Optical sensor systems that integrate Long-Period-Gratings (LPG) as the detection arm have been proven to be highly sensitive and reliable in many applications. With increasing public recognition of threats from bacteria-induced diseases and their potential outbreak among densely populated communities, an intrinsic, low-cost biosensor device that can perform quick and precise identification of the infection type is in high demand to respond to such challenging situations and control the damage those diseases could possibly cause. This dissertation describes the development of a biosensor platform that utilizes polymer thin films, known as ionic self-assembled multilayer (ISAM) films, to be the sensitivity- enhancing medium between an LPG fiber and specific, recognition layer. With the aid of cross- linking reactions, monoclonal antibodies (IgG) or DNA probes are immobilized onto the surface of the ISAM-coated fiber, which form the core component of the biosensor. By immersing such biosensor fiber into a sample suspension, the immobilized antibody molecules will bind the specific antigen and capture the target cells or cell fragments onto the surface of the fiber sensor, resulting in increasing the average thickness of the fiber cladding and changing the refractive index of the cladding. This change occurring at the surface of the fiber results in a decrease of optical power emerging from the LPG section of the fiber. By comparing the transmitted optical power before and after applying the sample suspension, we are able to determine whether or not certain bacterial species have attached to the surface of the fiber, and as a consequence, we are able to determine whether or not the solution contains the targeted bacteria. This platform has the potential for detection of a wide range of bacteria types. In our study, we have primarily investigated the sensitivity and specificity of the biosensor to methicillin- resistant Staphlococcus aureus (MRSA). The data we obtained have shown a sensitive threshold at as low as 102 cfu/ml with pure culture samples. A typical MRSA antibody-based biosensor assay with MRSA sample at this concentration has shown optical power reduction of 21.78%. In a detailed study involving twenty-six bacterial strains possessing the PBP2a protein that enables antibiotic resistance and sixteen strains that do not, the biosensor system was able to correctly identify every sample in pure culture samples at concentration of 104 cfu/ml. Further studies have also been conducted on infected mouse tissues and clinical swab samples from human ears, noses, and skin, and in each case, the system was in full agreement with the results of standard culture tests. However, the system is not yet able to correctly distinguish MRSA and non-MRSA infections in clinical swab samples taken from infected patient wounds. It is proposed that nonspecific binding due to insufficient blocking methods is the key issue. Other bacterial strains, such as Brucella and Francisella tularensis have also been studied using a similar biosensor platform with DNA probes and antibodies, respectively, and the outcomes are also promising. The Brucella DNA biosensor is able to reflect the existence of 3 Brucella strains at 100 cfu/ml with an average of 12.2% signal reduction, while negative control samples at 106cfu/ml generate an average signal reduction of -2.1%. Similarly, the F. tularensis antibodies biosensor has shown a 25.6% signal reduction to LVS strain samples at 100 cfu/ml, while for negative control samples at the same concentration, it only produces a signal reduction of 0.05%. In general, this biosensor platform has demonstrated the potential of detecting a wide range of bacteria in a rapid and relatively inexpensive manner. / Ph. D.

A colonização nasal por Staphylococcus aureus e a infecção pelo HIV representam problemas de saúde pública de preocupação mundial. O objetivo geral foi identificar os fatores de risco para a colonização nasal por Staphylococcus aureus em pessoas vivendo com HIV/aids. Para tanto, foi realizado um estudo tipo caso-controle, com pessoas vivendo com HIV/aids internadas nas unidades especializadas na assistência às doenças infecciosas de um hospital de ensino no interior paulista. A coleta de dados ocorreu de janeiro/2013 a fevereiro/2015, por meio de entrevista individual contemplando dados sociodemográficos e clínicos, além da coleta da secreção nasal com auxílio do swab em meio Stuart, ambos nas primeiras 24 horas de internação. As amostras foram encaminhadas e processadas pelo Laboratório de Microbiologia da própria instituição. Os critérios de inclusão foram: ter idade acima de 18 anos, ser soropositivo ao HIV, estar internado. Nas análises estatísticas foram realizados os testes qui-quadrado de Pearson, Exato de Fisher, t-Student, Wilcoxon e Regressão Logística Univariada e Multivariada, por meio do software SAS®. Os dados estão apresentados em tabelas e figuras. O presente estudo foi aprovado pelo Comitê de Ética em Pesquisa da Escola de Enfermagem de Ribeirão Preto (No CAAE 38990114.5.0000.5393) e pela instituição co-participante (No CAAE 38990114.5.3001.5440). Participaram do estudo 240 pessoas vivendo com HIV/aids, sendo 120 Casos e 120 Controles, houve predominância do sexo masculino em 65,0% dos Casos e 55,0% dos Controles, 35,8% dos Casos estavam na faixa etária de 30 a 39 anos e 45,8% dos Controles tinham idade de 40 a 49 anos, a etnia predominante foi a branca para Casos e Controles, 74,2% e 64,2%, respectivamente. Os grupos foram homogêneos entre si em relação ao sexo, etnia e escolaridade. A média do tempo de diagnóstico foi de 9 anos para Casos e 8,8 anos para Controles. O modelo final de regressão logística evidenciou como fatores de risco associados à colonização nasal por Staphylococcus aureus em pessoas vivendo com HIV/aids, ser da etnia branca, p=0,05 (OR:1,85; IC95% 1,00 - 3,57); ter carga viral >40 cópias/mL, p= 0,03 (OR: 2,90; IC95% 1,15 - 7,30); estar com contagem de LT-CD4+ <200 células/mm3 p=0,001 (OR: 2,71; IC95% 1,53 - 4,81); e apresentar doença oportunista p=0,014 (OR: 2,09; IC95% 1,20 - 3,67). Além disso, foi evidenciado como fator de proteção para a colonização nasal pelo Staphylococcus aureus em pessoas vivendo com HIV/aids o uso de antirretroviral p=0,008 (OR: 0,45; IC95% 0,25 - 0,81). Concluímos que a colonização nasal por Staphylococcus aureus nas pessoas vivendo com HIV/aids foi associada aos fatores: etnia, carga viral, contagem de LT-CD4+ , infecção oportunista e uso de antirretroviral / Staphylococcus aureus nasal colonization and HIV infection represent public health problems of global concern. The overall objective was to identify the risk factors for nasal colonization by Staphylococcus aureus in people living with HIV / AIDS. Therefore, a case-control study was conducted, with people living with HIV / AIDS hospitalized at the units specialized in infectious disease care at a teaching hospital in the interior of São Paulo. Data were collected from January / 2013 to February / 2015 by means of an individual interview, including sociodemographic and clinical data, as well as the collection of nasal secretions with the aid of swab in Stuart\'s medium, both during the first 24 hours of hospitalization. The samples were sent and processed by the Laboratory of Microbiology of the institution itself. The inclusion criteria were: to be over 18 years of age, to be known as infected HIV, to be hospitalized. Statistical analyzes were performed using the Pearson chi-square test, Fisher\'s exact test, Student t-test, Wilcoxon test, and Univariate and Multivariate logistic regression using the SAS® software. The data are presented in tables and figures. The present study was approved by the Research Ethics Committee of the Ribeirão Preto College of Nursing (CAAE 38990114.5.0000.5393) and by the co- participating institution (CAAE 38990114.5.3001.5440). A total of 240 people living with HIV / AIDS participated in the study, of which 120 were Cases and 120 Controls; 65.0% of Cases and 55.0% of Controls were male: 35.8% of Cases were in the age group of 30 at 39 years and 45.8% of the Controls were aged from 40 to 49 years, the predominant ethnicity was white for Cases and Controls, 74.2% and 64.2%, respectively. The groups were homogeneous among themselves in relation to gender, ethnicity and schooling. The mean time of diagnosis was 9 years for Cases and 8.8 years for Controls. The final logistic regression model showed that the risk factors associated with Staphylococcus aureus nasal colonization in people living with HIV / AIDS were white, p = 0.05 (OR: 1.85, 95% CI: 1.00 - 3.57); having viral load> 40 copies / mL, p = 0.03 (OR: 2.90; IC95% 1.15 - 7.30); being with LT-CD4+ <200 cells / mm3 p = 0.001 (OR: 2.71; IC95% 1.53 - 4.81); and present opportunistic disease p = 0.014 (OR: 2,09; IC95% 1,20 - 3,67). In addition, it was also obtained by the final regression final model that the use of antiretroviral therapy is a protection factor of p = 0.008 (OR: 0.45; 95% CI 0.25 - 0.81) for nasal colonization by Staphylococcus aureus. We conclude that nasal colonization by Staphylococcus aureus in people living with HIV/AIDS was associated with factors: ethnicity, viral load, LT-CD4+ count, opportunistic infection, and antiretroviral use

MRSA é um importante agente de colonização e infecção em pacientes hepatopatas e transplantados de fígado. Este estudo tem como objetivo avaliar a clonalidade e a virulência de isolados MRSA de pacientes hepatopatas atendidos no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. De agosto de 2010 a janeiro de 2012, foram coletados swabs nasais e inguinais de 190 pacientes (126 pré-transplante e 64 de pós-transplante). Isolados de MRSA foram identificados fenotipicamente e foi realizada detecção de genes de virulência, caracterização do tipo de SCCmec, análise de polimorfismo genômico por PFGE e técnica de microarranjo. Além disso, determinou-se a CIM para dez antimicrobianos pelo método de microdiluição em caldo. MRSA foi detectado em 20% dos pacientes pelo método de cultura e em 82% por PCRs. Apenas três pacientes colonizados desenvolveram infecção após o transplante. Entre os 69 isolados de MRSA, 42,0% (29/69) apresentaram SCCmec tipo II, 20,3% (14/69) SCCmec tipo I, 20,3% (14/69) SCCmec tipo III, 13,0% (9/69) SCCmec tipo IVa, 2,9% (2/69) SCCmec tipo IV e 1,5% (1/69) SCCmec tipo V. O gene tst foi detectado em 5,8% (4/69) dos isolados MRSA e todos eles foram definidos como SCCmec tipo I. Outros genes identificados por PCR foram: lukD (89,9%; 62/69), lukE (89,9%; 62/69), clf (91,3%; 63/69) e fnbA (89,9%; 62/69). A análise por PFGE dos 69 isolados mostrou a presença de um clone predominante chamado cluster A em 36,2% (25/69) e este cluster apresentou 84,6% de similaridade com o clone NewYork/Japan (BK2464). O dendrograma demonstrou também a presença de um cluster relacionado com BEC (Clone Endêmico Brasileiro) HSJ216. Atualmente o tipo de SCCmec mais prevalente em nosso hospital é o tipo II. Neste estudo, observou-se a presença de isolados virulentos tanto em pacientes hepatopatas como em pacientes transplantados. Nossos resultados mostraram que o clone predominante (cluster A) apresentou diferentes genes de virulência (genes fnbA, clf e lukD-lukE) e foi resistente a pelo menos seis diferentes drogas, além de ser caracterizado como HA-MRSA SCCmec tipo II. Em conclusão, a técnica de microarranjo permite a genotipagem e detecção de genes estafilocócicos clinicamente relevantes, e pode, na maioria dos casos, ser utilizada como uma importante ferramenta para a triagem da virulência e resistência a antimicrobianos em isolados de MRSA / MRSA is an important agent of colonization and infection in patients with liver disease and liver transplant. This study aims to evaluate clonality and virulence of MRSA isolates from liver diseases patients treated at Hospital of Clinics Faculty of Medicine from University of Sao Paulo. From August 2010 to January 2012, we collected nasal and groin swabs from 190 patients (126 pre-liver and 64 post-liver). MRSA isolates were identified phenotypically and the detection of virulence genes, characterization of SCCmec type, microarray and genomic polymorphism analysis by PFGE were done. In addition, it was determined the MIC for ten antibiotics by broth microdillution method. MRSA was detected in 20% patients by culture method and 82% by PCR. Only three patients colonized developed infection post-transplantation. Among the 69 MRSA isolates, 42.0% (29/69) had type II SCCmec, 20.3% (14/69) SCCmec type I, 20.3% (14/69) SCCmec type III, 13.0% (9/69) SCCmec type IVa, 2.9% (2/69) SCCmec type IV and 1.5% (1/69) SCCmec type V. The tst gene was detected in 5.8% (4/69) of MRSA isolates and all of them were defined as SCCmec type I. Other genes were identified by PCR: lukD (89.9%; 62/69), lukE (89.9%; 62/69), clf (91.3%; 63/69) and fnbA (89.9%; 62/69). The PFGE analysis of 69 isolates showed the presence of a predominant cluster named cluster A in 36.2% (25/69) and this cluster had 84.6% similarity with New York/Japan clone (BK2464). Dendrogram also demonstrated presence of one cluster related with BEC (Brazilian Endemic Clone) HSJ216. Currently the most prevalent SCCmec type in our hospital is type II. In this study, we observed virulent isolates in pre and post-transplantation patients. Our results showed that the predominant clone (cluster A) had different virulence genes (genes fnbA, clf and lukD-lukE) and was resistant to at least six different drugs, in addition to being characterized as HA-MRSA SCCmec type II. In conclusion, microarray profiling allows genotyping and detection of clinically relevant staphylococcal genes, and can, in most cases, be used as an important tool to screening virulence and antibiotic resistance genes in MRSA isolates

Na última década, Staphlylococcus aureus resistentes à meticilina não multidroga resistente (NM-MRSA) tem sido descrito como um importante agente de infecção de corrente sanguínea em nosso serviço. Este estudo de coorte prospectivo, realizado entre fevereiro de 2009 e janeiro de 2010 na unidade neonatal, avaliou 403 recém-nascidos (RN), suas 382 mães e 148 profissionais da área da saúde (PS). Duzentos e dezessete NB (54%), 187 mães (48%) e 87 PS (59%) foram colonizados por S. aureus (SA). A colonização por S. aureus resistente à meticilina (MRSA) foi maior entre RN (15%) do que entre mães (4.7%) e PS (3.4%). Embora a transmissão da mãe para seu RN tenha ocorrido, na maior parte dos casos, a mãe não foi a responsável pela colonização do RN. Houve dois padrões predominantes de polimorfismo do DNA por eletroforese em campo pulsado (PFGE) entre os RN, e algumas mães e PS foram colonizados por eles. Fatores estatisticamente associados com colonização por MRSA foram baixo nível de escolaridade materna (fator de risco - OR: 2.99; 95%CI: 1.10-8.07) e rinossinusite materna (fator protetor - OR: 0.33; 95%CI: 0.12-0.88). Entre os Rn que permaneceram hospitalizados mais do que 72 horas, o aleitamento materno foi protetor (OR: 0.22; 95%CI: 0.05-0.98). Todos os isolados foram NM-MRSA, portavam poucos fatores de virulência e Staphylococcal Cassete Chromossome mec (SCCmec) tipos IVa e IVd predominaram. Embora não tenham ocorrido casos de infecção, a transmissão nosocomial de MRSA claramente ocorreu na unidade neonatal e aponta para a necessidade de implementação de práticas de controle de infecção, como higienização das mãos para prevenção de infecção cruzada. Outras práticas de promoção à saúde, básicas, mas abrangentes, podem ser fundamentais, como educação e aleitamento materno / In the last decade non-multiresistant methicillin-resistant S. aureus (NM-MRSA) has been described as an important agent in bloodstream infections in our hospital. This prospective cohort study, conducted from February 2009 through January 2010 in the neonatal unit, evaluated 403 newborns (NB), their 382 mothers and 148 health care workers (HCW). 217 NB (54%), 187 mothers (48%) and 87 HCW (59%) were colonized by S. aureus (SA). Methicillin-resistant S. aureus (MRSA) colonization was greater among NB (15%) than mothers (4.7%) and HCW (3.4%). Although mother-to-NB transmission occurred, in most cases mothers were not responsible for NB colonization. There were two predominant PGFE patterns among the NB and some mothers and HCW became colonized by them. Factors significantly associated with MRSA carriage by NB were lower level of maternal schooling (risk factor: OR: 2.99; 95%CI: 1.10-8.07) and maternal rhinosinusitis (protective factor: OR: 0.33; 95%CI: 0.12-0.88). Among NB who remained hospitalized for more than 72 hours, breast feeding was protective (OR: 0.22; 95%CI: 0.05-0.98). All the isolates were NM-MRSA, carried few virulence factors and Staphylococcal Cassete Chromossome mec (SCCmec) types IVa and type IVd predominated. Although there were no cases of infection, nosocomial transmission of MRSA clearly occurred in the neonatal unit and this highlights the need for infection control practices such as hand hygiene to prevent cross-dissemination. Other healthcare practices, which are very basic but also ample in scope, may play a role, such as general education of women and breast feeding

Made available in DSpace on 2014-07-29T15:25:19Z (GMT). No. of bitstreams: 1 Tese Marlene Andrade Martins.pdf: 838189 bytes, checksum: 6a399180bc5d72130785d31bc207a701 (MD5) Previous issue date: 2012-04-05 / INTRODUCTION: In recent years, there has been the emergence of multidrug-resistant strains from patients seen in primary lesions with venous ulcers. OBJECTIVES: To determine the frequency and susceptibility profile of Staphylococcus sp.; verify the prevalence of methicillin resistant Staphylococcus aureus (MRSA) and minimum inhibitory concentration (MIC) of isolates; detect MLSB resistance in Staphylococcus sp. isolated from venous ulcers; describe the frequency of clinical signs and symptoms indicative of infection of venous ulcers (2005); identify the clinical stage of infection, to determine the relationship between clinical signs and symptoms of infection and culture results found for Staphylococcus sp. METHODS: Were evaluated 69 people with 98 ulcers in the period of October/09 to October/2010. The isolates resistant to cefoxitin and/or oxacillin (disk diffusion) were subjected to confirmatory test for detection of MIC, using tapes of oxacillin (E-test ®). The phenotypic detection of the inducible resistance to the MLSB group was performed by the D-test. The clinical signs and symptoms were investigated in accordance with criteria established by the document Identifying criteria for wound infection (EWMA, 2005). Were used the software Statistics Package for Social Sciences for Windows ® (SPSS 17.0) for data processing. For association analysis were used the Chi-square or Fisher's exact tests, adopting a significance level of 5% (&#945; = 0.05). Legal ethical aspects have been respected. RESULTS: The prevalence of S. aureus was 83% and 15% of CoNS. Were identified 28% of MRSA and 47% MRCoNS. Among S. aureus, 69.6% were resistant to erythromycin, 69.6% to clindamycin, 69.6% to gentamicin and 100% to ciprofloxacin. 74% of MRSA showed high level resistance to oxacillin, MIC &#8805; 256 &#956;g/mL, and in 65.2% predominated MLSBc constitutive resistance. Of the two MRSA isolates with sensibility to clindamycin, just one was positive for D-test. Signs and symptoms of infection more frequent were: discoloration of the wound and increase the volume of exudate. The stage III of infection was identified in 70 (71.4%) ulcers. An association among friable granulation tissue and cultures positive for Staphylococcus sp. (P = 0.004) and increase the local temperature of the skin and multiresistant Staphylococcus (p = 0.002). CONCLUSIONS: Staphylococcus sp. multidrug-resistant were isolates of venous ulcers in people treated in primary care. The results confirm that the MRSA isolates, beyond resistance to beta-lactams, also exhibit cross-resistance to other antimicrobials such as clindamycin, erythromycin, ciprofloxacin, and gentamicin. Remain a challenge to find indicators of infection for venous ulcers / INTRODUÇÃO: Nos últimos anos, tem sido observada a ocorrência de cepas multirresistentes provenientes de pacientes atendidos na atenção primária com lesões de etiologia venosa. OBJETIVOS: Determinar nos isolados de úlceras venosas a frequência e o perfil de suscetibilidade de Staphylococcus spp. aos antimicrobianos e, assim determinar a concentração inibitória mínima (MIC); detectar a resistência MLSB e correlacionar a positividade das culturas com os sinais e sintomas clássicos de infecção. Adiciona-se a classificação do estágio clínico de infecção das úlceras segundo critérios de EWMA, 2005. MÉTODOS: Foram avaliadas 69 pessoas com 98 úlceras no período de outubro/09 a outubro/2010. Os isolados resistentes a cefoxitina e/ou oxacilina (disco-difusão) foram submetidos ao teste confirmatório para detecção da CIM, empregando fitas de oxacilina (E-test®). Realizou-se a detecção fenotípica da resistência induzível ao grupo MLSB por meio do D-test. Utilizou-se o software Statistics Package for the Social Sciences for Windows® (SPSS 17.0), para processamento dos dados. A análise de associação envolveu os testes Qui-quadrado ou Exato de Fisher s, adotando-se o nível de significância de 5% (&#945;=0,05). Aspectos éticos legais foram atendidos. RESULTADOS: A prevalência de S. aureus foi de 83% e de 15% de CoNS. Identificou-se 28% de MRSA e 47% de MRCoNS. Entre o S. aureus, 69,6% apresentaram resistência a eritromicina, 69,6% a clindamicina, 69,6% a gentamicina e 100% a ciprofloxacina. 74% dos MRSA apresentaram elevado nível de resistência a oxacilina, MIC &#8805; 256 &#956;g/mL, e em 65,2% predominou a resistência constitutiva MLSBc. Dentre os dois isolados MRSA com sensibilidade à clindamicina, apenas um foi positivo para o D-teste. Os sinais e sintomas de infecção mais frequentes foram: descoloração do leito da lesão e aumento do volume do exsudato. O estágio III de infecção representou 70 (71,4%) das úlceras. Verificou-se associação entre tecido de granulação friável e culturas positivas para Staphylococcus spp. (p=0,004) e, aumento da temperatura local da pele com a ocorrência de Staphylococcus multirresistentes (p=0,002). CONCLUSÕES: Staphylococcus multirresistentes foram isolados de úlceras venosas em pessoas atendidas na atenção primária. Os resultados apresentados confirmam que os isolados MRSA, além da resistência aos beta-lactâmicos, também apresentam resistência cruzada a outros antimicrobianos. Cabe a preocupação com o aumento dessa resistência microbiana o que exige além da vigilância epidemiológica, também a priorização das políticas publicas em saúde por meio de programas efetivos de prevenção e controle. Ainda, permanece o desafio acerca dos indicadores de infecção para as úlceras venosas.

Introdução: a coleta de culturas de vigilância é uma das estratégias utilizadas no controle de infecções causadas por Staphylococcus aureus, especialmente S. aureus resistente a meticilina (MRSA). Estas culturas são utilizadas para determinar portadores assintomáticos e prevenir a disseminação do patógeno para outros pacientes através da tomada de medidas de isolamento do portador. Neste contexto, tem-se demonstrado que a descolonização de portadores pode reduzir o risco de infecções estafilocócicas em certas ocasiões. O sítio anatômico mais comumente analisado são as narinas anteriores, mas continuamos a nos questionar se seria necessária a cultura de outros sítios anatômicos para este fim. Objetivos: este estudo objetivou avaliar o desempenho de diferentes sítios de cultura de vigilância em determinar a colonização de gestantes e recém-nascidos (RN) e determinar os fatores associados a colonização nasal por S. aureus. Metodologia: este é um estudo descritivo, desenvolvido no Hospital das Clínicas de São Paulo, Brasil, um hospital terciário universitário. Os pacientes envolvidos no estudo são gestantes durante trabalho de parto e seus recém-nascidos. A coleta de material de seu em quatro sítios anatômicos para os recém-nascidos: narinas anteriores, orofaringe, períneo e umbigo, no momento do parto, no terceiro dia e semanalmente. Para as gestantes, foram coletados quatro sítios anatômicos: narinas anteriores, anus, períneo e orofaringe. Apenas a primeira cultura positiva foi considerada, os pacientes colonizados nas narinas foram comparados àqueles colonizados apenas em sítios extranasais e os fatores de risco para colonização por S. aureus foram determinados. Resultados: foram incluídas 392 gestantes e 382 recém-nascidos. A colonização materna por S. aureus foi 53% (MSSA 49% e MRSA 9%). A colonização de RN foi 47% (MSSA 39% e MRSA 9%). Entre os RN, o melhor sítio de coleta foi o umbigo (64% para MSSA e 68% para MRSA) e a melhor associação foi narinas anteriores mais umbigo (86% para MSSA e 91% para MRSA). Entre as gestantes o melhor sítio foi narinas anteriores (MSSA 59% e MRSA 67%) e a melhor associação de sítios foi narinas anteriores mais orofaringe (83% para MSSA e 80% para MRSA). Dentre os fatores de risco, apenas o número de moradores na mesma residência foi associado à colonização materna por S. aureus (2,0+0,6 vs 3,6+1,8; p: 0,04). Conclusão: nosso estudo confirma a necessidade da coleta de vários sítios para assegurar a sensibilidade das culturas de vigilância. Não há fatores associados a colonização nasal que distinguem portadores nasais dos colonizados em sítios extranasais. Os programas de controle de infecção baseados em culturas de vigilância nasal podem ser comprometidos / Introduction: Surveillance cultures are one of the strategies used to control Staphylococcus aureus infections, especially methicillin-resistant S. aureus (MRSA). These cultures are used to determine asymptomatic carriers and prevent spread of the organism to other patients by putting carriers under isolation precautions. Also some authors demonstrated that decolonization of carriers can reduce the risk of staphylococcal infections under certain conditions. The most commonly cultured body sites are the anterior nares but the challenge remains to determine whether routine culturing of other body sites is necessary. Objectives: the study objective to evaluate the performance of surveillance cultures at various body sites in determining S.aureus colonization in pregnant women and their newborns (NB) and determine factors associated with nasal colonization. Methods: This is a descriptive study, developed on Hospital das Clinicas, São Paulo, Brazil, a tertiary-care university hospital. Patients enrolled: pregnant women during labor and their newborns. Material collection: For NB four sites were evaluated: nares, oropharynx, perineum and umbilical stump at birth, 3rd day and weekly. For pregnant women four sites during labor: anterior nares, anus, perineum and oropharynx. Only the first positive culture was considered. Nasally colonized patients were compared with colonized only extra-nasally and risk factors to S. aureus colonization were determined. Results: 392 pregnant women and 382 NB were included. S. aureus colonization was 53% among pregnant women (MSSA 49% and MRSA 4%). S. aureus colonization among NB was 47% (MSSA 39% and MRSA 9%). For NB patients, the best body site was the umbilical stump (64% for MSSA and 68% for MRSA). The best combination in NB was nares plus umbilical stump (86% for MSSA and 91% for MRSA). Among pregnant women, the best body site was the anterior nares (MSSA 59% and MRSA 67%). The best combination was nares plus oropharynx, (83% for MSSA and 80% for MRSA). Only the smaller number of household members was associated with MRSA carriage in pregnant women (2.2±0.6 vs 3.6±1.8; p: 0.04). Conclusion: Our study confirms the need for multiple culture sites to assure sensitivity. No features distinguish nasal carriers from only extra-nasal colonized people. Control programs relying mainly on nasal surveillance cultures may be compromised

Depuis quelques années et dans plusieurs pays, un nouveau type de Staphylococcus aureus résistant à la méthicilline (SARM), le séquence type (ST) 398, a été fréquemment retrouvé chez les porcs et chez les fermiers en contact avec ces porcs. Au Canada, très peu d’informations sont disponibles concernant le SARM d’origine porcine. Une première étude dans notre laboratoire a permis de récolter 107 isolats de SARM provenant de deux abattoirs porcins du Québec. Le présent travail vise à caractériser les gènes de virulence et de résistance aux antibiotiques de ces SARM, d’étudier leur formation de biofilm en relation avec la spécificité du groupe agr et de vérifier la localisation plasmidique et la transférabilité de ces gènes à des souches de SARM d’origine humaine. Plusieurs souches ont démontré différents patrons phénotypiques de résistance aux antibiotiques. Vingt-quatre souches représentatives de ces isolats ont été soumises à une caractérisation plus approfondie par une étude génotypique en utilisant une biopuce à ADN et un grand nombre de gènes de virulence a été détecté codant pour des entérotoxines staphylococcales, des leucocidines, des hémolysines, des auréolysines, des facteurs d’immunoévasion, des superantigènes, des facteurs d’adhésion et des facteurs impliqués dans la formation de biofilm. Des gènes de résistance envers les aminoglycosides, les macrolides, les lincosamides, les tétracyclines et les biocides ont été également détectés par biopuce et leur localisation plasmidique a par la suite été déterminée. La transférabilité de ces gènes de souches porcines à des souches de SARM d’origine humaine a été démontrée par conjugaison bactérienne; ainsi le transfert horizontal de certains gènes de résistance aux antibiotiques et de virulence a été observé. Ces travaux de recherche apportent une meilleure connaissance de la résistance aux antibiotiques et de la virulence des SARM d’origine porcine et de leur potentiel de contribution à l’émergence de certaines résistances et facteurs de virulence chez le SARM d’origine humaine. / In recent years and in several countries, a new type of methicillin-resistant Staphylococcus aureus (MRSA), the sequence type (ST) 398, has been frequently found in pigs and in farmers in contact with these pigs. In Canada, little information is available concerning MRSA from pigs. A previous study in our laboratory identified 107 MRSA isolates from two pig slaughterhouses in Quebec. This study was conducted to determine antimicrobial resistance and virulence genes of MRSA from abattoir pig, to study their biofilm formation in relation with agr specificity groups and to evaluate horizontal transfer of genes to a MRSA of human clinical origin. Different phenotypic patterns of antimicrobial resistance were observed in these MRSA and a representative subset of these isolates was selected for further characterization. Twenty-four porcine MRSA were characterized by a DNA microarray, the StaphyType of CLONDIAG. Our results demonstrated that the MRSA strains from the abattoirs contain several antimicrobial resistance genes responsible for macrolide and tetracycline resistance and virulence genes encoding staphylococcal enterotoxins, hemolysins, leukocidins, aureolysin, superantigens, immunoevasion, adhesion, and biofilm development. This study presents the first evidence that horizontal transfer of some of these genes can occur between MRSA of porcine and human origin. We also report for the first time biofilm formation in Livestock Associated-MRSA of porcine origin associated with agr group II. It is possible that biofilm formation favors colonization, persistence as well as zoonotic potential. This research provides a better understanding of antimicrobial resistance and virulence of MRSA from pigs and their potential contribution to the emergence of some resistance and virulence factors in MRSA of human origin.

Le Staphylococcus aureus résistant à la méthicilline (SARM) est un pathogène important qui a été identifié comme agent d‟infection chez les animaux d‟élevage et les travailleurs exposés à ces animaux. Au Canada, très peu d‟informations sont disponibles concernant les SARMs d‟origine porcine. L‟objectif de cette étude était de déterminer la prévalence des SARMs provenant de porcs à l‟abattoir, de caractériser leur résistance aux antibiotiques ainsi que d‟évaluer le niveau de séroconversion des porcs envers le S. aureus chez les animaux porteurs ou non du SARM. Un total de 107 isolats ont été identifiés positifs aux SARMs sur 660 échantillons. La prévalence de SARMs à l‟abattoir A était de 30,8% et de 23,8% à l‟abattoir B. La susceptibilité aux antibiotiques a été déterminée en utilisant la méthode de micro-dilution de Sensititre. Tous les isolats ont démontré une sensibilité envers la ciprofloxacine, la gatifloxacine, la gentamicine, la lévofloxacine, le linézolide, la quinupristine/dalfopristine, la rifampicine, la streptomycine, le triméthoprime/sulfaméthoxazole et la vancomycine. De la résistance a été observée envers la daptomycine (0,93%), l‟érythromycine (29%), la clindamycine (29%), la tétracycline (98,1%). De plus, 30% des SARMs isolés étaient résistants à plus de deux antibiotiques autres que les β-lactamines. Par typage, deux clones prédominants ont été obtenus ainsi que deux types de SCCmec (type V et possiblement un nouveau type comprenant les cassettes III et IVb). 15 clones ont été identifiés par typage MLVA, comprenant les clones prédominants VI (40.1%; 43/107) et XI (17.7%; 19/107). Deux souches de SARMs ont été caractérisées par biopuce à ADN et des gènes d‟antibiorésistance, de typage (SCCmec et MLST) et de virulence ont été identifiés. Sans considération pour le site de colonisation, les porcs SA-/MRSA- (n=34) et les porcs SA+ (n=194) montrent, respectivement, des taux de séroconversion de 20.6% et 32.5%. Les porcs colonisés par un SARM à un site de iv prélèvement et non colonisés par un SA à l‟autre site (n=18) montrent une séroconversion (5.6%) significativement (P < 0.05) plus faible comparativement aux porcs colonisés par SA à un ou deux sites de prélèvement et n‟ayant pas de SARM. Nos résultats démontrent que les porcs provenant d‟abattoir peuvent être colonisés par des SARMs multi-résistants aux antibiotiques. De plus, ces SARMs sont possiblement capable de coloniser leurs hôtes sans stimuler la production d‟anticorps et ce par l‟atténuation de la réponse immunitaire ou par la colonisation de porcs qui sont moins immunocompétents. / Methicillin-resistant Staphylococcus aureus (MRSA) found in food producing animals is a major public health concern. Transmission to humans has been reported and MRSA represents a reservoir of antimicrobial resistance genes. Little is known on how MRSA successfully establishes colonization and how it is able to persist in the host. This study was conducted to determine the occurrence and the antimicrobial resistance profile of MRSA from abattoir pigs and their level of seroconversion toward S. aureus (SA). A total of 107 isolates were identified as MRSA from 660 samples. Antimicrobial susceptibilities were determined by broth microdilutions. Fifteen clones were identified by MLVA with clones VI (40.1%; 43/107) and XI (17.7%; 19/107) being the most predominant. All MRSA isolates were pvl-, tst-, eta- and etb-negative. Most isolates were SCCmec type V (70.1%; 75/107). All MRSA isolates were susceptible to ciprofloxacin, gatifloxacin, gentamicin, levofloxacin, linezolid, quinupristin/dalfopristin, rifampin, streptomycin, trimethroprim/sulfamethoxazole and vancomycin. However, resistance was observed toward clindamycin (29%), daptomycin (0.9%), erythromycin (29%) and tetracycline (98.1%). Multi-resistance was confirmed in MRSA since 28% of all isolates were resistant toward three antimicrobials other than β-lactams. The effect of MRSA carriage on seroconversion was examined to see whether the host responded differently to MRSA or SA colonization. The presence of SA-specific antibodies in pig serums was measured for each animal using indirect ELISA and a mixture of two widespread SA antigens (IsdH [HarA] and IsdB). Regardless of the colonization site, SA-/MRSA- pigs (n=34) and SA+ pigs (n=194) showed 20.6% and 32.5% seroconversion, respectively. Notably, pigs colonized by MRSA at one body site and no SA at the other sampling site (n=18) showed a significantly lower (5.6%) seroconversion (P < 0.05) compared to pigs colonized by SA at one or both vi sites without MRSA. The findings of the study show that the nares and axillae of abattoir pigs can harbor MRSA strains with multiple antimicrobial resistances. In addition, these MRSA were possibly able to colonize the host either without stimulating antibody production, by attenuating the immune response or by colonizing pigs that are less immunocompetent. This may explain the success of MRSA colonization and persistence in pigs. Further studies are required to better elucidate MRSA colonization in abattoir pigs and their public health risk.

Antibiotic resistant pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant enterococci (VRE), are an increasing burden on healthcare systems. Hospital acquired infections with these organisms leads to higher morbidity and mortality compared with the sensitive strains of the same species and both VRE and MRSA are on the rise worldwide including in Australian hospitals. Emerging community infectious diseases are also having an impact on hospitals. The Severe Acute Respiratory Syndrome virus (SARS Co-V) was noted for its propensity to spread throughout hospitals, and was contained largely through social distancing interventions including hospital isolation. A detailed understanding of the transmission of these and other emerging pathogens is crucial for their containment. The statistical inference and mathematical models used in this thesis aim to improve understanding of pathogen transmission by estimating the transmission rates of contagions and predicting the impact of interventions. Datasets used for these studies come from the Princess Alexandra Hospital in Brisbane, Australia and Shanxi province, mainland China. Epidemiological data on infection outbreaks are challenging to analyse due to the censored nature of infection transmission events. Most datasets record the time on symptom onset, but the transmission time is not observable. There are many ways of managing censored data, in this study we use Bayesian inference, with transmission times incorporated into the augmented dataset as latent variables. Hospital infection surveillance data is often much less detailed that data collected for epidemiological studies, often consisting of serial incidence or prevalence of patient colonisation with a resistant pathogen without individual patient event histories. Despite the lack of detailed data, transmission characteristics can be inferred from such a dataset using structured HiddenMarkovModels (HMMs). Each new transmission in an epidemic increases the infection pressure on those remaining susceptible, hence infection outbreak data are serially dependent. Statistical methods that assume independence of infection events are misleading and prone to over-estimating the impact of infection control interventions. Structured mathematical models that include transmission pressure are essential. Mathematical models can also give insights into the potential impact of interventions. The complex interaction of different infection control strategies, and their likely impact on transmission can be predicted using mathematical models. This dissertation uses modified or novel mathematical models that are specific to the pathogen and dataset being analysed. The first study estimates MRSA transmission in an Intensive Care Unit, using a structured four compartment model, Bayesian inference and a piecewise hazard methods. The model predicts the impact of interventions, such as changes to staff/patient ratios, ward size and decolonisation. A comparison of results of the stochastic and deterministic model is made and reason for differences given. The second study constructs a Hidden Markov Model to describe longitudinal data on weekly VRE prevalence. Transmission is assumed to be either from patient to patient cross-transmission or sporadic (independent of cross-transmission) and parameters for each mode of acquisition are estimated from the data. The third study develops a new model with a compartment representing an environmental reservoir. Parameters for the model are gathered from literature sources and the implications of the environmental reservoir are explored. The fourth study uses a modified Susceptible-Exposed-Infectious-Removed (SEIR) model to analyse data from a SARS outbreak in Shanxi province, China. Infectivity is determined before and after interventions as well as separately for hospitalised and community symptomatic SARS cases. Model diagnostics including sensitivity analysis, model comparison and bootstrapping are implemented.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that is endemic in hospitals all over the world. It has more recently emerged as a serious threat to the general public in the form of community-acquired MRSA. MRSA has been implicated in a wide variety of diseases, ranging from skin infections and food poisoning to more severe and potentially fatal conditions, including; endocarditis, septicaemia and necrotising pneumonia. Treatment of MRSA disease is complicated and can be unsuccessful due to the bacterium's remarkable ability to develop antibiotic resistance. The considerable economic and public health burden imposed by MRSA has fuelled attempts by researchers to understand the evolution of virulent and antibiotic resistant strains and thereby improve epidemiological management strategies. Central to MRSA transmission management strategies is the implementation of active surveillance programs, via which unique genetic fingerprints, or genotypes, of each strain can be identified. Despite numerous advances in MRSA genotyping methodology, there remains a need for a rapid, reproducible, cost-effective method that is capable of producing a high level of genotype discrimination, whilst being suitable for high throughput use. Consequently, the fundamental aim of this thesis was to develop a novel MRSA genotyping strategy incorporating these benefits. This thesis explored the possibility that the development of more efficient genotyping strategies could be achieved through careful identification, and then simple interrogation, of multiple, unlinked DNA loci that exhibit progressively increasing mutation rates. The baseline component of the MRSA genotyping strategy described in this thesis is the allele-specific real-time PCR interrogation of slowly evolving core single nucleotide polymorphisms (SNPs). The genotyping SNP set was identified previously from the Multi-locus sequence typing (MLST) sequence database using an in-house software package named Minimum SNPs. As discussed in Chapter Three, the genotyping utility of the SNP set was validated on 107 diverse Australian MRSA isolates, which were largely clustered into groups of related strains as defined by MLST. To increase the resolution of the SNP genotyping method, a selection of binary virulence genes and antimicrobial resistance plasmids were tested that were successful at sub typing the SNP groups. A comprehensive MRSA genotyping strategy requires characterisation of the clonal background as well as interrogation of the hypervariable Staphylococcal Cassette Chromosome mec (SCCmec) that carries the β-lactam resistance gene, mecA. SCCmec genotyping defines the MRSA lineages; however, current SCCmec genotyping methods have struggled to handle the increasing number of SCCmec elements resulting from a recent explosion of comparative genomic analyses. Chapter Four of this thesis collates the known SCCmec binary marker diversity and demonstrates the ability of Minimum SNPs to identify systematically a minimal set of binary markers capable of generating maximum genotyping resolution. A number of binary targets were identified that indeed permit high resolution genotyping of the SCCmec element. Furthermore, the SCCmec genotyping targets are amenable for combinatorial use with the MLST genotyping SNPs and therefore are suitable as the second component of the MRSA genotyping strategy. To increase genotyping resolution of the slowly evolving MLST SNPs and the SCCmec binary markers, the analysis of a hypervariable repeat region was required. Sequence analysis of the Staphylococcal protein A (spa) repeat region has been conducted frequently with great success. Chapter Five describes the characterisation of the tandem repeats in the spa gene using real-time PCR and high resolution melting (HRM) analysis. Since the melting rate and precise point of dissociation of double stranded DNA is dependent on the size and sequence of the PCR amplicon, the HRM method was used successfully to identify 20 of 22 spa sequence types, without the need for DNA sequencing. The accumulation of comparative genomic information has allowed the systematic identification of key MRSA genomic polymorphisms to genotype MRSA efficiently. If implemented in its entirety, the strategy described in this thesis would produce efficient and deep-rooted genotypes. For example, an unknown MRSA isolate would be positioned within the MLST defined population structure, categorised based on its SCCmec lineage, then subtyped based on the polymorphic spa repeat region. Overall, by combining the genotyping methods described here, an integrated and novel MRSA genotyping strategy results that is efficacious for both long and short term investigations. Furthermore, an additional benefit is that each component can be performed easily and cost-effectively on a standard real-time PCR platform.