non-O157 Shiga Toxigenic Escherichia Coli Research Articles

Genomic Profiling of Non-O157 Shiga Toxigenic Escherichia coli-Infecting Bacteriophages from South Africa.

Non-O157 Shiga toxigenic Escherichia coli (STEC) are one of the most important food and waterborne pathogens worldwide. Although bacteriophages (phages) have been used for the biocontrol of these pathogens, a comprehensive understanding of the genetic characteristics and lifestyle of potentially effective candidate phages is lacking. In this study, 10 non-O157-infecting phages previously isolated from feedlot cattle and dairy farms in the North-West province of South Africa were sequenced, and their genomes were analyzed. Comparative genomics and proteomics revealed that the phages were closely related to other E. coli-infecting Tunaviruses, Seuratviruses, Carltongylesviruses, Tequatroviruses, and Mosigviruses from the National Center for Biotechnology Information GenBank database. Phages lacked integrases associated with a lysogenic cycle and genes associated with antibiotic resistance and Shiga toxins. Comparative genomic analysis identified a diversity of unique non-O157-infecting phages, which could be used to mitigate the abundance of various non-O157 STEC serogroups without safety concerns.

Differential Survival of Non-O157 Shiga Toxigenic Escherichia coli in Simulated Cattle Feedlot Runoff.

Environmental survival time is important when evaluating adverse health outcomes from foodborne pathogens. Although outbreaks associated with manure-impacted irrigation or runoff water are relatively infrequent, their broad scope, regulatory importance, and severe health outcomes highlight the need to better understand the environmental survival of manure-borne pathogens. Shiga toxigenic Escherichia coli (STEC) are excreted in feces and persist in the environment until they die or recolonize a new host. Surface waters contaminated with manure-borne STEC can infect humans through drinking and recreational water use or irrigated crops that are minimally cooked. In this study, manure-impacted water microcosms mimicking beef cattle feedlot runoff were used to assess survival of STEC strains representing seven STEC serotypes (O26, O45, O103, O111, O121, O145, and O157) and persistence of target O antigen genes. Microcosms were sampled over the course of 1 year, and the entire experiment was repeated in a second year. Culture and polymerase chain reaction (PCR)-based techniques were used for detection and enumeration. Serotype-specific survival results were observed. Both STEC O26 and O45 declined slowly and remained culturable at 24 months. In contrast, STEC O121 and O145 decreased rapidly (-0.84 and -1.99 log10 abundance per month, respectively) and were unculturable by months 4 and 5, but detectable by PCR for a mean of 4.5 and 8.3 months, respectively. STEC O103, O111, and O157 remained culturable for a mean of 11.6, 5.5, and 15 months and detectable by PCR for a mean of 12, 13.8, and 18.6 months after inoculation, respectively. Results document that some STEC serotypes have the biological potential to survive in manure-impacted waters for extended periods of time when competing microflora are eliminated. Serotype-specific differences in survival of target bacteria and persistence of target genes were observed in this sample set, with STEC O26 and O45 strains appearing the most robust in these microcosm studies.

Variability in growth responses of non-O157 EHEC isolates in leafy vegetables, sprouted seeds and soil extracts occurs at the isolate level.

Foods of plant origin are recognised as a major source of foodborne pathogens, in particular for Shigatoxigenic Escherichia coli (STEC). Most work for STEC and plant-based fresh produce has focused on the most prevalent outbreak serogroup, O157. However, non-O157 STEC is an emerging hazard, and as such it is important to characterise aspects within this group that reflect their ability to colonise alternative hosts and habitats relevant to horticultural production. Growth kinetics were quantified for a diverse set of clinical enterohaemorrhagic E. coli isolates in extracts made from different tissues of spinach, lettuce or sprouted seeds, or from soil, to represent association with ready-to-eat fresh produce production. For leafy vegetables, spinach apoplast supported the fastest rates of growth and lettuce root extracts generated the slowest growth rates. Growth rates were similar for the majority of isolates in fenugreek or alfalfa sprouted seed extracts. Monosaccharides were the major driver of bacterial growth. No correlations were found for growth rates between different serotypes or for Shigatoxin gene carriage. Thus, growth rates varied in a plant-dependent and isolate-dependent manner, for all plant or soil extracts tested, indicative of isolate-specific differences in metabolic flexibility. These findings are relevant for risk assessment of non-O157 STEC.

Characterization of Bacteriophages Targeting Non-O157 Shiga Toxigenic Escherichia coli

Non-O157 Shiga toxigenic Escherichia coli (STEC) are an important group of foodborne pathogens, implicated in several outbreaks and recalls in the past 2 decades. It is therefore crucial to devise effective control strategies against these pathogens. Bacteriophages present an attractive alternative to conventional pathogen control methods in the food industry. Bacteriophages, targeting non-O157 STEC (O26, O45, O103, O111, O121, O145), were isolated from beef cattle operations in Oklahoma. Their host range and lytic ability were determined against several ( n = 21) non-O157 STEC isolates, by using the spot-on-lawn assay. Isolated phages were purified, and their morphology was determined under a transmission electron microscope. Infection kinetics of selected phages ( n = 19), particularly adsorption rate, rise period, latent period, and burst size, were determined. Phages were also evaluated for stability at a wide pH range (1 to 11) and temperature range (-80 to 90°C). In total, 45 phages were isolated and classified into Myoviridae, Siphoviridae, or Tectiviridae. The phages had a latent period between 8 and 37 min, a rise period between 19 and 40 min, and a large burst size (12 to 794 virions per infected cell), indicating high lytic activity. Tested phages were stable at pH 5 to 9 for 24 h, whereas a decrease in phage titer was observed at pHs 1, 2, and 11. Phages were stable at 40 and 60°C, except for O103-specific phages. At 70°C, all the phages lost viability after 20 min, except three phages targeting O26 and O121 and one phage targeting O45 and O111 STEC, which remained viable for 60 min. All the phages lost activity after 10 min at 90°C, except one each of O26 and O121 STEC-infecting phages that remained viable for 60 min. Phages remained stable for 90 days under refrigerated (4°C) and frozen (-20 and -80°C) storage. Characterization of phages, targeting diverse non-O157 STEC serotypes, could help in the development of effective biocontrol strategies for this group of pathogens in the food industry.

Detection and molecular characterization of sorbitol fermenting non-O157 Escherichia coli from goats

Shiga toxigenic Escherichia coli (STEC) O157 and several other serogroups of non-O157 STEC strains present as commensalism bacteria in small ruminants that possess a high potential of attaining pathogenic virulent genetic elements. The pathogenicity of non-O157 strains is emerging progressively in animals as well as in humans especially in the rural areas as they get transmitted through unsanitary practices of living, consumption of uncooked meat and milk, human-livestock close proximity as well as within livestock pathogenic bacterial transmission. The present study was carried out to determine the prevalence of non-O157 E.coli isolates and characterize based on clinical history, antibiotic sensitivity testing, multiplex PCR (mPCR) detection of virulence genes, genotype identification using pulse field gel electrophoresis (PFGE) and polyacrylamide gel electrophoretic separation of antigenic proteins to determine their prevalence, virulence and genetic comparison between host and environment for epidemiological significance. A total of 300 E.coli isolates were recovered from rectal swabs of goats and their surrounding environment over a period of one year (2016–2017) by selective isolation. Among which 50 isolates were confirmed to be from the non-O157 E.coli family. The mPCR analysis of these 50 isolates revealed the presence of two or more virulent genes, viz., hylA (90%), fliC (74%), eaeA (56%), stx1 (48%) and stx2 (22%).Four isolates exhibited multidrug-resistance to amoxiclav, doxycycline, ciprofloxacin and ceftriazone. The PFGE fingerprint profile showed six major clusters at 100% similarity from the 50 isolates. The major antigenic proteins identified from the isolates were stx1A, stx2B and fliC. This study has significant implications for understanding the molecular diversity of emerging pathotypes of non-O157 in young goats in terms of virulence and epidemiological aspects.

Comparative Effect of Heat Shock on Survival of O157:H7 and Non-O157 Shiga Toxigenic Escherichia coli and Salmonella in Lean Beef with or without Moisture-Enhancing Ingredients

Thermal tolerance of pathogenic bacteria has been shown to increase after exposure to sublethal elevated temperatures, or heat shock. We evaluated the effect of heat shock at 48°C on thermal tolerance (D55°C) of cocktails of O157 and non-O157 Shiga toxigenic Escherichia coli (STEC) and Salmonella in lean ground beef with or without moisture-enhancing ingredients. Beef was moisture enhanced to 110% (w) with a 5% NaCl-2.5% sodium tripolyphosphate (w/w) brine. Meat, with or without added brine, was inoculated (∼108 CFU/g) and heat shocked at 48°C for 0, 5, or 30 min, followed by isothermal heating at 55°C. Inoculated control samples were unenhanced and were not subject to heat shock. From the linear portion of the log CFU per gram surviving cells over time plots, D55°C-values (minutes) were calculated. D55°C was 20.43, 28.78, and 21.15 min for O157, non-O157, and Salmonella controls, respectively. Overall, heat shock significantly increased D55°C, regardless of pathogen (P < 0.05). After 30 min of heat shock, D55°C increased 89 and 160% for O157 STEC, 32 and 49% for non-O157 STEC, and 29 and 57% for Salmonella, in unenhanced and enhanced samples, respectively, relative to the pathogen control. D55°C for Salmonella was the same or significantly less than for O157 and non-O157 STEC, regardless of heat shock, and was significantly less than for O157 and non-O157 STEC in all trials with moisture-enhanced meat (P < 0.05). Moisture-enhancing ingredients significantly increased D55°C, regardless of pathogen (P < 0.05). We suggest that thermal processes validated against Salmonella may not prove effective against STEC in all cases and that regulators of the beef industry should focus attention on STEC in nonintact moisture-enhanced beef products.

Molecular based prevalence of shigatoxigenic Escherichia coli in rectal swab of apparently healthy cattle in Mymensingh district, Bangladesh

Objective: Cattle are regarded as the principle reservoir of O157 and non-O157 shigatoxigenic Escherichia coli (STEC). Spreading of the STEC to human is primarily happens through contaminated meat, milk and their byproducts. The present study was aimed to explore the occurrence of STEC in the rectal swab of apparently healthy cattle. Materials and methods: A total of 60 E. coli isolates that were previously isolated from the rectal swab of cattle were used in this study. DNA were extracted from the isolates and screened by PCR to detect E. coli stx (stx1, stx2), ehxA and rfbO157 genes. Representative amplicons of the PCR products were sequenced. The prevalence of the STEC was determined based on the detection of STEC specific stx genes. The prevalence data were further analyzed by SPSS to elucidate any difference among different demographic groups of the study population. Results: Overall, 43.33% (n=26/60) of the isolates were found carrying stx genes. Based on the presence of stx and ehxA genes, 6 different types of STEC were identified, of which 20% (n=12/26) were carrying both stx1 and stx2 genes. None of the isolates was positive for rfbO157. The PCR amplicons were sequenced, and the nucleotide sequences were deposited in GenBank (accession: KM596779-KM596784). Conclusion: In this study, non-O157 STEC were found highly prevalent in the local cattle. This study suggests that the apparently healthy cattle may act as a potential source of STEC infection for humans.

Characterization of Shiga Toxigenic Escherichia coli O157 and Non-O157 Isolates from Ruminant Feces in Malaysia.

Shiga toxigenic Escherichia coli (STEC) O157 and several other serogroups of non-O157 STEC are causative agents of severe disease in humans world-wide. The present study was conducted to characterize STEC O157 and non-O157 serogroups O26, O103, O111, O121, O45, and O145 in ruminants in Malaysia. A total of 136 ruminant feces samples were collected from 6 different farms in Peninsular Malaysia. Immunomagnetic beads were used to isolate E. coli O157 and non-O157 serogroups, while PCR was used for the detection and subtyping of STEC isolates. STEC O157:H7 was isolated from 6 (4%) feces samples and all isolates obtained carried stx 2c, eaeA-γ1, and ehxA. Non-O157 STEC was isolated from 2 (1.5%) feces samples with one isolate carrying stx 1a, stx 2a, stx 2c, and ehxA and the other carrying stx 1a alone. The presence of STEC O157 and non-O157 in a small percentage of ruminants in this study together with their virulence characteristics suggests that they may have limited impact on public health.

Thermal Tolerance of O157 and non-O157 Shiga Toxigenic Strains of Escherichia coli, Salmonella, and Potential Pathogen Surrogates, in Frankfurter Batter and Ground Beef of Varying Fat Levels

The non-O157 Shiga toxigenic Escherichia coli (STEC) serogroups most commonly associated with illness are O26, O45, O103, O111, O121, and O145. We compared the thermal tolerance (D55°C) of three or more strains of each of these six non-O157 STEC serogroups with five strains of O157:H7 STEC in 7% fat ground beef. D55°C was also determined for at least one heat-tolerant STEC strain per serogroup in 15 and 27% fat ground beef. D55°C of single-pathogen cocktails of O157 and non-O157 STEC, Salmonella, and potential pathogen surrogates, Pediococcus acidilactici and Staphylococcus carnosus, was determined in 7, 15, and 27% fat ground beef and in frankfurter batter. Samples (25 g) were heated for up to 120 min at 55°C, survivors were enumerated, and log CFU per gram was plotted versus time. There were significant differences in D55°C across all STEC strains heated in 7% fat ground beef (P < 0.05), but no non-O157 STEC strain had D55°C greater than the range observed for O157 STEC. D55°C was significantly different for strains within serogroups O45, O145, and O157 (P < 0.05). D55°C for non-O157 STEC strains in 15 and 27% fat ground beef were less than or equal to the range of D55°C for O157. D55°C for pathogen cocktails was not significantly different when measured in 7, 15, and 27% fat ground beef (P ≥ 0.05). D55°C of Salmonella in frankfurter batter was significantly less than for O157 and non-O157 STEC (P < 0.05). Thermal tolerance of pathogen cocktails in ground beef (7, 15, or 27% fat) and frankfurter batter was significantly less than for potential pathogen surrogates (P < 0.05). Results suggest that thermal processes in beef validated against E. coli O157:H7 have adequate lethality against non-O157 STEC, that thermal processes that target Salmonella destruction may not be adequate against STEC in some situations, and that the use of pathogen surrogates P. acidilactici and S. carnosus to validate thermal processing interventions in ground beef and frankfurter batter would be of limited utility to processors.

Thermal Tolerance Characteristics of Non-O157 Shiga Toxigenic Strains of Escherichia coli (STEC) in a Beef Broth Model System Are Similar to Those of O157:H7 STEC

The non-O157 Shiga toxigenic Escherichia coli (STEC) serogroups most commonly associated with illness are O26, O45, O103, O111, O121, and O145. In the United States, these serogroups are considered adulterants in raw nonintact beef. To begin to understand the behavior of these pathogens in meat systems, we compared the thermal tolerance of acid-adapted cells of non-O157 STEC and O157:H7 STEC in a beef-derived broth. D58°C-values were determined for at least three strains per serogroup, and D54.6°C-values and D63.6°C-values were determined for one strain per serogroup. Each strain was grown to stationary phase in brain heart infusion broth (BHIB; pH 7.0) and inoculated into prewarmed BHIB in a shaking water bath for thermotolerance experiments at 54.6, 58.0, or 63.6°C (three trials per strain). Samples were heated for up to 160 min at 54.6°C, 3 min at 58.0°C, or 45 s at 63.6°C, with periodic sampling followed by rapid cooling and plating on modified Levine's eosin methylene blue agar. For each strain and temperature, the log CFU per milliliter was plotted versus time, and D-values were determined. Across all strains, the least and most heat tolerant STEC serogroups at 58°C were O145 and O157, respectively. D58°C-values in BHIB ranged from 0.44 min for an O145 strain to 1.42 min for an O157:H7 strain. D58°C-values for O157 STEC strains were significantly higher than those for at least one strain in each of the non-O157 STEC serogroups (P < 0.05) except for serogroup O103. At 54.6°C, the most heat-resistant STEC strain belonged to serogroup O103 and was significantly more heat tolerant than the O157:H7 strains (P < 0.05). Grouping the strains, there were no significant differences in heat tolerance between O157 and non-O157 STEC at 63.6°C (P ≥ 0.05). The z-values for non-O157 STEC strains were comparable to those for O157:H7 STEC strains (P ≥ 0.05), ranging from 4.10 to 5.21°C. These results suggest that thermal processing interventions that target destruction of E. coli O157:H7 may have adequate lethality against non-O157 STEC.

Prolonged and mixed non-O157 Escherichia coli infection in an Australian household

An Australian family was identified through a Public Health follow up on a Shiga-toxigenic Escherichia coli (STEC) positive bloody diarrhoea case, with three of the four family members experiencing either symptomatic or asymptomatic STEC shedding. Bacterial isolates were submitted to stx sequence sub-typing, multi-locus variable number tandem repeat analysis (MLVA), multi-locus sequence typing (MLST) and binary typing. The analysis revealed that there were multiple strains of STEC being shed by the family members, with similar virulence gene profiles and the same serogroup but differing in their MLVA and MLST profiles. This study illustrates the potentially complicated nature of non-O157 STEC infections and the importance of molecular epidemiology in understanding disease clusters.

Changes in cell surface properties of shiga toxigenic Escherichia coli by Quercus infectoria G. Olivier.

The effects of Quercus infectoria (family Fagaceae) nutgalls on cell surface properties of Shiga toxigenic Escherichia coli (STEC) were investigated with an assay of microbial adhesion to hydrocarbon. The surface of bacterial cells treated with Q. infectoria exhibited a higher level of cell surface hydrophobicity (CSH) toward toluene than did the surface of untreated cells. With 50% ethanolic extract, the CSH of the three strains of STEC O157:H7 treated with 4X MIC of the extract resulted in moderate or strong hydrophobicity, whereas at 2x MIC and MIC, the CSH of only one strain of E. coli O157:H7 was significantly affected. The 95% ethanolic extract had a significant effect on CSH of all three strains at both 4X MIC and 2X MIC but not at the MIC. The effect on bacterial CSH was less pronounced with the other STEC strains. At 4X MIC, the 50% ethanolic extract increased the CSH of all non-O157 STEC strains significantly. At 2X MIC and 4X MIC, the 95% ethanolic extract affected the CSH of E. coli O26:H11 significantly but did not affect E. coli O111 :NM or E. coli O22. Electro microscopic examination revealed the loss of pili in the treated cells. The ability of Q. infectoria extract to modify hydrophobic domains enables this extract to partition the lipids of the bacterial cell membrane, rendering the membrane more permeable and allowing leakage of ions and other cell contents, which leads to cell death. Further studies are required to evaluate the effects of Q. infectoria extract in food systems or in vivo and provide support for the use of this extract as a food additive for control of these STEC pathogens.

Fate of non O157 Shiga toxigenic Escherichia coli in composted cattle manure

To determine the fate of Shiga toxigenic Escherichia coli (STEC) non-O 157 in composted manure from naturally colonized cattle, fresh manure was obtained from three cows carrying non-O157 STEC strains possessing the stx2 gene. Two composting systems were used: a 0.6m deep cave opened in the soil and an one meter high solid manure heap in a pyramidal architecture. Every day, for the 10 first days, and every five days for a month, one manure sample from three different points in both systems was collected and cultured to determine the presence of E. coli and the presence of the stx 2 gene in the cells. The temperature was verified at each sampling. STEC non-O157 E. coli cells survived for 8, 25 and 30 days at 42, 40 and 38ºC, respectively, in the deep cave and 4, 4 and 7 days at 65, 58 and 52ºC, respectively, in the heap, during the composting manure. Temperature and indigenous microorganisms appear to contribute to pathogen disappearance in the composting system. It is concluded that both composting systems were efficient to eliminate STEC cells. Land application of composted manure should minimize environmental risk associated with the dissemination of the pathogen.

Detection of Shiga-toxigenic Escherichia coli (STEC) in fresh seafood and meat marketed in Mangalore, India by PCR.

To study the incidence of Shiga-toxigenic Escherichia coli (STEC) in seafoods from India. Escherichia coli isolated from various seafoods such as fresh fish, clams and water were screened for the presence of stx, hlyA and rfbO157 genes by PCR; 5% of clams and 3% of fresh fish samples were positive for non-O157 STEC. STEC is prevalent in seafoods in India, and non-O157 serotype is more common. Seafood could be a vehicle for transmission of STEC even in tropical countries.