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.

Pathogenomes and variations in Shiga toxin production among geographically distinct clones of Escherichia coli O113:H21.

Infections with globally disseminated Shiga toxin-producing Escherichia coli (STEC) of the O113:H21 serotype can progress to severe clinical complications, such as hemolytic uremic syndrome (HUS). Two phylogeographically distinct clonal complexes have been established by multi locus sequence typing (MLST). Infections with ST-820 isolates circulating exclusively in Australia have caused severe human disease, such as HUS. Conversely, ST-223 isolates prevalent in the US and outside Australia seem to rarely cause severe human disease but are frequent contaminants. Following a genomic epidemiology approach, we wanted to gain insights into the underlying cause for this disparity. We examined the plasticity in the genome make-up and Shiga toxin production in a collection of 20 ST-820 and ST-223 strains isolated from produce, the bovine reservoir, and clinical cases. STEC are notorious for assembly into fragmented draft sequences when using short-read sequencing technologies due to the extensive and partly homologous phage complement. The application of long-read technology (LRT) sequencing yielded closed reference chromosomes and plasmids for two representative ST-820 and ST-223 strains. The established high-resolution framework, based on whole genome alignments, single nucleotide polymorphism (SNP)-typing and MLST, includes the chromosomes and plasmids of other publicly available O113:H21 sequences and allowed us to refine the phylogeographical boundaries of ST-820 and ST-223 complex isolates and to further identify a historic non-shigatoxigenic strain from Mexico as a quasi-intermediate. Plasmid comparison revealed strong correlations between the strains' featured pO113 plasmid genotypes and chromosomally inferred ST, which suggests coevolution of the chromosome and virulence plasmids. Our pathogenicity assessment revealed statistically significant differences in the Stx2a-production capabilities of ST-820 as compared to ST-223 strains under RecA-induced Stx phage mobilization, a condition that mimics Stx-phage induction. These observations suggest that ST-820 strains may confer an increased pathogenic potential in line with the strain-associated epidemiological metadata. Still, some of the tested ST-223 cultures sourced from contaminated produce or the bovine reservoir also produced Stx at levels comparable to those of ST-820 isolates, which calls for awareness and for continued surveillance of this lineage.

Molecular epidemiology of Shiga toxin-producing O113:H21 isolates from cattle and meat.

The serotype O113:H21 is considered one of the relevant non-O157 STEC serotypes associated with severe human infections. Due to the increased detection of O113 strains and their relationship with clinical cases, which emphasizes the importance of this serogroup as an emerging pathogen, our aim was to determine the characteristics of STEC O113:H21 strains circulating in bovine cattle and retail meat from Argentina. For this purpose, we determined the presence and combinations of various virulence genes (and their variants) related to adhesion and toxicity in a collection of 34 isolates. Their genetic relatedness using multiple-locus variable-number tandem repeat analysis (MLVA) was also studied. Subtyping of stx genes indicated that O113:H21 strains circulating in Argentina mainly present stx2a alone or together with stx2c or, less frequent, with stx2d , all of which are subtypes associated with human disease. We found plasmid markers, such as saa, ehxA and subA, in a higher proportion than previous studies, and five variants of saa, two of which were novel ones. In relation to MLVA subtyping, we detected a limited diversity among the isolates considering that several loci were not discriminative and, that in some farms, the same clone seemed to remain circulating throughout the year. The O113:H21 strains studied harbour several toxin and adhesion genes (saa, espP, fimCD, ehaA, iha, hcpA, elfA, lpfO113, ecpA, subA, cdt-V) and Stx subtypes associated with human disease. Results also highlighted that subtyping of stx and saa is useful to discriminate O113:H21 strains that share virulence genes. In conclusion, this study shows that a number of O113:H21 strains that occur in foods and bovines could be pathogenic for humans. This situation calls for further attention in the prevention and control of foodborne disease caused by these strains.

Toxins of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing Escherichia coli.

Studies on Shiga toxin-producing Escherichia coli (STEC) typically examine and classify the virulence gene profiles based on genomic analyses. Among the screened strains, a subgroup of STEC which lacks the locus of enterocyte effacement (LEE) has frequently been identified. This raises the question about the level of pathogenicity of such strains. This review focuses on the advantages and disadvantages of the standard screening procedures in virulence profiling and summarizes the current knowledge concerning the function and regulation of toxins encoded by LEE-negative STEC. Although LEE-negative STEC usually come across as food isolates, which rarely cause infections in humans, some serotypes have been implicated in human diseases. In particular, the LEE-negative E. coli O104:H7 German outbreak strain from 2011 and the Australian O113:H21 strain isolated from a HUS patient attracted attention. Moreover, the LEE-negative STEC O113:H21 strain TS18/08 that was isolated from minced meat is remarkable in that it not only encodes multiple toxins, but in fact expresses three different toxins simultaneously. Their characterization contributes to understanding the virulence of the LEE-negative STEC.