Increasing carbapenem resistance among clinical isolates of E. coli and K. pneumoniae has become a serious public health problem over the last decade. Molecular epidemiology studies have shown that there is a global dissemination of epidemic clones of carbapenem-resistant E. coli and K. pneumoniae. Besides, successful epidemic plasmids were reported to disseminate carbapenemase genes in Enterobacteriaceae. The wide spread of carbapenem-resistant E. coli and K. pneumoniae limits treatment options of the infection, poses severe challenges to clinical professionals and threatens our health. Recently, carbapenem-resistant E. coli and K. pneumoniae are increasingly reported in Hong Kong. In 2012, our group has documented the emergence of carbapenem-resistant clinical isolates in Hong Kong. The findings of the previous study showed that 26.1% of the Enterobacteriaceae isolates were confirmed to produce carbapenemase. Notably, a novel IncX3 plasmid was found to be involved in the dissemination of blaNDM-1 gene. However, the previous findings fail to explicate the carbapenem resistance mechanisms of the remaining non-carbapenemase producing isolates. Further investigation is needed to elucidate the situation. Firstly, we investigated the carbapenem resistance mechanism of carbapenem-resistant E. coli and K. pneumoniae isolates recovered from the Hong Kong West Cluster hospitals from 2010 to 2012. PCRs were used to detect carbapenemase genes (blaNDM, blaKPC, blaIMP, blaVIM and blaOXA-48), blaCTX-M ESBL genes and blaAmpC genes. SDS-PAGE was used to detect porin loss. Among the 92 isolates in this study, only nine (9.8 %) isolates were detected with carbapenemase genes. The blaCTX-M and/or blaAmpC β-lactamase genes plus porin loss were detected in 47 non-carbapenemase-producing isolates (16 E. coli and 31 K. pneumoniae). The resistance determinant profiles of these 16 E. coli included: blaCTX-M + porin loss (n= 10), blaCIT + porin loss (n = 1), blaCTX-M + blaCIT/DHA + porin loss (n = 5). The resistance determinant profiles of the 31 K. pneumoniae included: blaCTX-M + porin loss (n= 4), blaDHA + porin loss (n = 7), blaCTX-M + blaCIT/DHA + porin loss (n = 20). The results showed that apart from acquired carbapenemases, the production of AmpC β-lactamase and/or ESBLs plus porin loss played a main role in the carbapenem resistance mechanism of the carbapenem-resistant E. coli and K. pneumoniae isolates. Secondly, we accessed the clonal relatedness of the isolates. Multi-locus sequence typing results showed that 55 (77.5%) K. pneumoniae isolates fall into the clonal complex 37. Our results suggest that the CC37 K. pneumoniae are associated with the acquisition of DHA-1 β-lactamase, CTXM-1-group β-lactamase and porin alterations which could confer a high-level of resistance to carbapenems resulting in their predominance in this study. Finally, we characterized the plasmids that carry carbapenemase gene by S1-PFGE, Southern blot, plasmid replicon typing and whole plasmid sequencing. A novel IncX3 plasmid was found to carry blaKPC gene. Together with the previously reported blaNDM-1 carrying IncX3 plasmids, it shows that IncX3 plasmids might be new epidemic plasmids involved in the dissemination of carbapenemase genes. These novel IncX3 plasmids are worrisome. Nationwide surveillance and more epidemiological study of IncX3 plasmids are needed. (Word / published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Microbiology / Master / Master of Philosophy

Characteristically, in infections that are caused by the zoonotic pathogen Bartonella naturally infected reservoir hosts are asymptomatic, where infected incidental, non-natural, hosts develop symptomatic disease. Cat-scratch disease (CSD) is a well known example. Bartonella infections in humans may be self-limiting or fulminant and affect different organ systems. The objectives of the present thesis were to (1) identify and characterise Bartonella infection cases in Sweden, (2) to investigate certain human populations regarding Bartonella infections, and (3) compare natural populations of different Bartonella species. Cases with typical and atypical CSD were recognised by using a combination of PCR and serology. Gene sequence comparisons of different genes in B. henselae isolates from the United States and Europe showed that ftsZ gene variation is a useful tool for Bartonella genotyping. Myocarditis was a common finding among Swedish elite orienteers succumbing to sudden unexpected cardiac death (SUCD). The natural cycle of Bartonella spp., the life style of orienteers, elevated antibody titres to Bartonella antigens, Bartonella DNA amplified from myocardium and the lack of another feasible explanation make Bartonella a plausible aetiological factor. The first reported case of Bartonella endocarditis (B. quintana) was identified in an immunocompromised patient who underwent heart valve replacement. The patient had been body louse-infested during his childhood. It is hypothesised that a chronic B. quintana infection was activated by the immunosuppression. There was no evidence of an ongoing trench fever (TF) epidemic in a Swedish homeless population, although an increased risk for exposure to Bartonella antigens was demonstrated. The lack of louse infestation might explain the absence of B. quintana bacteremia and low B. quintana antibody titres. Comparisons of genetic loci and the whole genomes of environmental B. grahamii isolates from the Uppsala region, Sweden displayed variants that were not related to specific host species but to geographic locality. Natural boundaries seemed to restrict gene flow.

Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-04-27Bitstream added on 2014-06-13T19:43:42Z : No. of bitstreams: 1 malaspina_ac_dr_arafcf.pdf: 775274 bytes, checksum: 18bf752c95a916660f1a3b09c97c8077 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / O estudo da epidemiologia molecular através de diferentes técnicas vigentes como Restriction Fragment Length Polymorphism (RFLP), Spacer Oligonucleotide Typing (Spoligotyping) e Mycobacterial Interspersed Repetitive Units (MIRU) revolucionou a compreensão da epidemiologia da tuberculose. Foram analisados isolados clínico de pacientes portadores de tuberculose pulmonar no município de Araraquara, entre os anos 2000 e 2006, através de genotipagem por RFLP e Spoligotyping (n=122). A técnica de RFLP permitiu a identificação de um índice de 26,4% de casos de tuberculose proveniente de infecção recente na cidade de Araraquara, no período de 2000 a 2006. Verificou-se relação epidemiológica em cerca de 68% dos casos envolvendo grupos genéticos identificados pelo RFLP. Destaca-se as transmissões intradomiciliares e a moradia em condomínios residenciais do CDHU. A técnica de RFLP permitiu a distinção de dois grandes grupos genéticos, A e B, dentre os isolados de Araraquara. Cerca de 73% dos spoligotipos obtidos foram identificados no Banco Internacional de Spoligotyping SpolDB4, sendo as famílias LAM, T e H as mais predominantes e cerca de 27% dos spoligotipos encontrados apresentaram perfis não descritos no SpolDB4. A associação do RFLP, Spoligotyping e MIRU não foi uma boa ferramenta para a análise genética dos isolados clínicos visando esclarecimentos epidemiológicos. Embora a genotipagem através do RFLP permita uma melhor discriminação entre os isolados bem como uma excelente associação entre amostras relacionadas epidemiologicamente, a técnica de Spoligotyping associada ao MIRU permite uma boa discriminação entre os isolados clínicos, da mesma forma que propicia uma satisfatória correlação entre casos ligados epidemiologicamente / Molecular epidemiology study using different current methods such as Restriction Fragment Length Polymorphism (RFLP), Spacer Oligonucleotide Typing (Spoligotyping) and Mycobacterial Interspersed Repetitive Units (MIRU) modified tuberculosis epidemiology comprehension. Clinical isolates from pulmonary tuberculosis patients from Araraquara, between 2000 and 2006, were genotyped through RFLP and Spoligotyping (n=122). RFLP provided the identification of a 26.4% level of tuberculosis cases caused by recent infection in the period. Epidemiologic association were verified in 68% of the cases in clusters identified by RFLP. Intradomiciliary transmissions and living in residential condominiums from CDHU were important factors. RFLP fingerprint provides the distinction of two big genetic groups, A and B, among Araraquara isolates. About 73% of spoligotypes were described in the International Spoligotyping Database SpolDB4 and LAM, T and H families were the most frequent, in the other hand, 27% of spoligotypes had unique profifes not described in SploDB4. RFLP, Spoligotyping and MIRU association was not a good tool to genotype clinical isolates in order to provide epidemiological understandings. Although RFLP fingerprinting allows a better discrimination as well as an excellent association among epidemiological related isolates, Spoligotyping associated with MIRU provides a good discrimination among clinical isolates as well as provides a satisfactory correlation among cases epidemiological connected

As the leading cause of bacterial gastroenteritis, Campylobacter represents a significant public health burden; however, our knowledge of its epidemiology in low- and middle-income countries remains limited. Recent studies have demonstrated the power of whole-genome sequencing (WGS) for public health microbiology. The primary aim of this thesis was to exploit WGS to improve our understanding of the epidemiology of Campylobacter from the Republic of South Africa, a middle-income country. In the first half of this thesis, in silico approaches were developed to evaluate diagnostic assays and methods of species identification. Large-scale analyses of publicly available WGS data identified a robust real-time PCR assay for the detection of Campylobacter jejuni and Campylobacter coli, the primary causes of human campylobacteriosis. Evaluation of in silico speciation methods demonstrated that the atpA gene and ribosomal multilocus sequence typing can be used to identify Campylobacter from WGS data. The second half of this thesis extended concepts developed in the first half to investigate the epidemiology of Campylobacter from animals and humans from South Africa. Isolates from a study of Campylobacter from free-range broiler carcasses belonged to the agriculture-associated ST-828 lineage, but were atypically homogenous and differed at only 46/1,513 (3%) loci, providing novel insights into clonal infections in chickens. Analyses of human disease isolates collected in Cape Town in 1991, 2011, and 2012 confirmed that the local epidemiology of Campylobacter is distinct from that of high-income countries: in addition to major agriculture-associated C. jejuni and C. coli lineages, a putative novel C. jejuni subsp. jejuni/C. jejuni subsp. doylei hybrid clade and genetically diverse C. jejuni subsp. doylei and C. upsaliensis isolates were identified. This work delivers further evidence of the utility of WGS for clinical microbiology, presents approaches that address general problems in Campylobacter diagnostics and public health microbiology, and provides insights into the epidemiology of this important group of pathogens in South Africa.

Methicillin-resistant Staphylococcus aureus (MRSA) is a successful pathogen which was historically found in hospital settings but now is a common cause of infection in communities. The rapid emergence of community-acquired MRSA (CA-MRSA) at the turn of the 21st century has established this bacterium’s presence throughout the globe and MRSA continues to be endemic in certain countries. Asia is the most populous continent in the world and also holds a high burden of MRSA infection. This presents a concern for both public health and the acquisition of antibiotic resistance in this region. This literature review describes how MRSA became a successful pathogen. It provides a systematic review of the recent literature on MRSA in East Asia to identify major MRSA clones by country as determined by their molecular characteristics. Also to identify notable genetic and epidemiological factors associated with these MRSA clones. The results of this survey provided evidence of the importance of using molecular categorization techniques to accurately distinguish MRSA strains that require specific antibiotic treatment methods. It also provided evidence of CA-MRSA clones invading hospital settings and traditional hospital-acquired MRSA (HA-MRSA) clones continuing to develop multi-drug resistance throughout East Asian countries. The results also detected novel MRSA strains across hospitals and reported the spread of major MRSA clones within and between countries. Strengthening existing surveillance systems and collaborative efforts between countries within Asia should be a priority to monitor the evolution and movement MRSA especially in the age of globalization and accessible travel.

Made available in DSpace on 2014-06-11T19:31:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T20:01:59Z : No. of bitstreams: 1 rodrigues_mvp_dr_botfm.pdf: 2095427 bytes, checksum: da1c8df4916765105802c2a6f424aa29 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / Os Staphylococcus aureus são os principais microrganismos causadores de infecções nosocomiais, com uma grande variedade de manifestações clínicas, a variação deste espectro de manifestações geralmente depende dos numerosos fatores de virulência que cada cepa produz, entretanto, a importância desses patógenos está inserida na combinação da virulência mediada por suas toxinas, seu caráter invasivo e seu perfil de resistência a antibióticos. O presente estudo teve por objetivos documentar a disseminação de clones endêmicos e identificar os fatores individuais relacionados à sua aquisição, e relacioná-los a prevalência de fatores de resistência e virulência em cepas de MRSA isoladas de culturas clínicas e/ou de vigilância de pacientes de um hospital de ensino. No presente trabalho 1078 amostras de Staphylococcus aureus provenientes de pacientes internados no Hospital Estadual Bauru foram identificadas e testadas fenotipicamente frente a oxacilina (1 g), cefoxitina (30 g), vancomicina (30 g), eritromicina (15 g) e gentamicina (10 g) sendo que dessas, 810 (75.1%) apresentaram perfil fenotípico para Staphylococcus aureus Resistentes a Meticilina – MRSA. A avaliação genotípica do perfil de resistência foi realizada através da amplificação do gene mecA pela técnica de PCR onde foi observado que das 443 amostras testadas foi detectado o gene mecA em 336 amostras (75.8%), e que dessas, 305 amostras (90.8%) apresentaram perfil SCCmec Tipo III ou Tipo IIIA, 6 amostras (1.8%) Tipo II e 25 amostras (7.4%) Tipo IV. A avaliação do perfil genotípico de virulência mostrou a seguinte distribuição, 15.6% (n=69) genes da Leucocidina de Panton Valentine (lukPV), 14% (n=62) gene da Síndrome do Choque Tóxico (tst), 31.8% (n=141) gene da Enterotoxina A (sea), 19.8% (n=88) gene da Enterotoxina B (seb), 38.1% (n=169) gene da... / Staphylococcus aureus are the most common microorganisms causing nosocomial infections, with a wide variety of clinical manifestations, the variation of the spectrum of manifestations generally depends upon the numerous virulence factors that each strain produces, however, the importance of these pathogens is included in the combination of virulence mediated by their toxins, its invasive nature and profile of antibiotic resistance. This study aimed to document the spread of endemic clones and identify the individual factors related to its acquisition, and relate them to the prevalence of resistance and virulence factors in strains of MRSA isolated from clinical cultures and / or surveillance patients a teaching hospital. In this study 1078 samples of Staphylococcus aureus from hospitalized patients in Bauru State Hospital were identified phenotypically and tested against oxacillin (1  g), cefoxitin (30  g), vancomycin (30  g), erythromycin (15  g) and gentamicin (10  g) and that these, 810 (75.8%) showed phenotypic profile for Methicillin Resistant Staphylococcus aureus - MRSA. The evaluation of genotypic resistance profiles was performed by amplification of the mecA gene by PCR where it was observed that of 443 samples tested was detected mecA gene in 336 samples (75.8%), and that these, 305 samples (90.8%) profile were SCCmec type III or type IIIA, six samples (1.8%) type II and 25 samples (7.4%) type IV. The evaluation of virulence genotype was distributed as follows, 15.6% (n = 69) genes of Panton Valentine Leukocidin (lukPV), 14% (n = 62) gene for toxic shock syndrome (tst), 31.8% (n = 141) gene of enterotoxin A (sea), 19.8% (n = 88) gene of enterotoxin B (seb), 38.1% (n = 169) gene of enterotoxin C (sec-1), 100% (n = 443) Delta Hemolysin gene (hld), 99.5% (n = 441) Alpha Hemolysin gene (hla), 0.9% (n = 4) Exfoliative toxin A (eta), 3.6% (n = 16) Exfoliative... (Complete abstract click electronic access below)