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Simple Summary: Following the "Replacement/Reduction/Refinement" policy, insects are proposed to replace mammals and birds as experimental models to study the virulence of bacterial pathogens and to identify their virulence properties. The aim of this study was to assess in larvae of the Galleria mellonella moth the virulence of the Shigatoxigenic and enteropathogenic Escherichia coli O80:H2 and the respective roles of two virulence properties: the pS88 plasmid-encoded invasiveness properties and the phage-encoded Shiga toxin 2d. The objectives were to compare: (i) the virulence of bovine Shigatoxigenic and enteropathogenic E. coli O80:H2; (ii) the roles of the pS88 plasmid and Shiga toxin 2d-encoding phage; (iii) the virulence of E. coli O80:H2 and O80:non-H2. The results and the conclusions are: (i) E. coli O80:H2 and O80:non-H2 are lethal at log5 and log6 concentrations; (ii) the pS88 plasmids are partially responsible for the virulence of E. coli O80:H2; (iii) the phage-encoded Stx2d toxin is entirely responsible for the virulence of the Shigatoxigenic Escherichia coli O80:H2; (iv) the virulence properties of E. coli O80:non-H2 could not be identified. As a general conclusion, G. mellonella larvae represent a useful model to study the virulence of bacterial pathogens but are limited in identifying their virulence properties. 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. [ABSTRACT FROM AUTHOR]

Shiga toxin (Stx) is the key virulence factor of Stx-producing Escherichia coli (STEC). All known Stxs (Stx1 and Stx2) are encoded by bacteriophages (Stx phages). Although the genetic diversity of Stx phages has frequently been described, systematic analyses of Stx phages in a single STEC lineage are limited. In this study, focusing on the O26:H11 STEC sequence type 21 (ST21) lineage, where the stx1a gene is highly conserved, we analysed the Stx1a phages in 39 strains representative of the entire ST21 lineage and found a high level of variation in Stx1a phage genomes caused by various mechanisms, including replacement by a different Stx1a phage at the same or different locus. The evolutionary timescale of events changing Stx1a phages in ST21 was also determined. Furthermore, by using an Stx1 quantification system developed in this study, we found notable variations in the efficiency of Stx1 production upon prophage induction, which sharply contrasted with the conserved iron regulated Stx1 production. These variations were associated with the Stx1a phage alteration in some cases but not in other cases; thus, Stx1 production in this STEC lineage was determined by differences not only in Stx1 phages but also in host-encoded factors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bacteriophages (or phages) play major roles in the evolution of bacterial pathogens via horizontal gene transfer. Multiple phages are often integrated in a host chromosome as prophages, not only carrying various novel virulence-related genetic determinants into host bacteria but also providing various possibilities for prophage-prophage interactions in bacterial cells. In particular, Escherichia coli strains such as Shiga toxin (Stx)-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains have acquired more than 10 prophages (up to 21 prophages), many of which encode type III secretion system (T3SS) effector gene clusters. In these strains, some prophages are present at a single locus in tandem, which is usually interpreted as the integration of phages that use the same attachment (att) sequence. Here, we present phages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Some of the phages integrated into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages in a single cell. The identified attB sequences in prophage genomes are apparently derived from host chromosomes. In addition, two or three different attB sequences are present in some prophages, which results in the generation of prophage clusters in various complex configurations. These phages integrating into prophages represent a medically and biologically important type of inter-phage interaction that promotes the accumulation of T3SS effector genes in STEC and EPEC, the duplication of Stx phages in STEC, and the conversion of EPEC to STEC and that may be distributed in other types of E. coli strains as well as other prophage-rich bacterial species. Author summary: Multiple prophages are often integrated in a bacterial host chromosome and some are present at a single locus in tandem. The most striking examples are Shiga toxin (Stx)-producing and enteropathogenic Escherichia coli (STEC and EPEC) strains, which usually contain more than 10 prophages (up to 21). Many of them encode a cluster of type III secretion system (T3SS) effector genes, contributing the acquisition of a large number of effectors (>30) by STEC and EPEC. Here, we describe prophages integrating into T3SS effector gene cluster-associated loci in prophages, which are widely distributed in STEC and EPEC. Two or three different attachment sequences derived from host chromosomes are present in some prophages, generating prophage clusters in various complex configurations. Of note, some of such phages integrating into prophages are Stx-encoding phages (Stx phages) and have induced the duplication of Stx phages. Thus, "prophage-in-prophage" represents an important inter-phage interaction as they can promote not only the accumulation of T3SS effectors in STEC and EPEC but also the duplication of Stx phages and the conversion of EPEC to STEC. [ABSTRACT FROM AUTHOR]

Among Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 strains, those producing Stx2a cause more severe diseases. Atypical STEC O157:H7 strains showing a β-glucuronidase-positive phenotype (GP STEC O157:H7) have rarely been isolated from humans, mostly from persons with asymptomatic or mild infections; Stx2a-producing strains have not been reported. We isolated, from a patient with bloody diarrhea, a GP STEC O157:H7 strain (PV15-279) that produces Stx2a in addition to Stx1a and Stx2c. Genomic comparison with other STEC O157 strains revealed that PV15-279 recently emerged from the stx1a/stx2c-positive GP STEC O157:H7 clone circulating in Japan. Major virulence genes are shared between typical (β-glucuronidase-negative) and GP STEC O157:H7 strains, and the Stx2-producing ability of PV15-279 is comparable to that of typical STEC O157:H7 strains; therefore, PV15-279 presents a virulence potential similar to that of typical STEC O157:H7. This study reveals the importance of GP O157:H7 as a source of highly pathogenic STEC clones. [ABSTRACT FROM AUTHOR]

No outbreaks caused by Escherichia coli –producing heat-labile enterotoxin LT2 have been reported to date. Here, we revealed that the E. coli O8:H8 strains isolated from patients in 2 independent diarrhea outbreaks were negative for any known virulence determinants in routine microbiological tests, were very closely related, and carried a prophage-encoded gene for a novel LT2 variant (LT2d) and the genes for colonization factor antigen III. We also showed that LT2d has a cytotonic activity similar to LT1. These data indicate the importance of E. coli strains producing LT2d as a human pathogen. [ABSTRACT FROM AUTHOR]