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% (α = 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 ≥ 256 μg/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% (α=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 ≥ 256 μg/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.

S.aureus resistente à meticilina(SARM) é um problema em centros de fibrose cística(FC). Foi desenvolvido um multiplex PCR(mPCR) para detecção do SARM em secreção respiratória de 106 pacientes com FC. Foram usados 3 pares de primers para amplificar os genes: mecA, coa, 16S rRNA. O mPCR detectou até 0,25pg de DNA de SARM e identificou 70/106(66,0%) pacientes com S.aureus e 28/106(26,4%) com SARM. O mPCR mostrou especificidade, sensibilidade, valores preditivos positivo e negativo de 87,8%, 84,4%, 50% e 97,5%, considerando a cultura como padrão-ouro. Os resultados discordantes foram testados com outros primers, confirmando os obtidos pelo mPCR em 82/84. O mPCR mostrou-se método rápido e confiável para detecção de SARM / Methicillin-resistant S.aureus(MRSA) is a significant concern in cystic fibrosis(CF) centers. A multiplex PCR(MPCR) was developed to detect MRSA in respiratory secretion of 106 CF patients. Three pairs of primers were used for amplification of genes: mecA, coa, 16S rRNA. MPCR detected 0.25pg of MRSA DNA and identified 70/106(66.0%) of patients with S.aureus and 28/106(26.4%) with MRSA. MPCR showed specificity, sensitivity, positive and negative predicted values at 87.8%, 84.4%, 50% and 97.5%, considering culture as the gold standard. Discrepant results were retested using different primers, and confirmed MPCR results in 82/84. The developed MPCR was found to be a rapid and reliable method for MRSA detection

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

Background Coagulase-negative staphylococci (CoNS) and in particular Staphylococcus epidermidis have emerged as major pathogens primarily causing nosocomial infections in patients with indwelling medical devices. These infections are often caused by multidrug-resistant strains of S. epidermidis (MDRSE). Other clinical entities due to CoNS are lower urinary tract infections (UTI) in women and native valve endocarditis. The purpose of this work was to investigate the frequency of antibiotic resistance and the molecular epidemiology of both hospital and community-associated isolates of S. epidermidis in order to examine if certain clones are related to MDRSE infections. Furthermore, we aimed to explore if specific clones of S. saprophyticus are associated with UTI in women. Methods A total of 359 hospital-associated methicillin-resistant isolates of CoNS obtained from 11 hospitals in northern Europe and 223 community-associated staphylococcal isolates were examined. Furthermore, 126 isolates of S. saprophyticus isolated from women with uncomplicated UTI from five different locations in northern Europe were analyzed. Pulsed-field gel electrophoresis (PFGE) was used for genotyping. Additionally, some of the S. epidermidis isolates were analyzed with multilocus sequence typing (MLST). Antibiotic susceptibility was determined for all isolates by the disc diffusion test. Results 293 of the 359 (82%) hospital-associated and 124 of the 223 (56%) community-associated isolates belonged to the species S. epidermidis. Among the hospital-associated S. epidermidis isolates, two dominating PFGE types (type A and B) were distinguished, comprising 78 (27%) and 51 (17%) isolates, respectively. Type A, which was detected in a Norwegian and eight Swedish hospitals, corresponded with a novel sequence type (ST215). Type B was discovered in a German, a Danish and seven Swedish hospitals and corresponded with ST2. In contrast, community-associated isolates of S. epidermidis were genetically extremely diverse with no predominating genotype, and showed a low rate of antibiotic resistance; only two (1.6%) methicillin-resistant strains were detected. Among 126 analyzed isolates of S. saprophyticus, 47 different PFGE profiles were identified. Several clusters of genetically highly related isolates were detected among isolates obtained from different locations and periods of time. Conclusion We have demonstrated the occurrence, persistence and potential dissemination of two multidrug-resistant S. epidermidis (MDRSE) genotypes, including a novel sequence type (ST215), within hospitals in northern Europe. Community-associated isolates of S. epidermidis showed a low rate of methicillin-resistance and were genetically heterogeneous. These results indicate that MDRSE by large are confined to the hospital setting in our region. Moreover, although the S. saprophyticus population was quite heterogeneous, indistinguishable isolates of S. saprophyticus causing lower UTI in women were identified in different countries 11 years apart, indicating the persistence and geographical spread of some clones of S. saprophyticus.

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.

Inledning: Antibiotikaresistens är ett av de största hoten mot global folkhälsa. Meticillinresistent Staphylococcus aureus, tidigare främst associerat med sjukhusvård, överförs nu mellan individer i samhället (samhällsförvärvad meticillinresistent staphylococcus aureus). Att identifiera riskfaktorer är centralt för att kunna bedriva effektivt preventivt arbete mot smittöverföring. Syfte: Syftet med uppsatsen var att identifiera och beskriva riskfaktorer associerade med förekomst av samhällsförvärvad meticillinresistent staphylococcus aureus. Metod: Litteraturstudie baserad på 20 internationella artiklar. Huvudfynden i artiklarna kategoriserades i teman. Resultat: Riskfaktorer på samhälls-, hushålls- och individnivå kunde identifieras, bland annat rörande klimat, tidigare antibiotikaanvändning och samsjuklighet.  Diskussion: Ett fåtal enkelt påverkbara riskfaktorer kunde identifieras. Av de identifierade riskfaktorerna är troligen inte alla generaliserbara till en svensk kontext. I flera tidigare studier framhålls att samhällsförvärvad meticillinresistent staphylococcus aureus främst drabbar individer som sedan tidigare är friska, vilket fynden i föreliggande uppsats delvis motsäger då samsjuklighet i exempelvis HIV, diabetes och fetma identifierades som riskfaktorer associerade med samhällsförvärvad meticillinresistent staphylococcus aureus. / Introduction: Antibiotic resistance a threat to global public health. Methicillin resistant staphylococcus aureus, previously primarily associated with hospital care, is now being transmitted in the community (community acquired methicillin resistant staphylococcus aureus). Identifying risk factors is central to enable effective preventive efforts against transmission of community acquired methicillin resistant staphylococcus aureus.   Aim: The aim of this essay was to identify and describe risk factors associated with occurrence of community acquired methicillin resistant staphylococcus aureus.    Methods: Literature study based on 20 international studies. The main results from the articles were categorized into themes. Results: Risk factors at community-, household- and individual level could be identified, for instance concerning climate, previous antibiotic treatment and comorbidity. Discussion: A few easily affectable risk factors could be identified. Perhaps not all of the identified risk factors are generalizable to a Swedish context. Previous research demonstrates that community acquired methicillin resistant staphylococcus aureus usually affects healthy individuals, which the findings in this essay partly contradict: comorbidity, for instance with HIV, diabetes or obesity, is a risk factor associated with community acquired methicillin resistant staphylococcus aureus.

Microbial resistance to the current available antibiotics is considered a global health problem, especially for the Multi-Drug Resistant pathogens (MDR) including methicillin resistant Staphylococcus aureus. Recently nanoparticles (NPs) have been involved in variety of antimicrobial applications due to their unique properties of antibacterial effects. However, the molecular mechanisms behind their antibacterial activity are still not fully understood. In this study, we produced silver Ag NPs (average size 27 nm) and silver-Titanium Ag-TiO2 NPs (average size 47 nm) using picosecond laser ablation. Our results showed that both laser NPs had obvious size-dependent antibacterial activity. The laser Ag NPs with a size of 19 nm and Ag-TiO2 NPs with a size 20 nm presented the highest bactericidal effect. The laser generated Ag and Ag-TiO2 NPs with concentrations 20, 30, 40, and 50 Î1⁄4g/ml showed strong antibacterial effect against three bacterial strains: E. coli, P. aeruginosa, and S. aureus, and induced the generation of reactive oxygen species (ROS), lead to cell membrane interruption, lipid peroxidation, DNA damages, glutathione depletion and the eventual cell death. Both types of laser NPs at two concentrations (2.5 and 20 Î1⁄4g/ml) showed low cytotoxicity to the in vitro cultured five types of human cells originated from the lung (A549), kidney (HEK293), Liver (HepG2), skin (HDFc) and blood vessel cells (hCAECs). The antibacterial activity of the laser generated Ag and Ag-TiO2 NPs had lasted for over one year depending on the degree of air exposure and storage conditions. Frequent air exposure increased particle oxidation and reduced the antibacterial durability of the laser generated Ag NPs. The laser generated Ag NPs had lower antibacterial activity when stored in cold compared to that stored at room temperature. The antibacterial activity of laser generated Ag and Ag-TiO2 NPs were also compared with four types of commercial based-silver wound dressings (Acticoat TM, Aquacel® Ag, Contreet ®Foam, and Urgotul® SSD) against E. coli to inform future application in this area. In conclusion, laser generated Ag and Ag-TiO2 NPs have strong bactericidal effect and low toxicity to human cells which could be a type of promising antibacterial agents for future hygiene and medical applications.