Genetic characterization of Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 from diarrhoeic and septicaemic calves and relatedness to human Shigatoxigenic E. coli O80:H2.

AIM: The purpose of this work was to identify and genetically characterize enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) O80:H2 from diarrhoeic and septicaemic calves in Belgium and to comparing them with human EHEC after whole genome sequencing. METHODS AND RESULTS: Ten EHEC and 21 EPEC O80 identified by PCR between 2009 and 2018 from faeces, intestinal content and a kidney of diarrhoeic or septicaemic calves were genome sequenced and compared to 19 human EHEC identified between 2008 and 2019. They all belonged to the O80:H2 serotype and ST301, harboured the eaeξ gene, and 23 of the 29 EHEC contained the stx2d gene. Phylogenetically, they were distributed in two major sub-lineages: one comprised a majority of bovine EPEC whereas the second one comprised a majority of stx2d bovine and human EHEC. CONCLUSIONS: Not only EPEC but also EHEC O80:H2 are present in diarrhoeic and septicaemic calves in Belgium and are genetically related to human EHEC. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings support the need to assess cattle as potential source of contamination of humans by EHEC O80:H2 and to understand the evolution of bovine and human EHEC and EPEC O80:H2.

Biofilm formation, invasiveness and colicinogeny in locus of enterocyte and effacement negative O113:H21 Shigatoxigenic Escherichia coli.

AIMS: Although Shiga toxins (Stx) are well-established virulence traits of O113:H21 Shigatoxigenic Escherichia coli (STEC) strains, a shortage in the knowledge of other virulence properties that may contribute to pathogenesis may exist in this serotype. This study investigated biofilm, invasiveness and colicinogeny capabilities in O113:H21 STEC isolated in Brazil, mostly from animal reservoirs. A search for genes that were reported to participate in the process of biofilm formation was also performed. METHODS AND RESULTS: The 34 O113:H21 STEC isolates analysed were assayed for biofilm production in polystyrene microplates. Genes for biofilm were investigated by PCR. Invasion of cell lineages was assessed in gentamicin protection assays and colicinogeny was investigated by phenotypic tests. Fifty per cent of the strains were biofilm formers, and 35% exhibited an invasive behaviour. The pattern of distribution of biofilm-related genes did not correlate with biofilm phenotypes observed, and a high percentage of the investigated strains were able to secrete colicins. CONCLUSION: Ability to form biofilm, invasiveness and colicinogeny is demonstrated for the first time in a collection of O113:H21 STEC. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to express three additional phenotypes besides Stx production may be a factor influencing the pathogenicity and persistence potential of O113:H21 STEC.

Escherichia coli O80 in Healthy Cattle: Absence of Shigatoxigenic and Enteropathogenic E. coli O80:H2 and (Phylo) Genomics of Non-Clonal Complex 165 E. coli O80.

The origin of human and calf infections by Shigatoxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli O80:H2 is still unknown. The aim of this study was to identify E. coli O80 in healthy cattle with an emphasis on melibiose non-fermenting E. coli O80:H2. Faecal materials collected from 149 bulls at 1 slaughterhouse and 194 cows on 9 farms were tested with O80 antigen-encoding gene PCR after overnight growth in enrichment broths. The 53 O80 PCR-positive broths were streaked on different (semi-)selective agar plates. Five E. coli colonies from 3 bulls and 11 from 2 cows tested positive with the O80 PCR, but no melibiose non-fermenting E. coli was isolated. However, these 16 E. coli O80 were negative with PCR targeting the fliCH2, eae, stx1, stx2 and hlyF genes and were identified by WGS to serotypes and sequence types O80:H6/ST8619 and O80:H45/ST4175. They were phylogenetically related to E. coli O80:H6 and O80:H45 isolated from different animal species in different countries, respectively, but neither to STEC and EPEC O80:H2/ST301, nor to other serotypes of the clonal complex 165. As a conclusion, healthy adult cattle were not identified as a source of contamination of humans and calves by STEC or EPEC O80:H2.

Virulence of Shigatoxigenic and Enteropathogenic Escherichia coli O80:H2 in Galleria mellonella Larvae: Comparison of the Roles of the pS88 Plasmids and STX2d Phage.

The invasiveness properties of Shigatoxigenic and enteropathogenic Escherichia coli (STEC and EPEC) O80:H2 in humans and calves are encoded by genes located on a pS88-like ColV conjugative plasmid. The main objectives of this study in larvae of the Galleria mellonella moth were therefore to compare the virulence of eight bovine STEC and EPEC O80:H2, of two E. coli pS88 plasmid transconjugant and STX2d phage transductant K12 DH10B, of four E. coli O80:non-H2, and of the laboratory E. coli K12 DH10B strains. Thirty larvae per strain were inoculated in the last proleg with 10 µL of tenfold dilutions of each bacterial culture corresponding to 10 to 106 colony-forming units (CFUs). The larvae were kept at 37 °C and their mortality rate was followed daily for four days. The main results were that: (i) not only the STEC and EPEC O80:H2, but also different E. coli O80:non-H2 were lethal for the larvae at high concentrations (from 104 to 106 CFU) with some variation according to the strain; (ii) the Stx2d toxin and partially the pS88 plasmid were responsible for the lethality caused by the E. coli O80:H2; (iii) the virulence factors of E. coli O80:non-H2 were not identified. The general conclusions are that, although the Galleria mellonella larvae represent a useful first-line model to study the virulence of bacterial pathogens, they are more limited in identifying their actual virulence properties.

Caracterización genotípica y biofilms de E. coli shigatoxigénica aisladas de casos clínicos humanos / Genotypic characterization and biofilms of shigatoxigenic E. coli isolated from human clinical cases

Resumen Escherichia coli shigatoxigénica (STEC) está involucrada en el desarrollo del síndrome urémico hemolítico, entre otras enfermedades que son de gran importancia para la salud pública e inocuidad alimentaria a nivel mundial. La capacidad de STEC de formar biofilms en los alimentos y en diferentes superficies podría conducir a la contaminación cruzada por el desprendimiento de las células bacterianas. El objetivo del presente trabajo fue detectar la presencia de genes que codifican factores de adherencia mediante la técnica de PCR y determinar la capacidad de formación de biofilms por medio de cultivo en microplacas de poliestireno de 96 pocillos y la técnica de cristal violeta, en cepas de STEC aisladas de muestras clínicas humanas en la ciudad de Mar del Plata, Argentina. El perfil de genes de adherencia más frecuente fue efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43,9%). Todas las cepas de STEC formaron biofilms con valores de densidad óptica entre 0,209 y 3,251 y el 54,4% (31/57) de las mismas fueron clasificadas como fuertes formadoras de biofilms. La capacidad de formación de biofilms de STEC constituye un riesgo evidente en la transmisión de este patógeno al ser humano a tener en cuenta para su vigilancia y control.

Abstract Shigatoxigenic Escherichia coli (STEC) is involved in the development of hemolytic uremic syndrome, among other diseases that are relevant to public health and food safety worldwide. The ability of STEC to form biofilms in food and on different surfaces could lead to cross-contamination by shedding bacterial cells. The aim of this work was to detect the presence of genes encoding adherence factors by the PCR technique and to determine the biofilm formation ability by culture in 96-well polystyrene microplates and the crystal violet technique, in STEC strains isolated from human clinical samples in Mar del Plata city, Argentina. The most frequent adherence gene profile was efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43.9%). All STEC strains formed biofilms with optical density values between 0.209 and 3.251. Also, the 54.4% (31/57) of STEC strains were classified as strong biofilm formers. The ability of STEC to form biofilms constitutes an evident risk in the transmission of this pathogen to humans, which must be taken into account for its surveillance and control.

Resumo A Escherichia coli shigatoxigênica (STEC) está envolvida no desenvolvimento da síndrome hemolítica urêmica, entre outras doenças relevantes para a saúde pública e segurança alimentar em todo o mundo. A capacidade do STEC de formar biofilmes nos alimentos e em diferentes superfícies poderia levar à contaminação cruzada através do desprendimento de células bacterianas. O objetivo do presente trabalho foi detectar a presença de genes que codificam fatores de aderência através da técnica PCR e determinar a capacidade de formação de biofilme por cultura em microplacas de poliestireno de 96 poços e da técnica de cristal violeta, em cepas STEC isoladas de amostras clínicas humanas na cidade de Mar del Plata, Argentina. O perfil de genes de aderência mais frequente foi efa1, iha, fimCD, ehaA, lpfA1-3, lpfA2-2, cah (43,9%). Todas as cepas de STEC formaram biofilmes com valores de densidade ótica entre 0,209 e 3,251. Também, os 54,4% (31/57) das estirpes STEC foram classificados como fortes formadores de biofilmes. A habilidade de formação de biofilmes de STEC constitui um risco evidente na transmissão deste patógeno ao humano, que deve ser levado em consideração para sua vigilância e controle.

Hybrids of Shigatoxigenic and Enterotoxigenic Escherichia coli (STEC/ETEC) Among Human and Animal Isolates in Finland.

Diarrhoeagenic Escherichia coli (DEC) cause serious foodborne infections in humans. Total of 450 Shigatoxigenic E. coli (STEC) strains isolated from humans, animals and environment in Finland were examined by multiplex PCR targeting the virulence genes of various DEC pathogroups simultaneously. One per cent (3/291) of the human STEC and 14% (22/159) of the animal and environmental STEC had genes typically present in enterotoxigenic E. coli (ETEC). The strains possessed genes encoding both Shiga toxin 1 and/or 2 (stx1 and/or stx2 ) and ETEC-specific heat-stable (ST) enterotoxin Ia (estIa). The identified stx subtypes were stx1a, stx1c, stx2a, stx2d and stx2g. The three human STEC/ETEC strains were isolated from the patients with haemolytic uraemic syndrome and diarrhoea and from an asymptomatic carrier. The animal STEC/ETEC strains were isolated from cattle and moose. The human and animal STEC/ETEC strains belonged to 11 serotypes, of which O2:H27, O15:H16, O101:H-, O128:H8 and O141:H8 have previously been described to be associated with human disease. Identification of multiple virulence genes offers further information for assessing the virulence potential of STEC and other DEC. The emergence of novel hybrid pathogens should be taken into account in the patient care and epidemiological surveillance.

Consumer preferences, internal color and reduction of shigatoxigenic Escherichia coli in cooked hamburgers.

The aim of this study was to relate consumer preferences and preparation of hamburgers to color change, internal temperature and reduction of shigatoxigenic Escherichia coli (STEC) serogroups O157 and the "Big Six" (O26, O45, O103, O111, O121, O145) under two ground beef packaging scenarios: 75% O2 MAP and vacuum. 75% O2 MAP hamburgers cooked to 60 °C core temperature appeared done and showed less internal red color (lower a*) than corresponding vacuum hamburgers. Similar STEC reduction (<4 log10) was found for both hamburgers at core temperatures ≤ 66 °C. In a representative survey (N=1046) most consumers reported to judge hamburger doneness by the color and many preferred undercooked hamburgers. Premature browning of 75% O2 MAP hamburgers represents a risk of foodborne illness, when considering consumers' food handling practices. The risk is even greater if such ground beef is prepared by consumers who prefer undercooked hamburgers and judge doneness by color.