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Introduction . Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that may cause diarrhoeal outbreaks and occasionally are associated with haemolytic-uraemic syndrome (HUS). We report on STEC O26:H11 associated with a cluster of four HUS cases in South Africa in 2017. Methodology . All case-patients were female and aged 5 years and under. Standard microbiological tests were performed for culture and identification of STEC from specimens (human stool and food samples). Further analysis of genomic DNA extracted from bacterial cultures and specimens included PCR for specific virulence genes, whole-genome sequencing and shotgun metagenomic sequencing. Results. For 2/4 cases, stool specimens revealed STEC O26:H11 containing eae, stx2a and stx2b virulence genes. All food samples were found to be negative for STEC. No epidemiological links could be established between the HUS cases. Dried meat products were the leading food item suspected to be the vehicle of transmission for these cases, as 3/4 case-patients reported they had eaten this. However, testing of dried meat products could not confirm this. Conclusion. Since STEC infection does not always lead to severe symptoms, it is possible that many more cases were associated with this cluster and largely went unrecognized.

Shiga toxin-producing Escherichia coli (STEC) O26:H11 is an emerging foodborne pathogen of growing concern. Since current strategies to control microbial contamination in foodstuffs do not guarantee the elimination of O26:H11, novel approaches are needed. Bacteriophages present an alternative to traditional biocontrol methods used in the food industry. Here, a previously isolated bacteriophage vB_EcoM_VR26 (VR26), adapted to grow at common refrigeration temperatures (4 and 8 °C), has been evaluated for its potential as a biocontrol agent against O26:H11. After 2 h of treatment in broth, VR26 reduced O26:H11 numbers (p < 0.01) by > 2 log10 at 22 °C, and ~3 log10 at 4 °C. No bacterial regrowth was observed after 24 h of treatment at both temperatures. When VR26 was introduced to O26:H11-inoculated lettuce, ~2.0 log10 CFU/piece reduction was observed at 4, 8, and 22 °C. No survivors were detected after 4 and 6 h at 8 and 4 °C, respectively. Although at 22 °C, bacterial regrowth was observed after 6 h of treatment, O26:H11 counts on non-treated samples were >2 log10 CFU/piece higher than on phage-treated ones (p < 0.02). This, and the ability of VR26 to survive over a pH range of 3–11, indicates that VR26 could be used to control STEC O26:H11 in the food industry.

Shiga toxin-producing Escherichia coli (STEC) are responsible for human infections, ranging from mild watery diarrhea to hemorrhagic colitis (CH) that may be complicated by hemolytic uremic syndrome (HUS). The main STEC virulence factor is Shiga toxin encoded by the stx gene, located in the genome of a bacteriophage integrated into the bacterial chromosome. The serotype O26:H11 is the second HUS-causing serotype worldwide (after O157:H7), and the first found in dairy products such as raw-milk cheeses. A small number of HUS cases identified each year in France are caused by serotype O26:H11. Stx phage induction is known to result in STEC lysis and release of new Stx phages particles. This phenomenon could negatively impact STEC screening in foods based on stx gene detection by PCR. Here, we evaluated the influence of physicochemical parameters related to cheese-making process on the induction rate of Stx phages from STEC O26:H11, including H2O2, NaCl, lactic acid and temperature. In addition, selective agents from the analytical STEC enrichment and detection procedure (XP CEN ISO/TS 13136) were tested, including novobiocin, acrifavin, cefixim-tellurite, and bile salts. An impact of H2O2 and NaCl on Stx phage induction was observed. Production of Stx phages was also observed during a real cheese-making process. By contrast, no significant effect could be demonstrated for the chemical agents of the STEC detection procedure when tested separately, except for acriflavin and novobiocin which reduced Stx1 phage production in some cases. In conclusion, these results suggest that the cheese-making process might trigger the production of Stx phages, potentially interfering with the analysis of STEC in food.

Shiga toxin-producing Escherichia coli (STEC) are responsible for human infections, ranging from mild diarrhea to hemolytic-uremic syndrome (HUS), sometimes with fatal outcome. Transmission of STEC to humans occurs mainly through the ingestion of contaminated food. The main virulence factor of STEC is the stx gene (encodes Shiga-toxin) located in the genome of a prophage. The PhD thesis was focused on STEC O26:H11, which is the second serotype causing HUS in the world, and the first one found in raw milk cheeses. The first objective was to characterize genetically STEC O26:H11 strains and their Stx phages (stx gene subtypes and insertion sites of Stx phages) in order to identify any differences between these strains according to their origin. The majority of the investigated food and bovine strains possessed stx1a subtype and their Stx phages were integrated into wrbA and yehV. Human strains possessed the stx1a and stx2a subtypes in equivalent proportions. Their Stx phages were also integrated into wrbA and yehV but unlike food and bovine strains, yecE site were identified as insertion site. All the human strains carrying an Stx2a phage integrated into wrbA and yecE caused HUS, which could indicate a high virulence. Studies showed that Attaching/Effacing E. coli (AEEC) O26:H11 strains were isolated from foods identified as "stx +" by PCR. Except for the absence of stx gene, these AEEC strains are similar to STEC. Their characterization showed, in this study, that the majority of them had intact insertion sites, in agreement with a possible loss of Stx phage by spontaneous excision. The stability of Stx phages was evaluated in STEC O26:H11. Presence/absence of Stx phages was quantified for each strain in the total bacterial population, showing that STEC were capable of losing their Stx phages. However, this instability was not related to the insertion sites. Several attempts to introduce Stx phages in AEEC strains in order to convert them into STEC were conducted but the results showed that it was difficult to infect these strains. The induction rate of Stx phages in vitro in STEC O26:H11 showed that, with mitomycin C, the Stx2 phages were more inducible than Stx1 phages. However no difference was found with the origin of the strains tested and the Stx phage integration site used. The morphological analysis of some Stx phages showed that Stx1 or Stx2 type was not related to a specific phage shape. Study of various stress related to the cheese-making process showed that the osmotic and oxidative stress related to the potential release of H2O2 by other bacteria, led to the induction of Stx phages. Because AEEC strains are frequently isolated from food qualified as "stx+" by PCR, the analytical STEC isolation procedure was studied for its ability to induce Stx phages. Production of Stx phages during the enrichment phase seemed possible from contaminated food. However, none of the components of this method, tested individually, could be identified as an inducer of Stx phages. This work highlighted the diversity of Stx phages from STEC O26:H11 isolated from humans and dairy sector. Differences in stx subtypes and Stx phages insertion sites present among the STEC O26:H11 strains were observed depending on the origin of the strains. Moreover the induction levels of Stx phages differed according to the stx subtypes. These differences might reflect the existence of distinct clones, with varying virulence potential, circulating in foods, cattle and patients, Les Escherichia coli producteurs de Shiga-toxines (STEC) sont responsables d'infections humaines, allant d'une diarrhée aqueuse bénigne pouvant se compliquer en syndrome hémolytique et urémique (SHU), parfois mortel. La transmission de STEC à l'Homme s'effectue principalement par l'ingestion d'aliments contaminés. Le principal facteur de virulence des STEC est le gène stx (codant la Shiga-toxine), localisé dans le génome d'un prophage. La thèse a été centrée sur les STEC O26:H11, deuxième sérotype à l'origine de SHU dans le monde, et premier retrouvé dans les fromages au lait cru.Le premier objectif était de caractériser génétiquement les STEC O26:H11 et leurs phages Stx (variants du gène stx et sites d'insertion des phages Stx) afin d'identifier d'éventuelles différences entre ces souches selon leur origine. La majorité des souches alimentaires et bovines étudiées possèdent le variant stx1a et leurs phages Stx sont intégrés dans wrbA et yehV. Les souches humaines possèdent les variants stx1a et stx2a en proportions équivalentes. Leurs phages Stx sont aussi intégrés dans wrbA et yehV mais à la différence des souches alimentaires et bovines, le site yecE a été identifié comme site d'insertion. Toutes les souches humaines qui possédaient un phage Stx2a intégré dans wrbA et yecE ont causé des SHU ; ce qui pourrait être un indicateur de haute virulence. Des études ont montré que des souches E. coli Attachant/Effaçant (AEEC) O26:H11 sont isolées à partir d'aliments identifiés comme « stx+ » par PCR. En dehors de l'absence du gène stx, ces souches AEEC sont similaires aux STEC. Leur caractérisation a montré ici que la majorité d'entre elles ont leurs sites d'insertion intacts, caractéristique compatible avec une perte de phage Stx par excision spontanée. La stabilité des phages Stx a donc été évaluée chez les STEC O26:H11. La présence/absence de phages Stx a été quantifiée par PCRq pour chaque souche au niveau de la population bactérienne entière montrant que les STEC ont la capacité de perdre leurs phages Stx. En revanche, cette instabilité n'est pas liée aux sites d'insertion. Plusieurs essais visant à introduire des phages Stx dans les souches AEEC pour les convertir en STEC ont été réalisés mais les résultats ont montré qu'il était difficile d'infecter ces souches. L'étude du taux d'induction des phages Stx in vitro chez les STEC O26:H11 a montré, qu'en présence de mitomycine C, les phages Stx2 étaient plus inductibles que les phages Stx1. En revanche, il n'a pas été mis en évidence de différences en fonction de l'origine des souches testées et du site d'intégration des phages Stx. L'analyse morphologique de quelques phages Stx a montré que le type Stx1 ou Stx2 n'était pas lié à une forme spécifique de phage. L'étude des stress relatifs à la technologie fromagère a montré que le stress salin et le stress oxydatif, lié à la libération potentielle d'H2O2 par d'autres bactéries, entrainaient l'induction des phages Stx. Comme des souches AEEC sont fréquemment isolées à partir d'aliments qualifiés de « stx+» par PCR, le processus analytique d'isolement des STEC a aussi été étudié. La production de phages Stx lors de la phase d'enrichissement semble possible à partir d'un aliment contaminé. En revanche, aucun composant de cette méthode, testé individuellement, n'a pu être identifié comme inducteur des phages Stx. Ces travaux ont permis d'acquérir des connaissances sur la diversité des phages Stx issus de STEC O26:H11 isolées chez l'Homme et dans la filière laitière. Des différences au niveau des variants du gène stx mais aussi des sites d'insertion des phages Stx ont pu être observées en fonction de l'origine des souches. De plus les niveaux d'induction des phages Stx diffèrent selon le variant du gène stx. Ces différences pourraient refléter l'existence de clones distincts, aux potentiels de virulence différents, circulant dans les aliments, chez les bovins et les patients

The Shiga-toxin producing Escherichia coli (STEC) may cause serious illness in human. Here we analyze O26:H11 strains known to be among the most reported STEC strains causing human infections. Genetic characterization of strains isolated from animal, food, and clinical specimens in Argentina showed that most carried either stx1a or stx2a subtypes. Interestingly, stx2a-positive O26:H11 rarely isolated from cattle in other countries showed to be an important proportion of O26:H11 strains circulating in cattle and food in our region. Seventeen percent of the isolates harbored more than one gene associated with antimicrobial resistance. In addition to stx, all strains contained the virulence genes eae-β, tir, efa, iha, espB, cif, espA, espF, espJ, nleA, nleB, nleC, and iss; and all except one contained ehxA, espP, and cba genes. On the other hand, toxB and espI genes were exclusively observed in stx2-positive isolates, whereas katP was only found in stx1a-positive isolates. Our results show that O26:H11 STEC strains circulating in Argentina, including those isolated from humans, cattle, and meat products, present a high pathogenic potential, and evidence that cattle can be a reservoir of O26:H11 strains harboring stx2a., Instituto de Genética Veterinaria