Isolation of Shiga toxigenic Escherichia coli from butcheries in Taquaritinga city, State of São Paulo, Brazil

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

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.

Prevalence and risk-factor analysis of Shiga toxigenic Escherichia coli in faecal samples of organically and conventionally farmed dairy cattle

Cattle are a natural reservoir for Shiga toxigenic Escherichia coli (STEC), however, no data are available on the prevalence and their possible association with organic or conventional farming practices. We have therefore studied the prevalence of STEC and specifically O157:H7 in Swiss dairy cattle by collecting faeces from approximately 500 cows from 60 farms with organic production (OP) and 60 farms with integrated (conventional) production (IP). IP farms were matched to OP farms and were comparable in terms of community, agricultural zone, and number of cows per farm. E. coli were grown overnight in an enrichment medium, followed by DNA isolation and PCR analysis using specific TaqMan assays. STEC were detected in all farms and O157:H7 were present in 25% of OP farms and 17% of IP farms. STEC were detected in 58% and O157:H7 were evidenced in 4.6% of individual faeces. Multivariate statistical analyses of over 250 parameters revealed several risk-factors for the presence of STEC and O157:H7. Risk-factors were mainly related to the potential of cross-contamination of feeds and cross-infection of cows, and age of the animals. In general, no significant differences between the two farm types concerning prevalence or risk for carrying STEC or O157:H7 were observed. Because the incidence of human disease caused by STEC in Switzerland is low, the risk that people to get infected appears to be small despite a relatively high prevalence in cattle. Nevertheless, control and prevention practices are indicated to avoid contamination of animal products.

Presence of activatable Shiga toxin genotype (stx2d) in Shiga toxigenic Escherichia coli from livestock sources

Stx2d is a recently described Shiga toxin whose cytotoxicity is activated 10- to 1,000-fold by the elastase present in mouse or human intestinal mucus. We examined Shiga toxigenic Escherichia coli (STEC) strains isolated from food and livestock sources for the presence of activatable stx(2d). The stx(2) operons of STEC were first analyzed by PCR-restriction fragment length polymorphism (RFLP) analysis and categorized as stx(2), stx(2c) (vha), stx(2c) (vhb), or stx(2d) (EH250). Subsequently, the stx(2c) (vha) and stx(2c) (vhb) operons were screened for the absence of a PstI site in the stx(2a) subunit gene, a restriction site polymorphism which is a predictive indicator for the stx(2d) (activatable) genotype. Twelve STEC isolates carrying putative stx(2d) operons were identified, and nucleotide sequencing was used to confirm the identification of these operons as stx(2d). The complete nucleotide sequences of seven representative stx(2d) operons were determined. Shiga toxin expression in stx(2d) isolates was confirmed by immunoblotting. stx(2d) isolates were induced for the production of bacteriophages carrying stx. Two isolates were able to produce bacteriophages phi1662a and phi1720a carrying the stx(2d) operons. RFLP analysis of bacteriophage genomic DNA revealed that phi1662a and phi1720a were highly related to each other; however, the DNA sequences of these two stx(2d) operons were distinct. The STEC strains carrying these operons were isolated from retail ground beef. Surveillance for STEC strains expressing activatable stx(2d) Shiga toxin among clinical cases may indicate the significance of this toxin subtype to human health.

In vitro studies on the colonization of bovine colonic mucosa by Shiga-toxigenic Escherichia coli (STEC)

This study investigated host-related factors that influence intestinal colonization by Shiga-toxigenic E. coli (STEC). A quantitative colonization assay was developed to comparatively measure attachment of STEC to bovine colonic tissues maintained in vitro. No differences were determined in colonization susceptibility between tissues derived from weaning calves and adult cattle, or for tissues from cattle fed grain and forage-based rations. Substrate conditions designed to represent various intra-enteric environments were tested for their effect on STEC/mucosal interaction. Under conditions corresponding to a well-fed ruminant (high volatile fatty acid and lactate concentrations, low pH), significantly less STEC colonized the mucosal surface of colonic biopsies. These results may help explain why fasted. poorly or intermittently fed cattle and pre-ruminant calves excrete STEC to a greater degree. Studies on the ecology of STEC within the ruminant gut help identify mechanisms to reduce their threat to public health.

Attachment of Shiga toxigenic Escherichia coli to beef muscle and adipose tissue

Shiga toxigenic Escherichia coli (STEC) serotypes are important foodborne pathogens that cause gastrointestinal disease worldwide. An understanding of how STEC strains attach to surfaces may provide insight into the potential persistence of and contamination with STEC in food environments. The initial attachment of a selection of STEC serotypes to beef muscle and adipose tissue was evaluated for isolates grown in planktonic and sessile culture. Initial experiments were performed to determine whether attachment differed among STEC strains and between the two modes of growth. Viable counts were obtained for loosely and strongly attached cells, and the strength of attachment (S-r) was calculated. All bacterial isolates grown in sessile culture attached in higher numbers to muscle and adipose tissue than did bacteria in planktonic cultures. For all attachment assays performed, mean concentrations for loosely attached cells were consistently higher than concentrations for strongly attached cells. The mean concentrations for strongly attached bacteria for planktonic and sessile cultures were significantly higher (P < 0.05) on adipose than on muscle tissue. However, some strains of STEC, particularly those from sessile culture, did not differ in their attachment to muscle or adipose tissue. S-r values were not significantly different (P > 0.05) among STEC isolates for all assays. No correlation was found between bacterial hydrophobicity and surface charge values (previously determined) and production of surface structures, viable counts, and S-r values. STEC grown in planktonic and sessile culture seems to behave differently with respect to attachment to muscle and adipose tissue. Cells in sessile culture may have a greater potential to strongly attach to meat surfaces.

Non-toxigenic Escherichia coli O157:H7 strain NCTC12900 causes attaching-effacing lesions and eae-dependent persistence in weaned sheep

Ruminants are regarded as a primary reservoir for Escherichia coli O157:H7, an important human pathogen. Intimin, encoded by the Locus of Enterocyte Effacement by E. coli O157:H7 organisms, has been cited as one bacterial mechanism of colonisation of the gastrointestinal tract. To confirm this and to test whether a non-toxigenic E. coli O157:H7 strain would colonise and persist in a sheep model, E. coli O157:H7 strain NCTC12900, that lacks Shiga toxin (stx) genes, was evaluated for use in a sheep model of persistence. Following oral inoculation of six-week-old sheep, persistent excretion of NCTC12900 was observed for up to 48 days. E. coli O157-associated attaching-effacing (AE) lesions were detected in the caecum and rectum of one six-week-old lamb, one day after inoculation. This is the first recorded observation of AE lesions in orally inoculated weaned sheep. Also, mean faecal excretion scores of NCTC12900 and an isogenic intimin (eae)-deficient mutant were determined from twenty-four six-week-old orally inoculated sheep. The eae mutant was cleared within 20 days and had lower mean excretion scores at all time points after day one post inoculation compared with the parental strain that was still being excreted at 48 days. Tissues were collected post mortem from animals selected at random from the study groups over the time course of the experiment. The eae mutant was detected in only 1/43 samples but the parental strain was recovered from 64/140 samples primarily from the large bowel although rumen, duodenum, jejunum, and ileum were culture positive especially from animals that were still excreting at and beyond 27 days after inoculation.

Occurrence of non-O157 Shiga toxin-producing Escherichia coli in dogs with diarrhea

Escherichia coli Shiga toxigênica (STEC) e E. coli Attaching- effacing (AEEC) têm sido associadas à doença diarréica em cachorros. Entre janeiro e dezembro de 2006, 92 cepas de E. coli isoladas de 25 cachorros diarréicos foram examinadas. As cepas foram analisadas para a detecção dos genes produtores de Shiga toxina (stx 1 e stx 2) e da intimina (eae). Por meio de PCR foi observado que sete cepas (7,6%) portavam o gene stx 1, cinco cepas (5,4%) carregavam o gene stx 2 e nenhum cepa apresentou ambos os genes associados. Nove cepas de E. coli (9,8%) apresentaram o gene eae isoladamente. Treze das cepas (62,0%) que apresentaram os genes stx ou eae também apresentaram a produção de a hemolisina. As cepas que apresentaram genes de virulência foram também examinadas em relação à resistência a 12 agentes antimicrobianos. As resistências mais comuns foram para cefalotina (85,7%), estreptomicina (81,0%), amoxicilina (71,4%) e gentamicina (71,4%).

Production of attaching-effacing lesions in ligated large intestine loops of 6-month-old sheep by Escherichia coli O157:H7

Shiga-toxigenic Escherichia coli O157:H7 (STEC O157:H7) is associated with potentially fatal human disease, and a persistent reservoir of the organism is present in some farm animal species, especially cattle and sheep. The mechanisms of persistent colonisation of the ruminant intestine by STEC O157:H7 are poorly understood but may be associated with intimate adherence to eukaryotic cells. Intimate adherence, as evidenced by induction of attaching-effacing (AE) lesions by STEC O157, has been observed in 6-day-old conventional lambs after deliberate oral infection but not in older animals. Thus, the present study used a ligated intestinal loop technique to investigate whether STEC O157:H7 and other attaching-effacing E. coli may adhere intimately to the sheep large intestinal mucosa. To do this, four STEC O157:H7 strains, one STEC 026:K60:H11 and one Shiga toxin-negative E. coli O157:H7 strain, suspended in either phosphate-buffered saline or Dulbecco's modified Eagle's medium, were inoculated into ligated spiral colon loops of each of two lambs. The loops were removed 6 h after inoculation, fixed and examined by light and electron microscopy. AE lesions on the intestinal mucosa were produced by all the inoculated strains. However, the lesions were sparse and small, typically comprising bacterial cells intimately adhered to a single enterocyte, or a few adjacent enterocytes. There was little correlation between the extent of intimate adherence in this model and the bacterial cell density, pre-inoculation growth conditions of the bacteria or the strain tested.

Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil

Pesquisou-se a ocorrência de Escherichia coli toxigênica, em queijo produzido com leite não pasteurizado, na Região Centro Oeste do Brazil. Foram utilizados 50 queijos adquiridos em diferentes supermercados. As amostras isoladas foram classificadas por sorogrupo, avaliadas em relação à sensibilidade para 13 agentes antimicrobianos e submetidas à reação em cadeia da polimerase para a presença de genes característicos de E. coli verotoxigênica (VTEC) e enterotoxigênica (ETEC). E. coli foi recuperada em 48(96,0%) dos queijos. Foram identificados os sorogrupos O125 (6,0%), O111 (4,0%), O55 (2,0%) e O119 (2,0%). Três (6,0%) amostras de E. coli foram classificadas como VTEC e uma (2,0%) como ETEC. Os maiores índices de resistência foram verificados para: cefalotina (60,0%), ácido nalidíxico (40,0%), doxiciclina (33,0%), tetraciclina (31,0%) e ampicilina (29,0%).

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)

Fate of non O157 Shigatoxigenic Escherichia coli in ovine manurecomposting

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

Shiga Toxigenic and Atypical Enteropathogenic Escherichia coli in the Feces and Carcasses of Slaughtered Pigs

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of geographical origin, host animal and stx gene on the virulence characteristics of Escherichia coli O26 strains

The influence of geographical origin, host animal and presence of the stx gene on the virulence of Escherichia coli O26 strains from ruminants was determined in this study. A clear association was found between the virulence profile and geographical origin of Shiga-toxigenic E. coli (STEC) O26 strains, with UK STEC O26 strains harbouring virtually identical profiles, whilst central European strains showed considerable heterogeneity in plasmid-encoded genes. The former group were also more likely to be non-motile and katP gene positive. Comparison of UK STEC and atypical enteropathogenic E. coli (aEPEC O26 strains showed that the presence of the stx1 gene was positively correlated with the presence of espP and katP genes and negatively associated with the presence of the yagP-yagT region and with rhamnose fermentation. In contrast to the uniform profiles of STEC O26 strains from ruminants in the UK, aEPEC O26 strains of bovine and ovine origin showed diverse profiles both within and between groups, and could not be separated into discrete groups. These results indicate that the characteristics of UK O26 strains from ruminants are distinct from those of O26 strains from ruminants and humans in other regions in central Europe. Such differences are expected to influence the zoonotic potential of this pathogen and the subsequent incidence of O26-associated human disease.