Detecção de Escherichia coli shigatoxigênica (STEC) e enteropatogênica (EPEC) em carcaças e fezes de suínos abatidos na região de Ribeirão Preto – SP e perfil de susceptibilidade dos isolados frente a diferentes antimicrobianos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção de Escherichia coli shigatoxigênica (STEC) e enteropatogênica (EPEC) isoladas de búfalos leiteiros no Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Eliminação de Escherichia coli Shigatoxigênica não-O157 em compostagem de esterco bovino

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização de Escherichia coli Shigatoxigênica isolada em estabelecimentos comerciais no município de Taquaritinga, S.P

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Detecção de Escherichia coli shigatoxigênica (STEC) e enteropatogênica (EPEC) em peixes de pisciculturas e de vida livre

Pós-graduação em Microbiologia Agropecuária - FCAV

Fate of non O157 Shiga toxigenic Escherichia coli in composted cattle manure

Determinou-se o tempo necessário para a eliminação de Escherichia coli Shigatoxigênica (STEC) não-O157 em esterco bovino composto, obtido de fezes frescas de três vacas portadoras de cepas STEC não-O157 que apresentavam o gene stx 2. Foram utilizados dois sistemas de compostagem, o primeiro foi um buraco de 0,6m escavado no solo e o segundo um monte apresentando uma arquitetura piramidal com um metro de altura. Todos os dias, durante os primeiros 10 dias e a cada cinco dias durante um mês, uma amostra de três pontos diferentes dos dois sistemas de compostagem foram coletadas e semeadas para determinar a presença de E. coli e a presença do gene stx 2 nas células, sendo que em cada coleta a temperatura do sistema de compostagem foi determinada. Células de STEC não-O157 sobreviveram por 8, 25 e 30 dias nas temperaturas de 42, 40 e 38ºC, respectivamente, no sistema enterrado no solo, enquanto que no sistema de monte as células foram detectadas por 4, 4 e 7 dias em temperaturas de 65, 58 e 52ºC, respectivamente. A temperatura e os microrganismos presentes na microbiota do sistema de compostagem parecem ser os responsáveis pela eliminação do patógeno. Pode-se concluir que os dois sistemas de compostagem utilizados mostraram-se eficientes na eliminação de células de STEC. A aplicação de esterco após compostagem deve diminuir o risco de contaminação ambiental e a disseminação do patógeno.

Análise de cepas de Escherichia coli Shiga toxigênica (STEC) e E. coli patogênica extraintestinal (ExPEC) isoladas de cachorros diarréicos atendidos em clínica privada no município de Ituverava, SP

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Escherichia coli potencialmente patogênica isoladas de ovinos saudáveis criados extensivamente e de carcaças em matadouros-frigoríficos no Estado de São Paulo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Quantificação de enterobactérias e Clostridium spp. e detecção molecular de Clostridium perfringens, Escherichia coli e Salmonella spp. em pontos de cadeia produtiva da carne de frango

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Shigatoxigenic Escherichia coli serogroups O157, O111 and O113 in feces, water and milk samples from dairy farms

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Campylobacter jejuni and campylobacter coli genotyping by high-resolution melting analysis of a flaA fragment

The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP. flaAHRManalysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.

Development of rapid and highly resolving combinatorial genotyping schemes for Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni followed by Campylobacter coli contribute substantially to the economic and public health burden attributed to food-borne infections in Australia. Genotypic characterisation of isolates has provided new insights into the epidemiology and pathogenesis of C. jejuni and C. coli. However, currently available methods are not conducive to large scale epidemiological investigations that are necessary to elucidate the global epidemiology of these common food-borne pathogens. This research aims to develop high resolution C. jejuni and C. coli genotyping schemes that are convenient for high throughput applications. Real-time PCR and High Resolution Melt (HRM) analysis are fundamental to the genotyping schemes developed in this study and enable rapid, cost effective, interrogation of a range of different polymorphic sites within the Campylobacter genome. While the sources and routes of transmission of campylobacters are unclear, handling and consumption of poultry meat is frequently associated with human campylobacteriosis in Australia. Therefore, chicken derived C. jejuni and C. coli isolates were used to develop and verify the methods described in this study. The first aim of this study describes the application of MLST-SNP (Multi Locus Sequence Typing Single Nucleotide Polymorphisms) + binary typing to 87 chicken C. jejuni isolates using real-time PCR analysis. These typing schemes were developed previously by our research group using isolates from campylobacteriosis patients. This present study showed that SNP + binary typing alone or in combination are effective at detecting epidemiological linkage between chicken derived Campylobacter isolates and enable data comparisons with other MLST based investigations. SNP + binary types obtained from chicken isolates in this study were compared with a previously SNP + binary and MLST typed set of human isolates. Common genotypes between the two collections of isolates were identified and ST-524 represented a clone that could be worth monitoring in the chicken meat industry. In contrast, ST-48, mainly associated with bovine hosts, was abundant in the human isolates. This genotype was, however, absent in the chicken isolates, indicating the role of non-poultry sources in causing human Campylobacter infections. This demonstrates the potential application of SNP + binary typing for epidemiological investigations and source tracing. While MLST SNPs and binary genes comprise the more stable backbone of the Campylobacter genome and are indicative of long term epidemiological linkage of the isolates, the development of a High Resolution Melt (HRM) based curve analysis method to interrogate the hypervariable Campylobacter flagellin encoding gene (flaA) is described in Aim 2 of this study. The flaA gene product appears to be an important pathogenicity determinant of campylobacters and is therefore a popular target for genotyping, especially for short term epidemiological studies such as outbreak investigations. HRM curve analysis based flaA interrogation is a single-step closed-tube method that provides portable data that can be easily shared and accessed. Critical to the development of flaA HRM was the use of flaA specific primers that did not amplify the flaB gene. HRM curve analysis flaA interrogation was successful at discriminating the 47 sequence variants identified within the 87 C. jejuni and 15 C. coli isolates and correlated to the epidemiological background of the isolates. In the combinatorial format, the resolving power of flaA was additive to that of SNP + binary typing and CRISPR (Clustered regularly spaced short Palindromic repeats) HRM and fits the PHRANA (Progressive hierarchical resolving assays using nucleic acids) approach for genotyping. The use of statistical methods to analyse the HRM data enhanced sophistication of the method. Therefore, flaA HRM is a rapid and cost effective alternative to gel- or sequence-based flaA typing schemes. Aim 3 of this study describes the development of a novel bioinformatics driven method to interrogate Campylobacter MLST gene fragments using HRM, and is called ‘SNP Nucleated Minim MLST’ or ‘Minim typing’. The method involves HRM interrogation of MLST fragments that encompass highly informative “Nucleating SNPS” to ensure high resolution. Selection of fragments potentially suited to HRM analysis was conducted in silico using i) “Minimum SNPs” and ii) the new ’HRMtype��� software packages. Species specific sets of six “Nucleating SNPs” and six HRM fragments were identified for both C. jejuni and C. coli to ensure high typeability and resolution relevant to the MLST database. ‘Minim typing’ was tested empirically by typing 15 C. jejuni and five C. coli isolates. The association of clonal complexes (CC) to each isolate by ‘Minim typing’ and SNP + binary typing were used to compare the two MLST interrogation schemes. The CCs linked with each C. jejuni isolate were consistent for both methods. Thus, ‘Minim typing’ is an efficient and cost effective method to interrogate MLST genes. However, it is not expected to be independent, or meet the resolution of, sequence based MLST gene interrogation. ‘Minim typing’ in combination with flaA HRM is envisaged to comprise a highly resolving combinatorial typing scheme developed around the HRM platform and is amenable to automation and multiplexing. The genotyping techniques described in this thesis involve the combinatorial interrogation of differentially evolving genetic markers on the unified real-time PCR and HRM platform. They provide high resolution and are simple, cost effective and ideally suited to rapid and high throughput genotyping for these common food-borne pathogens.

Highly discriminatory single-nucleotide polymorphism interrogation of Escherichia coli by use of allele-specific real-time PCR and eBURST analysis

In total, 782 Escherichia coli strains originating from various host sources have been analyzed in this study by using a highly discriminatory single-nucleotide polymorphism (SNP) approach. A set of eight SNPs, with a discrimination value (Simpson's index of diversity [D]) of 0.96, was determined using the Minimum SNPs software, based on sequences of housekeeping genes from the E. coli multilocus sequence typing (MLST) database. Allele-specific real-time PCR was used to screen 114 E. coli isolates from various fecal sources in Southeast Queensland (SEQ). The combined analysis of both the MLST database and SEQ E. coli isolates using eight high-D SNPs resolved the isolates into 74 SNP profiles. The data obtained suggest that SNP typing is a promising approach for the discrimination of host-specific groups and allows for the identification of human-specific E. coli in environmental samples. However, a more diverse E. coli collection is required to determine animal- and environment-specific E. coli SNP profiles due to the abundance of human E. coli strains (56%) in the MLST database.