina e levofloxacina, para os quais apenas resistência intermediária foi observada (21,6% e 1,1% respectivamente). Todas as amostras foram susceptíveis à vancomicina. Considerando as linhagens resistentes 14 (25,9%) amplificaram o gene específico, enquanto que 40 (74,1%) mostraram-se resistentes à oxacilina por outros mecanismos. Nossos resultados suscitam reflexões relacionadas à sobrevivência de microrganismos potencialmente patogênicos e linhagens resistentes aos antimicrobianos, carreando importantes marcadores de resistência e sua possível disseminação via RSS, contribuindo para seu trânsito intra-hospitalar.
It is accepted that health-care waste may act as vehicle for spreading potentially pathogenic microorganisms and their genetic resistance markers. Thus, considering the risks for human and environmental health associated with the phenomenon of microbial drug resistance, our objectives were to identify Coagulase-negative Staphylococcus (CoNS) strains isolated from the sanitary landfill disposal of healthcare waste of Juiz de Fora, MG; to determine the antimicrobial susceptibility patterns against antimicrobials of clinical and microbiological relevance; and to evaluate the occurrence of mecA gene. Bacterial strains presumptively characterized as CoNS (n= 109) were identified using a commercial system and the antimicrobial susceptibility patterns determined by the agar dilution technique, according to the recommendations of Clinical and Laboratory Standards Institute (CLSI). Only 26.6% of the bacterial samples were identified as follows: S. sciuri (31%), S. epidermidis (27.6%), S. lentus (20.7%), S. vitulinus (10.3%) S. saprophyticus (6.9%), S. haemolyticus (3.5%). The other strains were identified as Staphylococcus spp. (73.4%). Considering the minimal inhibitory concentrations of the antimicrobials, penicillin and oxacillin drugs were the less effective drugs (61.4% resistance) followed by erythromycin and azithromycin (27.3 and 22.7% resistance, respectively). The most effective antimicrobials were gentamicin and levofloxacin, for which only intermediate resistance was observed (21.6 and 1.1% respectively). All samples were susceptible to vancomycin. Considering the resistant strains, 14 (25,9%) showed to harbour mecA gene and in 40 (74,1%) were resistant to oxacillin by other mechanisms. Our results raise considerations related to the survival of potentially pathogenic microorganisms and strains resistant to antibiotics harboring genetic markers. It is possible that these bacteria might spread via health-care waste, contributing dissemination into hospitals.

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Funda??o Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro
Coagulasenegative staphylococci (SCN) take part of normal microbiota. Although it has been considered saprophytic, nowadays there is a concern about its pathogenic potential.Nevertheless, all advance in SCN identification assays, these microorganisms are continually neglected in laboratorial routine of infectious diseases, because of the wide range of species. In spite of the difficulties, it is necessary to make an appropriated species identification in order to differentiate the potential pathogenic agents and to determine its antimicrobial susceptibility pattern. Resistance in SCN is related to the presence of mecA gene, which expresses a heterogeneous pattern that can be detected through diverse phenotypics tests. In this study, 72 SCN isolates obtained from external ear conducts of dogs, bovine mastitis and human nosocomial infections were evaluated. Staphylococcus xylosus was the most prevalent microorganism in animal and S. cohnii subsp.urealyticus in human sample. The antimicrobial resistance patterns were evaluated through disc diffusion test, and a high level of resistance to penicillin and ampicillin were detected. The most efficient antibiotics evaluated were gentamicin, vancomicin and the association ampicillin+sulbactam. Oxacillin resistance was phenotypically detected by modified disc diffusion test, agar screen, broth microdilution and agar dilution. Presence of mecA gene where detected in 5,55% of the isolates by Polimerase Chain Reaction (PCR). Correlation with the detection of mecA gene was used as gold standard for evaluation of sensitivity and specificity of phenotypical assays. The low number of mecA positives isolates did not allowed the association between cefoxitin and oxacillin resistance as a test to detect the presence this gene. The broth microdilution and agar dilution tests presented the best accuracy in phenotypic detection of the oxacillin resistance.
Os estafilococos coagulasenegativos (ECN) fazem parte da microbiota normal da pele e apesar de terem sido considerados sapr?fitas por muito tempo, o seu significado cl?nico como agente etiol?gico tem aumentado com o passar dos anos. No entanto, apesar de todo avan?o nas t?cnicas de identifica??o dos ECN e do conhecimento destes como agentes etiol?gicos em diversos processos infecciosos, estes microrganismos muitas vezes s?o negligenciados na rotina laboratorial, devido a enorme diversidade de esp?cies encontradas. A identifica??o das esp?cies de ECN, embora de dif?cil realiza??o para a maioria dos laborat?rios cl?nicos, ? necess?ria para diferenciar o potencial patog?nico e o perfil de resist?ncia de cada isolado. A resist?ncia ? oxacilina em ECN ? mediada pelo gene mecA e usualmente heterog?nea, sendo detectada por v?rios m?todos fenot?picos. Neste estudo, foram avaliados 72 isolados de ECN provenientes de amostras do conduto auditivo de c?es da ra?a Beagles, de mastite bovina e de infec??es humanas. Staphylococcus xylosus foi o microrganismo mais isolado, nas amostras animais, e S. cohnii subsp.urealyticus em humanos, dentro de uma ampla gama de esp?cies identificadas. O perfil de resist?ncia aos antimicrobianos em isolados de animais e humanas foi avaliado atrav?s da t?cnica de difus?o em disco, na qual detectouse um elevado n?vel de resist?ncia ? penicilina e ampicilina. A gentamicina, vancomicina e associa??o ampicilina+sulbactam foram eficientes frente aos isolados testados. A avalia??o da resist?ncia ? oxacilina foi realizada atrav?s da difus?o em disco modificada, ?gar screen, microdilui??o em caldo e em ?gar. A presen?a do gene mecA foi determinada pelo m?todo da Rea??o em Cadeia de Polimerase (PCR), sendo 5,55% dos isolados mecA positivos. Os resultados fenot?picos de resist?ncia a cefoxitina e a oxacilina foram correlacionados com a detec??o do gene mecA, que foi utilizado como padr?o ouro para avalia??o da sensibilidade e especificidade das t?cnicas utilizadas. O reduzido n?mero de isolados mecA + n?o permitiu estabelecer o grau de correla??o entre a cefoxitina e a oxacilina como m?todo de predi??o do gene, embora ambas tenha apresentado o mesmo percentual de resist?ncia. O teste fenot?pico que apresentou melhor acur?cia na detec??o da resist?ncia ? oxacilina foi a microdilui??o em caldo.

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha
Resumo: A alta frequência de Estafilococos Coagulase-Negativa (CoNS) na pele de indivíduos saudáveis e em doenças associadas ao sangue, associados à seleção de cepas resistentes devido a uso indiscriminado de antimicrobianos, tornou mais estreitos os limites entre o ambiente hospitalar e o comunitário quanto à distribuição de cepas. Objetivou-se, com este estudo, caracterizar isolados de CoNS de origem hospitalar e comunitária da cidade de Botucatu-SP quanto ao perfil clonal, analisar os aspectos de resistência à oxacilina pela aferição de metodologia de detecção, e investigar os determinantes de heterorresistência à vancomicina nessas cepas. As espécies estudadas incluíram S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. lugdunensis, S. capitis, S. saprophyticus, S. pasteuri, S. simulans e S. xylosus. O teste de disco-difusão (TDD) com discos de oxacilina e cefoxitina, fitas de Etest impregnadas com oxacilina e pesquisa do gene mecA por PCR em tempo real foram realizadas. A triagem em ágar com 6 e 8 µg/ml de vancomicina, microdiluição em caldo para aferição da Concentração Inibirtória Mínima (MIC), microscopia eletrônica de transmissão para verificar espessamento da parede celular e alterações fenotípicas por testes bioquímicos foram realizadas. O perfil clonal foi determinado por PFGE (Pulsed-Field Gel Eletrophoresis) e para clones de S. epidermidis, o MLST (Multilocus Sequence Typing). S. epidermidis apresentou alta diversidade clonal, mas presença de clusters no ambien... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The high frequency of Coagulase-Negative Staphylococci (CoNS) on the skin of healthy individuals and in bloodstream infections, together with the selection of resistant strains, has narrowed the boundaries between the hospital and the community environment for the distribution of strains. This study aimed to characterize CoNS isolated from clinical and colonization specimens of patients and individuals from Botucatu-SP, to compare their clonal profile, to analyze the determination of oxacillin resistance by the evaluation of the methodology of detection, and to investigate the determinants of reduced susceptibility to vancomycin in those strains. CoNS species included S. epidermidis, S. haemolyticus, S. warneri, S. hominis, S. lugdunensis, S. capitis, S. saprophyticus, S. pasteuri, S. simulans and S. xylosus. The disc diffusion test (DDT) using oxacillin and cefoxitin discs was employed, Etest strips impregnated with oxacillin and mecA gene detection by real-time PCR were used. An agar screening with 6 and 8 µg/ml of vancomycin, the broth microdiluition method for the Minimal Inhibitory Concentration (MIC), the transmission eletronic microscopy for evaluation of cellwall thickening and phenotypic modifications by biochemical tests were performed. Clonal profile was determined by PFGE (Pulsed-Field Gel Eletrophoresis) and, for S. epidermidis clones, MLST (Multilocus Sequence Typing). S. epidermidis presented high clonal diversity, despite some clusters circulating within hospi... (Complete abstract click electronic access below)
Doutor

Staphylococcus aureus est une bactérie commensale et pathogène qui s’est rapidement adaptée à la pression sélective des antibiotiques entraînant la diffusion de souches résistantes à la méticilline (MRSA). En Belgique, des souches appelées Healthcareassociated (HA)-MRSA sont devenues endémiques dans les hôpitaux, causant de nombreuses épidémies durant les années 90.

Parallèlement, des cas d’infections communautaires par des souches appelées Community-associated (CA)-MRSA produisant la leucocidine de Panton-Valentine (PVL) ont été rapportées en Europe, en Australie et aux USA, chez des patients jeunes sans contact avec les institutions de soins. Depuis 2005, une prévalence élevée de portage de MRSA a été observée chez les porcs, les éleveurs et les vétérinaires aux Pays-Bas et dans les pays voisins. Ces souches dites Livestock-associated (LA)-MRSA montrent une origine génétique distincte des souches humaines précédemment décrites.

Nos travaux de recherche ont porté sur l’épidémiologie et la caractérisation moléculaire de la colonisation et l’infection par S. aureus dans diverses populations humaines en Belgique afin d’élucider :1) l’évolution de la distribution spatio-temporelle des clones épidémiques de MRSA parmi les patients hospitalisés au cours de la période de 1995 à 2003 ;2) les mécanismes moléculaires de résistance aux antibiotiques associés à ces clones ;3) les relations phylogénétiques entre souches de S. aureus sensibles et résistantes à la méticilline parmi les patients hospitalisés afin d’identifier l’origine probable des clones épidémiques ;4) la prévalence et les facteurs de risque de portage de MRSA parmi les résidents des maisons de repos et l’origine de ces souches ;5) l’origine des souches de CA-MRSA et les profils cliniques des infections associées ;6) la prévalence et les facteurs de risque associés au portage de MRSA et la diffusion des souches de MRSA dans les fermes d’élevage porcin.

Des enquêtes multicentriques nous ont permis de caractériser les souches de S. aureus résistantes et sensibles à la méticilline affectant les patients hospitalisés (4 enquêtes, 1995 -2003), les patients ambulants (1 enquête, 2002-2004), les résidents dans des maisons de repos et de soins (1 enquête en 2005) et les habitants des fermes d’élevage porcin (1 enquête en 2007). Ces souches ont été caractérisées par détermination du type de cassette mec (SCCmec typing), séquençage d’un gène hypervariable (spa typing) et de 7 gènes de ménage (multi-locus sequence typing, MLST), combinées à l’analyse par électrophorèse en champs pulsé (PFGE) du profil de acrorestriction génomique. Les gènes codant pour trois classes d’antibiotiques et les toxines, PVL, TSST-1 et exfoliatines, ont été recherchés par PCR.

L’étude des souches de MRSA a montré une diversification importante des clones dans les hôpitaux, avec le remplacement d’un clone multi-résistant par des clones plus sensibles aux antibiotiques. La distribution des gènes de résistance ainsi que du gène TSST-1 était fortement liée au génotype. Les S. aureus sensibles à la méticilline (MSSA) montraient une plus grande diversité génotypique que les MRSA. La majorité des MRSA épidémiques appartient à des génotypes endémiques de MSSA, suggérant la possibilité d’émergence locale de MRSA par acquisition récente de l’élément mobile SCCmec.

D’autres souches de génotypes pandémiques pourraient avoir été importées en Belgique. L’enquête dans les maisons de repos et de soins a montré une prévalence élevée de résidents porteurs de MRSA. L’exposition aux antibiotiques, les lésions cutanées, la perte de mobilité, l’absence de formulaire thérapeutique et de procédures d’hygiène pour le contrôle du MRSA, associée à un nombre élevé de médecins, augmentaient significativement le risque de portage. La présence des mêmes génotypes dans les hôpitaux et les maisons de repos d’une même province suggère des transferts locaux de MRSA entre patients résidant dans et circulant entre ces secteurs de soins.

Les souches de CA-MRSA productrices de toxine PVL importées ou acquises en Belgique appartiennent à divers génotypes. La présence de MRSA de même lignée clonale mais présentant des profils de virulence différents clonale dans les institutions de soins et dans la population générale suggère que ces souches ont évolué de manière divergente dans des environnements différents.

Nous avons documenté une prévalence très élevée de porteurs de MRSA de génotype ST398 chez les éleveurs de porcs. Le réservoir de ce clone est très probablement d’origine animale, la transmission à l’homme ayant lieu par contact avec des animaux d’élevage ou de compagnie et potentiellement, par voie alimentaire.

En conclusion, S. aureus est un pathogène capable de s’adapter dans des environnements très différents. Les souches épidémiques résistantes aux antibiotiques, qu’elles soient d’origine hospitalière, communautaire ou animale, appartiennent à un nombre limité de génotypes bien établis dans chaque écosystème au niveau local ou régional. L’étude approfondie de la dynamique de transmission des MRSA, et de leur profil de résistance dans la communauté, les secteurs des soins aigus et chroniques et l’élevage, est indispensable pour définir les stratégies cliniques et de santé publique pour adapter les schémas thérapeutiques et endiguer l’endémie d’infections à MRSA.

Doctorat en Sciences médicales
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Na última década houve uma clara mudança na etiologia da sepse. Dados recentes do National Healthcare Safety Network (NHSN) mostram que os cocos Gram-positivos têm ultrapassado os bacilos Gram-negativos como os principais agentes etiológicos e os Estafilococos coagulase negativa (ECN) se tornaram os agentes mais frequentes. Entretanto a maioria dos laboratórios clínicos não identifica esses microrganismos a nível de espécies devido à necessidade de realização de uma série de provas bioquímicas. A aplicação de técnic as de biologia molecular, entre elas a reação em cadeia da polimerase (PCR) para a identificação de bactérias se mostrou uma técnica promissora devido a sua rapidez, acurácia e sensibilidade. Assim, este trabalho objetivou avaliar a eficiência, acurácia e sensibilidade da técnica de PCR multiplex para a detecção do gênero e de espécies de Staphylococcus spp. mais encontradas em hemoculturas, do operon ica de produção de biofilme e da presença do gene mecA de resistência à oxacilina diretamente dos frascos de hemocultivo. Foram analisadas 371 amostras de hemoculturas, positivas para o gênero Staphylococcus obtidas de pacientes atendidos no HC da Faculdade de Medicina de Botucatu (FMB). As amostras foram isoladas e identificadas por testes bioquímicos convencionais e simultaneamente o DNA bacteriano foi extraído diretamente das hemoculturas e destes realizou-se o PCR multiplex. Das 371 amostras estudadas 85 (23,0%) foram de S. aureus e 286 (77,0%) ECN. Dos ECN 152 (53,1%) foram identificados como S. epidermidis, 36 (12,6%) S. haemolyticus, 36 (12,6%) S. hominis, 23 (8,0%) S. capitis, 18 (6,3%) S. warneri, 7 S. caprae, 5 S. simulans, dois S. lugdunensis, dois S. cohnii, um S. saprophyticus, um S. schleiferi, um S. xylosus e concomitantemente um S. haemolyticus e S. hominis, e um S. haemolyticus e S. epidermidis. Das 85 amostras de S. aureus, 43 (50,6%) se mostraram resistentes à oxacilina pelo ...
A clear shift in the etiology of sepse has occurred in the last decade. Recent data from the National Healthcare Safety Network (NHSN) show that Gram-positive cocci have exceeded Gram-negative bacilli as the major etiological agents. In this respect, coagulase-negative staphylococci (CoNS) have become the most frequent. However, most clinical laboratories do not identify these microorganisms to species level due to the need for a series of biochemical tests. The application of molecular biology techniques, including the polymerase chain reaction (PCR), for bacterial identification proved to be promising due to their speed, accuracy and sensitivity. The aim of this study was to evaluate the efficiency, accuracy and sensitivity of multiplex PCR for the detection of Staphylococcus spp. frequently found in blood cultures, investigation of the ica operon involved in biofilm formation and the mecA gene encoding oxacillin resistance, and direct detection of major CoNS species in blood culture flasks. A total of 371 blood culture samples positive for Staphylococcus spp. obtained from patients seen at the Hospital of the Botucatu School of Medicine (FMB) were analyzed. The strains were isolated and identified by conventional biochemical tests. Simultaneously, bacterial DNA was extracted directly from the blood cultures for multiplex PCR. S. aureus was isolated from 85 (23%) of the 371 samples studied and CoNS from 286 (77%). Among the latter, 152 (53.1%) isolates were identified as S. epidermidis, 36 (12.6%) as S. haemolyticus, 36 (12.6%) as S. hominis, 23 (8%) as S. capitis, 18 (6.3%) as S. warneri, 7 as S. caprae, 5 as S. simulans, two as S. lugdunensis, two as S. cohnii, one as S. saprophyticus, one as S. schleiferi, and one as S. xylosus. S. haemolyticus and S. hominis were concomitantly identified in one sample and S. haemolyticus and S. epidermidis in another. Forty-three (50.6%) of the 85 S. aureus strains were resistant to oxacillin in ...

Orientador: Maria de Lourdes Ribeiro de Souza da Cunha
Coorientador: Alessandro Lia Mondelli
Banca: Carlos Magno Castelo Branco Fortaleza
Banca: Antônio Carlos Pignatari
Resumo: Na última década houve uma clara mudança na etiologia da sepse. Dados recentes do National Healthcare Safety Network (NHSN) mostram que os cocos Gram-positivos têm ultrapassado os bacilos Gram-negativos como os principais agentes etiológicos e os Estafilococos coagulase negativa (ECN) se tornaram os agentes mais frequentes. Entretanto a maioria dos laboratórios clínicos não identifica esses microrganismos a nível de espécies devido à necessidade de realização de uma série de provas bioquímicas. A aplicação de técnic as de biologia molecular, entre elas a reação em cadeia da polimerase (PCR) para a identificação de bactérias se mostrou uma técnica promissora devido a sua rapidez, acurácia e sensibilidade. Assim, este trabalho objetivou avaliar a eficiência, acurácia e sensibilidade da técnica de PCR multiplex para a detecção do gênero e de espécies de Staphylococcus spp. mais encontradas em hemoculturas, do operon ica de produção de biofilme e da presença do gene mecA de resistência à oxacilina diretamente dos frascos de hemocultivo. Foram analisadas 371 amostras de hemoculturas, positivas para o gênero Staphylococcus obtidas de pacientes atendidos no HC da Faculdade de Medicina de Botucatu (FMB). As amostras foram isoladas e identificadas por testes bioquímicos convencionais e simultaneamente o DNA bacteriano foi extraído diretamente das hemoculturas e destes realizou-se o PCR multiplex. Das 371 amostras estudadas 85 (23,0%) foram de S. aureus e 286 (77,0%) ECN. Dos ECN 152 (53,1%) foram identificados como S. epidermidis, 36 (12,6%) S. haemolyticus, 36 (12,6%) S. hominis, 23 (8,0%) S. capitis, 18 (6,3%) S. warneri, 7 S. caprae, 5 S. simulans, dois S. lugdunensis, dois S. cohnii, um S. saprophyticus, um S. schleiferi, um S. xylosus e concomitantemente um S. haemolyticus e S. hominis, e um S. haemolyticus e S. epidermidis. Das 85 amostras de S. aureus, 43 (50,6%) se mostraram resistentes à oxacilina pelo ...
Abstract: A clear shift in the etiology of sepse has occurred in the last decade. Recent data from the National Healthcare Safety Network (NHSN) show that Gram-positive cocci have exceeded Gram-negative bacilli as the major etiological agents. In this respect, coagulase-negative staphylococci (CoNS) have become the most frequent. However, most clinical laboratories do not identify these microorganisms to species level due to the need for a series of biochemical tests. The application of molecular biology techniques, including the polymerase chain reaction (PCR), for bacterial identification proved to be promising due to their speed, accuracy and sensitivity. The aim of this study was to evaluate the efficiency, accuracy and sensitivity of multiplex PCR for the detection of Staphylococcus spp. frequently found in blood cultures, investigation of the ica operon involved in biofilm formation and the mecA gene encoding oxacillin resistance, and direct detection of major CoNS species in blood culture flasks. A total of 371 blood culture samples positive for Staphylococcus spp. obtained from patients seen at the Hospital of the Botucatu School of Medicine (FMB) were analyzed. The strains were isolated and identified by conventional biochemical tests. Simultaneously, bacterial DNA was extracted directly from the blood cultures for multiplex PCR. S. aureus was isolated from 85 (23%) of the 371 samples studied and CoNS from 286 (77%). Among the latter, 152 (53.1%) isolates were identified as S. epidermidis, 36 (12.6%) as S. haemolyticus, 36 (12.6%) as S. hominis, 23 (8%) as S. capitis, 18 (6.3%) as S. warneri, 7 as S. caprae, 5 as S. simulans, two as S. lugdunensis, two as S. cohnii, one as S. saprophyticus, one as S. schleiferi, and one as S. xylosus. S. haemolyticus and S. hominis were concomitantly identified in one sample and S. haemolyticus and S. epidermidis in another. Forty-three (50.6%) of the 85 S. aureus strains were resistant to oxacillin in ...
Mestre

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Alguns dos agentes patogênicos de mastite bovina são relevantes em relação à qualidade do leite, bem como, para a saúde pública. Foram estudadas dez fazendas localizadas em cinco regiões do estado de São Paulo. Foram examinadas 1.148 vacas, correspondentes a 4.592 glândulas mamárias avaliadas pelos testes de tamis e CMT. Foram colhidas 1.318 amostras de leite, das vacas positivas na triagem para avaliação microbiológica e contagem de células somáticas (CCS). A frequência dos agentes variou de 0,3 a 36,4%. Do total de isolados de Staphylococcus spp (36,4%), 48,7% corresponderam a estafilococos coagulase negativa (SCN), 34,2% S. aureus e 15,9% outros estafilococos coagulase positiva (SCP). Streptococcus spp foram isolados de 23,3% das amostras, dos quais 41,7% Streptococcus. agalactiae, 41,1% Streptococcus dysgalactiae e 17,3% Streptococcus uberis. Corynebacterium spp. foram isolados em 31,8% dos casos de mastite. As amostras de leite das glândulas mamárias infectadas apresentaram CCS significativamente mais elevada que as negativas. Dos 48,7% de SCN foram identificadas 18 espécies: S. warneri, S. epidermidis, S. hyicus, S. xylosus, S. haemolyticus, S. auriculares, S. cohnii subsp cohnii, S. lugdunensis, S. pasteuri, S. saccharolyticus, S. saprophyticus subsp bovis, S. schleiferi subsp scheleiferi, S. simulans, S. saccharolyticus, S. capitis, S. saprophyticus subsp saprophyticus, S. sciuri subsp sciuri e S. chromogenes. As fazendas, II (27,3%) e III (26,7%) apresentaram maior frequência de SCN, enquanto as fazendas I (13,3%) e X (44,4%) revelaram maior prevalência de SCP sendo as diferenças significantes. Foram avaliados por PCR 263 estafilococos para detecção de gene codificadores das enterotoxinas clássicas. Entre os SCN foram detectados: sea (35,5%) seb (7,1%), sec (6,5%), sed (1,8%) e associações destes genes. Em SCP foram detectados: sea (9,5%) seb (4,4%)...
The purpose of this study was to assess the occurrence of mastitis cases in ten Brazilian dairy herds located in five regions in São Paulo state, to characterize the main etiological agents and, to proceed staphylococcal isolates identification to species level, to perform detection by PCR assays of enterotoxin encoded genes aiming the awareness of their potential capability in producing the classical enterotoxins and, mecA a methicilin resistance gene and, evaluated resistance toward antimicrobials. A total of 4,592 mammary glands of 1,148 dairy cows were examined by strip cup and CMT. From these 1,318 milk samples were collected for microbiological exams. It was isolated 263 (19.9%) staphylococci from mastitis cases and they were identified, as being: S.aureus (34.2%), other CPS (15.9%) respectively, S. intermedius (15.2%), S.hyicus(12.9%) and, S. schleiferi subsp coagulans(3.8%), and CNS (48.7%). Among these 128 CNS isolates eighteen species were identified: S. xylosus, S. haemolyticus, S. auriculares; S. cohnii subsp cohnii, S. lugdunensis, S. pasteuri, S. saccharolyticus, S. saprophyticus subsp bovis, S. schleiferi subsp scheleiferi, S. simulans, S. capitis, S. saprophyticus subsp saprophyticus, S. sciuri subsp sciuri and, S. chromogenes. The more frequently isolated species were: S.warneri (31.3%), S. epidermidis (14.8%) and S.hyicus (12.5%). The milk samples from infected mammary glands showed higher CCS than the negatives. PCR assay was used to determine the presence of classical enterotoxin codifying genes (sea, seb, sec and sed). Among CNS the occurrence of enterotoxin classical genes was determined as: 35.1% for sea, 7.1% for seb, 6.5% for sec, 1.8% for sed) 5.3% for both sea and seb, 3.6% for both sea, sec and sed, 1.8% for both sec and sed. Whereas among CPS isolates the occurrence of enterotoxin... (Complete abstract click electronic access below)

Orientador: Helio Langoni
Banca: Márcio Garcia Ribeiro
Banca: Nilson Robeti Benites
Resumo: Alguns dos agentes patogênicos de mastite bovina são relevantes em relação à qualidade do leite, bem como, para a saúde pública. Foram estudadas dez fazendas localizadas em cinco regiões do estado de São Paulo. Foram examinadas 1.148 vacas, correspondentes a 4.592 glândulas mamárias avaliadas pelos testes de tamis e CMT. Foram colhidas 1.318 amostras de leite, das vacas positivas na triagem para avaliação microbiológica e contagem de células somáticas (CCS). A frequência dos agentes variou de 0,3 a 36,4%. Do total de isolados de Staphylococcus spp (36,4%), 48,7% corresponderam a estafilococos coagulase negativa (SCN), 34,2% S. aureus e 15,9% outros estafilococos coagulase positiva (SCP). Streptococcus spp foram isolados de 23,3% das amostras, dos quais 41,7% Streptococcus. agalactiae, 41,1% Streptococcus dysgalactiae e 17,3% Streptococcus uberis. Corynebacterium spp. foram isolados em 31,8% dos casos de mastite. As amostras de leite das glândulas mamárias infectadas apresentaram CCS significativamente mais elevada que as negativas. Dos 48,7% de SCN foram identificadas 18 espécies: S. warneri, S. epidermidis, S. hyicus, S. xylosus, S. haemolyticus, S. auriculares, S. cohnii subsp cohnii, S. lugdunensis, S. pasteuri, S. saccharolyticus, S. saprophyticus subsp bovis, S. schleiferi subsp scheleiferi, S. simulans, S. saccharolyticus, S. capitis, S. saprophyticus subsp saprophyticus, S. sciuri subsp sciuri e S. chromogenes. As fazendas, II (27,3%) e III (26,7%) apresentaram maior frequência de SCN, enquanto as fazendas I (13,3%) e X (44,4%) revelaram maior prevalência de SCP sendo as diferenças significantes. Foram avaliados por PCR 263 estafilococos para detecção de gene codificadores das enterotoxinas clássicas. Entre os SCN foram detectados: sea (35,5%) seb (7,1%), sec (6,5%), sed (1,8%) e associações destes genes. Em SCP foram detectados: sea (9,5%) seb (4,4%)... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The purpose of this study was to assess the occurrence of mastitis cases in ten Brazilian dairy herds located in five regions in São Paulo state, to characterize the main etiological agents and, to proceed staphylococcal isolates identification to species level, to perform detection by PCR assays of enterotoxin encoded genes aiming the awareness of their potential capability in producing the classical enterotoxins and, mecA a methicilin resistance gene and, evaluated resistance toward antimicrobials. A total of 4,592 mammary glands of 1,148 dairy cows were examined by strip cup and CMT. From these 1,318 milk samples were collected for microbiological exams. It was isolated 263 (19.9%) staphylococci from mastitis cases and they were identified, as being: S.aureus (34.2%), other CPS (15.9%) respectively, S. intermedius (15.2%), S.hyicus(12.9%) and, S. schleiferi subsp coagulans(3.8%), and CNS (48.7%). Among these 128 CNS isolates eighteen species were identified: S. xylosus, S. haemolyticus, S. auriculares; S. cohnii subsp cohnii, S. lugdunensis, S. pasteuri, S. saccharolyticus, S. saprophyticus subsp bovis, S. schleiferi subsp scheleiferi, S. simulans, S. capitis, S. saprophyticus subsp saprophyticus, S. sciuri subsp sciuri and, S. chromogenes. The more frequently isolated species were: S.warneri (31.3%), S. epidermidis (14.8%) and S.hyicus (12.5%). The milk samples from infected mammary glands showed higher CCS than the negatives. PCR assay was used to determine the presence of classical enterotoxin codifying genes (sea, seb, sec and sed). Among CNS the occurrence of enterotoxin classical genes was determined as: 35.1% for sea, 7.1% for seb, 6.5% for sec, 1.8% for sed) 5.3% for both sea and seb, 3.6% for both sea, sec and sed, 1.8% for both sec and sed. Whereas among CPS isolates the occurrence of enterotoxin... (Complete abstract click electronic access below)
Mestre

博士
國立成功大學
生物醫學工程學系
104
MALDI-TOF Phenotypic identification of species of Staphylococcuscan be difficult. The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a cost-effective and rapid method for microbial identification. However, different score cutoff values have been used for identification of staphylococci. This study aimed toevaluate two score cutoffs (2.0 and 1.7) and replicate number onthe identification of staphylococci. A collection of 440 clinical isolates (11 species) identified by 16S rRNA or gap or tuf gene sequencing was evaluated in duplicate.At a cutoff of 1.7, the rates of species, genus, and unreliableidentifications were99.3%, 0.2%, and0.5% respectively, while the respective values were 93.4%, 5.7%, and 0.9% when a cutoff of 2.0 was used. Comparing the two cutoffs (1.7 vs 2.0), the species identification rate was significantly higher (99.3% vs 93.4%, P〈0.05) and the genus identification rate was significantly lower(0.2% vs 5.7%, P〈0.05) at the lower cutoff. Furthermore, 144 reference strains (37 species) of staphylococci wereanalyzed. The species identification rates were 88.9% and 77.1% at cutoffs of 1.7 and 2.0 (P〈 0.05), respectively, and the respective rates for genus identification were 3.5% and 13.9% (P〈0.05).Furthermore, a duplicate test resulted in higher species identification rates than a single test at a cutoff of 1.7 (99.3% vs 92.7%, P〈0.05)or 2.0 (93.4% vs 83.9%, P〈0.05). In conclusion, a cutoff of 1.7 and a duplicate test are recommended in routine practice for identification of staphylococci using the Bruker MALDI-TOF MS. Oligonucleotid Array AlthoughS. arureus is an important pathogen in clinical settings, coagulase-negative staphylococci (CoNS) can causea wide spectrum of infections inimmunosuppressed patients, neonates, and people with prosthetic implants. Phenotypic identification of CoNScan be not reliable and treatment of infections caused by CoNS is challenging as many species in this group carry genes for multiple antibiotic resistances. The aim of the study was to develop an oligonucleotide array to identify 30 species of staphylococci and to detect mecA gene in them. A total of 129 target reference strains and 434 clinical isolates of staphylococci were evaluated by the array. The species names of all reference strains and clinical isolates were reconfirmed by sequencing of genes of 16S rRNA, gaportuf.Species names determined by gene sequencing and methicillin susceptibility determined by the disc diffusion methodwere considered the gold standard. The array correctly identified100% (129/129) of allstaphylococcal reference strains, and 98.9% (186/188) of clinical isolates of S. aureus and 98% (241/246) of CoNS. Comparing with the disc diffusion method, mecAdetection by the array had a sensitivity of 99% and a specificity of 98.9% in clinical isolatesof S. aureus and the respective values inclinical isolates of CoNS were 97.2% and 93.7%. In conclusion, the current array with a turnaround time of 5 hhas a good performance for the identification of staphylococci and for the detection of mecA. Application of the array in direct clinical specimensneeds further investigation.

This thesis is an investigation of two common skin conditions of pigs: exudative epidermitis (EE) and ear necrosis (EN). The cause of exudative epidermitis and risk factors are well understood, however the study was prompted because of reports of treatment failure. A survey of veterinary practitioners (n=15) and pork producers (n=58) was conducted to determine which treatments are commonly used. Amongst farmer respondents topical treatments were often used and in serious cases injectable penicillin G was administered. Thirty farms with a history of EE were visited and skin samples taken from affected pigs. The antimicrobial resistance pattern for isolates of Staphylococcus hyicus and Staphylococcus aureus revealed that almost all isolates were resistant to penicillin G and ampicillin. In addition, certain isolates of S. hyicus as well as S. aureus were shown to possess the mecA gene which is associated with resistance to methicillin. The presence of widespread resistance to penicillin G among staphylococci isolates suggests a reason for poor treatment response. The presence of the mecA gene in staphylococci other than S. aureus recovered from pigs has not been reported before and is of interest from a public health standpoint. A second study investigated EN. The causative agent(s) and the associated risk factors are not well understood. Eleven case farms were visited and skin biopsies and oral swabs taken from pigs in early, mid and late stages of the disease. Bacteriological culturing was performed for staphylococci and spirochetes as well as histological examination of the biopsy samples. Farm-level risk factors were assessed on 14 case farms and 9 control farms. Staphylococci were generally recovered in abundance from the majority of samples but spirochetes were not cultured and only identified microscopically in a small number of tissue samples. Histology revealed that the disease appeared to occur first as a lesion on the epidermal surface that caused tissue damage and led to subsequent invasion of the dermis. This pathogenesis was consistent with the hypothesis that staphylococci colonize the skin surface and produce exfoliating toxins. Ear biting was noted to be commonly present and may be an important contributing factor.
Ontario Pork Animal Health Strategic Initiative Fund Ontario Ministry of Agriculture, Food and Rural Affairs(OMAFRA) Ontario Veterinary College, University of Guelph

Staphylococcus aureus is an important opportunistic pathogen that can cause a wide variety of diseases from mild to life-threatening conditions. S. aureus can colonize many parts of the human body but the anterior nares are the primary ecological niche. Its clinical importance is due to its ability to resist almost all classes of antibiotics available together with its large number of virulence factores. MRSA (Methicillin-Resistant S. aureus) strains are particularly important in the hospital settings, being the major cause of nosocomial infections worldwide. MRSA resistance to β-lactam antibiotics involves the acquisition of the exogenous mecA gene, part of the SCCmec cassette. Fast and reliable diagnostic techniques are needed to reduce the mortality and morbidity associated with MRSA infections, through the early identification of MRSA strains. The current identification techniques are time-consuming as they usually involves culturing steps, taking up to five days to determine the antibiotic resistance profile. Several amplification-based techniques have been developed to accelerate the diagnosis. The aim of this project was to develop an even faster methodology that bypasses the DNA amplification step. Gold-nanoprobes were developed and used to detect the presence of mecA gene in S. aureus genome, associated with resistance traits, for colorimetric assays based on non-crosslinking method. Our results showed that the mecA and mecA_V2 gold-nanoprobes were sensitive enough to discriminate the presence of mecA gene in PCR products and genomic DNA (gDNA) samples for target concentrations of 10 ng/μL and 20 ng/μL, respectively. As our main objective was to avoid the amplification step, we concluded that the best strategy for the early identification of MRSA infection relies on colorimetric assays based on non-crosslinking method with gDNA samples that can be extracted directly from blood samples.

Introdução e Objetivos: Staphylococcus aureus é considerada uma das bactérias Grampositivo mais frequentemente isolada na comunidade e no ambiente hospitalar, estando associada a diversas infeções. As estirpes multirresistentes a antibióticos (Staphylococcus aureus resistente à meticilina − MRSA) representam mundialmente uma das maiores causas de infeções nosocomiais, representando elevadas taxas de mortalidade. Analisaram-se diferentes superfícies do equipamento de medicina dentária para avaliar a presença de Staphylococcus aureus sensível à meticilina (MSSA) e/ou MRSA. Materiais e Métodos: 354 Amostras foram recolhidas com zaragatoas Copon Liquid Amies Elution eSwab, de seis superfícies do equipamento de medicina dentária em seis áreas de atendimento antes e após consulta a pacientes e cultivadas em meio seletivo chromlD® MRSA/chromlD S. aureus. Estirpes MRSA/MSSA foram confirmadas por PCR. Resultados: A percentagem de contaminados foi de: MRSA – 17,5%, sendo que 5,9% correspondem a amostras recolhidas antes do atendimento e 11,6% após; MSSA – 39.3%, com 14,1% amostras recolhidas antes do atendimento e 25,2% após. As amostras não contaminadas correspondem a 55,6% da amostra total. A prevalência de MRSA/MSSA foi significativamente maior após atendimento de pacientes. Clínica de Pacientes Especiais representa a área de atendimento mais contaminada (MRSA – 41,7%; MSSA – 58,3%) e a cuspideira (MRSA – 27,1%; MSSA – 59,3%) a superfície mais contaminada. Conclusões: As clínicas dentárias são reservatórios de transmissão de MRSA/MSSA, contribuindo para potenciais infeções nosocomiais, sendo os pacientes uma possível porta de entrada destas bactérias. Contudo, os protocolos de desinfeção aplicados nestas clínicas são suficientes para o controlo da infeção por estes microrganismos.
Introduction and Objectives: Staphylococcus aureus is one of the most commonly found Gram-positive bacteria in the community and in the hospital environment, being associated with several infections. The multidrug-resistant strains (methicillin-resistant Staphylococcus aureus − MRSA) represent worldwide one of the major causes of nosocomial infections, representing high mortality indexes. Several dental medicine equipment surfaces were analysed to evaluate the presence of methicillin-sensitive Staphylococcus aureus (MSSA) and/or MRSA. Materials and Methods: 354 Samples were collected with Copon Liquid Amies Elution eSwab swabs from six surfaces of dental medicine equipment in six treatment areas before and after patient consultation and cultured in selective medium chromlD® MRSA / chromlD S. aureus. MRSA/MSSA strains were confirmed by PCR. Results: The percentage of contamination was: MRSA − 17.5%; where 5.9% correspond to samples collected before treatment and 11.6% after; MSSA − 39.3%, with 14.1% of samples collected before care and 25.2% after. Uncontaminated samples were 55,6% of the total number of samples. The prevalence of MRSA/MSSA was significantly higher after patient care. Special Patient clinic represented the most contaminated service area (MRSA − 41.7%, MSSA − 58.3%) and dental spittoon (MRSA − 27.1%; MSSA − 59.3%) the most contaminated surface. Conclusions: Dental clinics are reservoirs for the transmission of MRSA/MSSA, contributing to potential nosocomial infections, and patients are a possible gateway for these bacteria. However, the disinfection protocols applied in these clinics are sufficient for the control of the infection by these microorganisms.