Heterogeneity in resistance to food-related stresses and biofilm formation ability among verocytotoxigenic Escherichia coli strains

bForming biofilms may be a survival strategy of Shiga toxin-producing Escherichia coli to enable it to persist in the environment and the food industry. Here, we evaluate and characterize the biofilm-forming ability of 39 isolates of Shiga toxin-producing Escherichia coli isolates recovered from human infection and belonging to seropathotypes A, B, or C. The presence and/or production of biofilm factors such as curli, cellulose, autotransporter, and fimbriae were investigated. The polymeric matrix of these biofilms was analyzed by confocal microscopy and by enzymatic digestion. Cell viability and matrix integrity were examined after sanitizer treatments. Isolates of the seropathotype A (O157:H7 and O157:NM), which have the highest relative incidence of human infection, had a greater ability to form biofilms than isolates of seropathotype B or C. Seropathotype A isolates were unique in their ability to produce cellulose and poly-N-acetylglucosamine. The integrity of the biofilms was dependent on proteins. Two autotransporter genes, ehaB and espP, and two fimbrial genes, z1538 and lpf2, were identified as potential genetic determinants for biofilm formation. Interestingly, the ability of several isolates from seropathotype A to form biofilms was associated with their ability to agglutinate yeast in a mannose-independent manner. We consider this an unidentified biofilmassociated factor produced by those isolates. Treatment with sanitizers reduced the viability of Shiga toxin-producing Escherichia coli but did not completely remove the biofilm matrix. Overall, our data indicate that biofilm formation could contribute to the persistence of Shiga toxin-producing Escherichia coli and specifically seropathotype A isolates in the environment. Shiga toxin-producing Escherichia coli (STEC) strains, including enterohemorrhagic E. coli (EHEC), are food-borne and waterborne human enteric pathogens responsible for infections. They are associated with important public health concerns in developed countries. Symptoms associated with STEC infections range from abdominal cramps and bloody diarrhea to postinfectious complications, such as hemolytic-uremic syndrome (HUS). HUS, a life-threatening complication of STEC infections, is a consequence of Shiga toxin production. According to the Public Health Agency of Canada (PHAC), more than 30,000 cases of STEC infections occur each year in Canada (1). Environmentally, the main reservoir for STEC is cattle. Transmission of STEC to humans typically is associated with the consumption of contaminated food such as undercooked beef, fresh produce, unpasteurized milk, or contaminated drinking water (2), but these pathogens also can be transmitted from person to person or via direct contact with animals or their feces.The 2003 Karmali seropathotype model classifies STEC into seropathotypes based on their reported incidence in human disease, outbreaks, and/or association with the development of severe symptoms in humans (3). Serotypes frequently responsible for hemorrhagic colitis (HC) and HUS, ...

Section: Discussionsupporting

confidence: 71%

Biofilm-Forming Abilities of Shiga Toxin-Producing Escherichia coli Isolates Associated with Human Infections

Vogeleer

Tremblay

Jubelin³

et al. 2016

“…E. coli strains have been shown to accumulate mutations in the rpoS gene when grown long term in the laboratory, in batch culture, in stab cultures, in chemostats run with poor carbon sources, and even in natural commensal, pathogenic, and environmental isolates from fermented sausage, human feces and urine, beaches, wastewater effluent, and animals (7,(26)(27)(28)(29)(30)(31)(32)(33)(34). When we compared the rpoS locus in the test strains with E. coli K-12 MG1655, various nucleotide substitution mutations were observed, but none of these resulted in a premature stop codon (Table 3).…”

Section: Discussionmentioning

confidence: 99%

“…A number of studies have shown that, in low-nutrient conditions, particularly when growth rates are very slow, mutations can accumulate within the rpoS open reading frame (ORF), resulting in the partial or complete loss of RpoS function and reduced stress tolerance (25,26). Mutations in rpoS have been reported in strains obtained from laboratory growth conditions as well as among natural isolates of E. coli (7,(26)(27)(28)(29)(30)(31)(32)(33)(34), Salmonella (35)(36)(37), Cronobacter (38), and Citrobacter (39). These mutations are thought to provide a selective advantage to microorganisms undergoing nutrient starvation due to a trade-off between stress resistance and growth (40).…”

mentioning

confidence: 99%

The General Stress Response Is Conserved in Long-Term Soil-Persistent Strains of Escherichia coli

Somorin

Abram

Brennan

et al. 2016

Although Escherichia coli is generally considered to be predominantly a commensal of the gastrointestinal tract, a number of recent studies suggest that it is also capable of long-term survival and growth in environments outside the host. As the extraintestinal physical and chemical conditions are often different from those within the host, it is possible that distinct genetic adaptations may be required to enable this transition. Several studies have shown a trade-off between growth and stress resistance in nutrient-poor environments, with lesions in the rpoS locus, which encodes the stress sigma factor RpoS ( S ). In this study, we investigated a unique collection of long-term soil-persistent E. coli isolates to determine whether the RpoS-controlled general stress response is altered during adaptation to a nutrient-poor extraintestinal environment. The sequence of the rpoS locus was found to be highly conserved in these isolates, and no nonsense or frameshift mutations were detected. Known RpoS-dependent phenotypes, including glycogen synthesis and ␥-aminobutyrate production, were found to be conserved in all strains. All strains expressed the full-length RpoS protein, which was fully functional using the RpoS-dependent promoter reporter fusion PgadX:: gfp. RpoS was shown to be essential for long-term soil survival of E. coli, since mutants lacking rpoS lost viability rapidly in soil survival assays. Thus, despite some phenotypic heterogeneity, the soil-persistent strains all retained a fully functional RpoS-regulated general stress response, which we interpret to indicate that the stresses encountered in soil provide a strong selective pressure for maintaining stress resistance, despite limited nutrient availability. IMPORTANCEEscherichia coli has been, and continues to be, used as an important indicator species reflecting potential fecal contamination events in the environment. However, recent studies have questioned the validity of this, since E. coli has been found to be capable of long-term colonization of soils. This study investigated whether long-term soil-persistent E. coli strains have evolved altered stress resistance characteristics. In particular, the study investigated whether the main regulator of genes involved in stress protection, the sigma factor RpoS, has been altered in the soil-persistent strains. The results show that RpoS stress protection is fully conserved in soil-persistent strains of E. coli. They also show that loss of the rpoS gene dramatically reduces the ability of this organism to survive in a soil environment. Overall, the results indicate that soil represents a stressful environment for E. coli, and their survival in it requires that they deploy a full stress protection response. E scherichia coli is a Gram-negative, facultative anaerobe, belonging to the Enterobacteriaceae family, which inhabits the intestinal tracts of humans, warm-blooded animals, and reptiles (1, 2). It can be transferred through water and sediments via feces and is almost universally used as an i...

“…Moreover, Alvarez-Ordóñez et al (42) confirmed strong acid stress resistance especially for O157:NM strains. These strains are associated with a higher incidence of progression to HUS than E. coli O157:H7 strains and have emerged as important pathogens since their first isolation in Germany in 1998 (2)(3)(4).…”

Section: Discussionmentioning

confidence: 90%

Fitness of Enterohemorrhagic Escherichia coli (EHEC)/Enteroaggregative E. coli O104:H4 in Comparison to That of EHEC O157: Survival Studies in Food and In Vitro

Böhnlein

Kabisch

Meske

et al. 2016

In 2011, one of the world's largest outbreaks of hemolytic-uremic syndrome (HUS) occurred, caused by a rare Escherichia coli serotype, O104:H4, that shared the virulence profiles of Shiga toxin-producing E. coli (STEC)/enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC). The persistence and fitness factors of the highly virulent EHEC/EAEC O104:H4 strain, grown either in food or in vitro, were compared with those of E. coli O157 outbreak-associated strains. The log reduction rates of the different EHEC strains during the maturation of fermented sausages were not significantly different. Both the O157:NM and O104:H4 serotypes could be shown by qualitative enrichment to be present after 60 days of sausage storage. Moreover, the EHEC/EAEC O104:H4 strain appeared to be more viable than E. coli O157:H7 under conditions of decreased pH and in the presence of sodium nitrite. Analysis of specific EHEC strains in experiments with an EHEC inoculation cocktail showed a dominance of EHEC/EAEC O104:H4, which could be isolated from fermented sausages for 60 days. Inhibitory activities of EHEC/EAEC O104:H4 toward several E. coli strains, including serotype O157 strains, could be determined. Our study suggests that EHEC/ EAEC O104:H4 is well adapted to the multiple adverse conditions occurring in fermented raw sausages. Therefore, it is strongly recommended that STEC strain cocktails composed of several serotypes, instead of E. coli O157:H7 alone, be used in food risk assessments. The enhanced persistence of EHEC/EAEC O104:H4 as a result of its robustness, as well as the production of bacteriocins, may account for its extraordinary virulence potential. IMPORTANCEIn 2011, a severe outbreak caused by an EHEC/EAEC serovar O104:H4 strain led to many HUS sequelae. In this study, the persistence of the O104:H4 strain was compared with those of other outbreak-relevant STEC strains under conditions of fermented raw sausage production. Both O157:NM and O104:H4 strains could survive longer during the production of fermented sausages than E. coli O157:H7 strains. E. coli O104:H4 was also shown to be well adapted to the multiple adverse conditions encountered in fermented sausages, and the secretion of a bacteriocin may explain the competitive advantage of this strain in an EHEC strain cocktail. Consequently, this study strongly suggests that enhanced survival and persistence, and the presumptive production of a bacteriocin, may explain the increased virulence of the O104:H4 outbreak strain. Furthermore, this strain appears to be capable of surviving in a meat product, suggesting that meat should not be excluded as a source of potential E. coli O104:H4 infection. The Shiga toxin-producing Escherichia coli (STEC) strains, which include enterohemorrhagic E. coli (EHEC) strains, are pathogenic E. coli strains with the potential to cause severe enteric and systemic disease in humans. These bacteria have accounted for various foodborne infections over recent decades. The pathogenesis of EHEC disease depends on the produ...