STEC Serotypes Research Articles

Occurrence, serotypes and virulence characteristics of Shiga toxin-producing and Enteropathogenic Escherichia coli isolates from dairy cattle in South Africa

Shiga toxin-producing and Enteropathogenic Escherichiacoli are foodborne pathogens commonly associated with diarrheal disease in humans. This study investigated the presence of STEC and EPEC in 771 dairy cattle fecal samples which were collected from 5 abattoirs and 9 dairy farms in South Africa. STEC and EPEC were detected, isolated and identified using culture and PCR. Furthermore, 339 STEC and 136 EPEC isolates were characterized by serotype and major virulence genes including stx1, stx2, eaeA and hlyA and the presence of eaeA and bfpA in EPEC. PCR screening of bacterial sweeps which were grown from fecal samples revealed that 42.2% and 23.3% were STEC and EPEC positive, respectively. PCR serotyping of 339 STEC and 136 EPEC isolates revealed 53 different STEC and 19 EPEC serotypes, respectively. The three most frequent STEC serotypes were O82:H8, OgX18:H2, and O157:H7. Only 10% of the isolates were classified as “Top 7” STEC serotypes: O26:H2, 0.3%; O26:H11, 3.2%; O103:H8, 0.6%; and O157:H7, 5.9%. The three most frequent EPEC serotypes were O10:H2, OgN9:H28, and O26:H11. The distribution of major virulence genes among the 339 STEC isolates was as follows: stx1, 72.9%; stx2, 85.7%; eaeA, 13.6% and hlyA, 69.9%. All the 136 EPEC isolates were eaeA-positive but bfpA-negative, while 46.5% carried hlyA. This study revealed that dairy cattle are a major reservoir of STEC and EPEC in South Africa. Further comparative studies of cattle and human STEC and EPEC isolates will be needed to determine the role played by dairy cattle STEC and EPEC in the occurrence of foodborne disease in humans.Please kindly check and confirm the country and city name in affiliation [6].This affiliation is correct.Please kindly check and confirm the affiliationsConfirmed. All Affiliations are accurate

Detection and Molecular Characterization of Shiga-toxin-producing Escherichia coli from Fecal Samples of Cattle Farms Al-Buḥayrah Governorate Egypt

Escherichia coli that produce the Shiga toxin (STEC) has become one of the most significant foodborne pathogens linked to serious illnesses. Our study was aimed to detect the existence, antimicrobial profiles, and virulence repertoire of different STEC serotypes from fecal samples of dairy cattle.
 We collected 19 fecal samples (12 diarrheic and 7 non-diarrheic) from dairy cattle. E. coli isolation and identification were performed. Shiga toxins genes were detected and the antimicrobial resistance was investigated.
 E. coli was detected at the rate of 13/19 (68.4%) from the fecal samples. The frequency of Stx2a represented 12/13 (92.3%), Stx2 represented 3/13 (23.14%), and Stx1d, Stx2f, Stx2g, and eae represented 2/13 (15.4%). While the virulence genes Stx1, Stx1c, Stx2c, Stx2d, Stx2e, and ehxA represented 0/13 (0%). The most effective antimicrobials were tulathithromycin 30 (53.84%) followed by ceftiofur 30 and marbofloxacin (46.16%), amikacin (15.38%), and the least resistance was recorded for nalidixic acid and oxytetracycline (7.70%). The resistant antibiotics with percentages reached to 100% were Amoxicillin/ Clavulanic acid, Ceftriaxone, Ciprofloxacin, Doxycycline, Erythromycin, Gentamicin, Ninezolid, Nitrofurantoin, Penicillin G, Streptomycin, and Trimethoprim/Sulfamethoxazole, followed by chloramphenicol, nalidixic acid, oxytetracycline, and Tylosine (92.30%), Amikacin and Ampicillin (84.62%), Ceftiofur (23.07%), Marbofloxacin (15.38%) and Tulathithromycin 30 (7.70%).
 The findings demonstrated that E. coli that are resistant to numerous groups of antimicrobials spread through feces and may pose a risk to human and animal health.

Phenotypic and genotypic characterization of Shiga toxin-producing Escherichia coli strains recovered from bovine carcasses in Uruguay.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that cause food-borne diseases in humans. Cattle and derived foodstuffs play a known role as reservoir and vehicles, respectively. In Uruguay, information about the characteristics of circulating STEC in meat productive chain is scarce. The aim was to characterize STEC strains recovered from 800 bovine carcasses of different slaughterhouses. To characterize STEC strains we use classical microbiological procedures, Whole Genome Sequencing (WGS) and FAO/WHO risk criteria. We analyzed 39 STEC isolated from 20 establishments. They belonged to 21 different O-groups and 13 different H-types. Only one O157:H7 strain was characterized and the serotypes O130:H11(6), O174:H28(5), and O22:H8(5) prevailed. One strain showed resistance in vitro to tetracycline and genes for doxycycline, sulfonamide, streptomycin and fosfomycin resistance were detected. Thirty-three strains (84.6%) carried the subtypes Stx2a, Stx2c, or Stx2d. The gene eae was detected only in two strains (O157:H7, O182:H25). The most prevalent virulence genes found were lpfA (n = 38), ompA (n = 39), ompT (n = 39), iss (n = 38), and terC (n = 39). Within the set of STEC analyzed, the majority (81.5%) belonged to FAO/WHO's risk classification levels 4 and 5 (lower risk). Besides, we detected STEC serotypes O22:H8, O113:H21, O130:H11, and O174:H21 belonged to level risk 2 associate with diarrhea, hemorrhagic colitis or Hemolytic-Uremic Syndrome (HUS). The only O157:H7 strain analyzed belonged to ST11. Thirty-eight isolates belonged to the Clermont type B1, while the O157:H7 was classified as E. The analyzed STEC showed high genomic diversity and harbor several genetic determinants associated with virulence, underlining the important role of WGS for a complete typing. In this set we did not detect non-O157 STEC previously isolated from local HUS cases. However, when interpreting this findings, the low number of isolates analyzed and some methodological limitations must be taken into account. Obtained data suggest that cattle constitute a local reservoir of non-O157 serotypes associated with severe diseases. Other studies are needed to assess the role of the local meat chain in the spread of STEC, especially those associated with severe diseases in humans.

Occurrence, Serotypes and Virulence Characteristics of Shiga-Toxin-Producing Escherichia coli Isolates from Goats on Communal Rangeland in South Africa.

Shiga-toxin-producing Escherichia coli is a foodborne pathogen commonly associated with human disease characterized by mild or bloody diarrhea hemorrhagic colitis and hemolytic uremic syndrome. This study investigated the occurrence of STEC in fecal samples of 289 goats in South Africa using microbiological culture and PCR. Furthermore, 628 goat STEC isolates were characterized by serotype (O:H) and major virulence factors by PCR. STEC was found in 80.2% (232/289) of goat fecal samples. Serotyping of 628 STEC isolates revealed 63 distinct serotypes including four of the major top seven STEC serogroups which were detected in 12.1% (35/289) of goats: O157:H7, 2.7% (8/289); O157:H8, 0.3%, (1/289); O157:H29, 0.3% (1/289); O103:H8, 7.6% (22/289); O103:H56, 0.3% (1/289); O26:H2, 0.3% (1/289); O111:H8, 0.3% (1/289) and 59 non-O157 STEC serotypes. Twenty-four of the sixty-three serotypes were previously associated with human disease. Virulence genes were distributed as follows: stx1, 60.6% (381/628); stx2, 72.7% (457/628); eaeA, 22.1% (139/628) and hlyA, 78.0% (490/628). Both stx1 and stx2 were found in 33.4% (210/628) of isolates. In conclusion, goats in South Africa are a reservoir and potential source of diverse STEC serotypes that are potentially virulent for humans. Further molecular characterization will be needed to fully assess the virulence potential of goat STEC isolates and their capacity to cause disease in humans.

Eugenol, citral, and hexanal, alone or in combination with heat, affect viability, biofilm formation, and swarming on Shiga-toxin-producing Escherichia coli.

Shiga-toxin-producing Escherichia coli strains are pathogenic for humans and cause mild to severe illnesses. In this study, the antimicrobial effect of citral, eugenol, and hexanal in combination with heat shock (HS) was evaluated in terms of the growth, biofilm formation, swarming, and expression of virulence genes of STEC serotypes (O157:H7, O103, O111, and O26). Eugenol was the most effective compound against the growth of E. coli strains (MBC = 0.58 to 0.73mg/mL), followed by citral (MBC = 0.86 to 1.26mg/mL) and hexanal (MBC = 2.24 to 2.52mg/mL). Biofilm formation and swarming motility have great variability between STEC strains. Natural compounds-alone or combined with HS-inhibited biofilm formation; however, swarming motility was induced by most treatments. The expression of the studied genes during biofilm formation and swarming under natural antimicrobials was affected but not in a uniform pattern. These treatments could be used to control contamination of STEC and inhibit biofilm formation.

Characterization of verotoxigenic E.coli and enteropathogenic E.coli isolated from infants with diarrhea in combination with antimicrobial resistance pattern in Minia, Egypt

Diarrheal disease is considered a significant health problem, principally in developing countries as it is one of the leading causes of morbidity and mortality especially in infants. Studies found that STES and EPEC strains are among the most predominant causative agents in acute diarrhea. The aim of the study was to investigate the presence of certain virulence factors and antibiotic resistances of STEC and EPEC originating from infants with diarrhea in Egypt. A total of 200 infants of different ages suffering from diarrhea between August 2017 and February 2019 were registered in this study. Standard cultural and biochemical methods were used to isolate E. coli species while; PCR was applied for the detection of virulence genes (stx1, stx2, eae and pfb A) for STEC and EPEC isolates, respectively. Eighty nine (44.5%) E. coli strains were isolated from 200 stool samples. A total of 40 (44.9%) strains were identified as STEC, and 1(1.12%) strain was atypical EPEC. Fourteen of the STEC serotypes were O157:H7. Susceptibilities of 40 STEC and 1 aEPEC isolates were determined for ten antimicrobial drugs. STEC O157:H7 strains and STEC Non-O157:H7 exhibited the highest resistance against cefazolin and no resistance against imipnem and Amikacin, respectively while, the isolated strain of aEPEC was sensitive to all the studied antibiotics except cefazolin. Results showed that STEC is one of the major causes of diarrhea that showed resistant to commonly used antibiotics so representatives must pay great attention to this matter in order to increase the health of the community.

Molecular profiling and antimicrobial resistance of Shiga toxin-producing Escherichia coli O26, O45, O103, O121, O145 and O157 isolates from cattle on cow-calf operations in South Africa

In this study, 140 cattle STEC isolates belonging to serogroups O157, O26, O145, O121, O103 and O45 were characterized for 38 virulence-associated genes, antimicrobial resistance profiles and genotyped by PFGE. The majority of isolates carried both stx1 and stx2 concurrently, stx2c, and stx2d; plasmid-encoded genes ehxA, espP, subA and saa but lacked katP and etpD and eaeA. Possession of eaeA was significantly associated with the presence of nle genes, katP, etpD, ureC and terC. However, saa and subA, stx1c and stx1d were only detected in eaeA negative isolates. A complete OI-122 and most non-LEE effector genes were detected in only two eaeA positive serotypes, including STEC O157:H7 and O103:H2. The eaeA gene was detected in STEC serotypes that are commonly implicated in severe humans disease and outbreaks including STEC O157:H7, STEC O145:H28 and O103:H2. PFGE revealed that the isolates were highly diverse with very low rates of antimicrobial resistance. In conclusion, only a small number of cattle STEC serotypes that possessed eaeA, had the highest number of virulence-associated genes, indicative of their high virulence. Further characterization of STEC O157:H7, STEC O145:H28 and O103:H2 using whole genome sequencing will be needed to fully understand their virulence potential for humans.

Serotypes and virulence profiles of Shiga toxin-producing Escherichia coli strains isolated during 2017 from human infections in Switzerland

Since 2015, the Swiss Federal Office of Public Health registered an increase of notifications of STEC, probably due to the adoption of culture independent stx screening tests in diagnostic laboratories. This study aimed to identify the serotypes and virulence genes of 120 STEC isolated from human clinical stx positive specimens during 2017 in order to estimate any changes in serotype distribution and toxin profiles of STEC compared to the time span 2010–2014. Culturing of STEC from stool samples was achieved using the streak plate technique on MacConkey agar. We performed O and H serotyping by PCR and by micro array. Virulence genes were identified and subtyped using molecular methods, including stx1 and stx2 subtypes, and the intimin encoding gene, eae. STEC were recovered from 27.5% of the stx positive samples. STEC O157:H7 accounted for 7.5% of all isolates, and STEC O80:H2, O91:H10/H14/H21, O103:H2/H11, and O26:H11 accounted for 36.9% of the non-O157 strains. Forty-five isolates with stx1 variants, 47 with stx2 variants and 28 isolates with both stx1 and stx2 variants were identified. Forty (33.3% of all isolates) carried the subtypes associated with high pathogenic potential, stx2a, stx2c, or stx2d. The eae gene for intimin was detected in 54 strains (45% of all strains). Compared to 2010–2014, our data show that the proportion of the so called "top five" serogroups, STEC O26, O111, O103, and O157 declined from 53.7% to 28.3% in 2017. The proportion of isolates with stx2a, stx2c, or stx2d decreased from 50.5% to 33.3%. We also observed an increase of STEC harbouring the low pathogenic subtypes stx2b and stx2e from 12.6% to 29.2%, and of eae negative STEC from 29.5% in 2010–2014 to 55% in 2017. Simultaneously, there was a sharp increase of the patients' median age from 24 years to 46.5 years. Clinical manifestations in the patients included abdominal pain without diarrhea (22.3%), diarrhea (77.7%), and the haemolytic-uremic syndrome (HUS) (7.4%). Our data show that a greater number and a wider range of STEC serotypes are detected by culture-independent testing, with implications for public health services.

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.

Ability of Shiga toxigenic Escherichia coli to survive within dry-surface biofilms and transfer to fresh lettuce

Biofilms are known to play important roles in bacterial survival and persistence in food-processing environments. This study aimed to determine the ability of the top 7 STEC serotypes to form biofilms on polystyrene (POL) and stainless steel (SS) plates and to quantify their survival and transfer from dry-surface biofilms to lettuce pieces. The ability of 14 STEC strains to form biofilms on these two materials at different exposure times and temperatures was assessed using crystal violet, Congo red and SEM. At 10 °C all serotypes were weak biofilm producers on both surfaces. In contrast, serotypes O45-040, O45-445, O103-102, O103-670 and O157-R508 were strong biofilm producers at 25 °C. Strains O103-102, O103-670, O111-CFS, O111–053 and O157:H7-R508 were expressers of curli. Under scanning electron microscopy, strains O103-670, O111-CFS, O157-R508, and O121-083 formed more discernible multilayer, mature biofilms on SS coupons. Regardless of the surface (POL/SS), all STEC strains were able to transfer viable cells onto fresh lettuce within a short contact time (2 min) to varying degrees (up to 6.35 log cfu/g). On POL, viable cell of almost all serotypes exhibited decreased detachment (p = 0.001) over 6 days; while after 30 days on SS, serotypes O45-040, O103-102, O103-670, O111-053, O111-CFS, O121-083, O145-231 O157:H7-R508 and O157:H7-122 were transferred to lettuce. After enrichment, all 14 STEC strains were recovered from dry-surface biofilms on POL and SS plates after 30 days. Results demonstrated that the top 7 STEC remained viable within dry-surface biofilms for at least 30 days, transferring to lettuce within 2 min of exposure and acting as a source of adulteration.

Genetic Variation in Shiga Toxin-producing Escherichia coli Recovered from Patients in Michigan and Connecticut

BackgroundShiga toxin-producing Escherichia coli (STEC) is a gram-negative foodborne pathogen that causes approximately 265,000 illnesses in the US annually. STEC O157 and six non-O157 STEC serotypes are most commonly associated with illness, though variation exists in the ability of different STEC types to cause disease. Consequently, we sought to examine genetic variation in virulence genes and clustered regularly interspaced repeat (CRISPR) loci among clinical strains of diverse lineages from different geographic locations.MethodsIsolates were collected from a sentinel surveillance in 2000–2006 by the Michigan Department of Health and Human Services (n = 44) and Connecticut Department of Public Health (n = 115). Whole genome sequencing was performed and genes for O-antigen (serotype), multilocus sequence typing (MLST) and virulence factors were extracted. CRISPRFinder and Geneious were used for CRISPR loci.ResultsA phylogenetic tree based on MLST found no geographic clustering of the strains. Similarly, no difference was observed for stx1 (MI: 89.6%, CT: 83.5%), stx2 (MI: 6.9%, CT: 7.1%), stx1/stx2 (MI: 3.4%, CT: 9.4%), ehxA (MI: 80.0%, CT: 81.2%) and eae (MI: 86.7%, CT: 94.1%) frequencies across geographical locations. Although the CRISPR loci were similar within related STs, which was independent of serotype, some variation was detected between locations.ConclusionThese data highlight the circulation of common non-O157 STEC lineages capable of causing disease in different populations. Strains had similar virulence gene profiles, though the diversity of the CRISPR loci varied across strains. The latter could be impacted by varying selective pressures that could affect disease frequencies and symptom severity. Continued surveillance of non-O157 STEC is needed to elucidate the genetic characteristics that are most important for disease severity.Disclosures All authors: No reported disclosures.

Comparative Characterization of Shiga Toxin Type 2 and Subtilase Cytotoxin Effects on Human Renal Epithelial and Endothelial Cells Grown in Monolayer and Bilayer Conditions.

Postdiarrheal hemolytic uremic syndrome (HUS) affects children under 5 years old and is responsible for the development of acute and chronic renal failure, particularly in Argentina. This pathology is a complication of Shiga toxin (Stx)-producing Escherichia coli infection and renal damage is attributed to Stx types 1 and 2 (Stx1, Stx2) produced by Escherichia coli O157:H7 and many other STEC serotypes. It has been reported the production of Subtilase cytotoxin (SubAB) by non-O157 STEC isolated from cases of childhood diarrhea. Therefore, it is proposed that SubAB may contribute to HUS pathogenesis. The human kidney is the most affected organ because very Stx-sensitive cells express high amounts of biologically active receptor. In this study, we investigated the effects of Stx2 and SubAB on primary cultures of human glomerular endothelial cells (HGEC) and on a human tubular epithelial cell line (HK-2) in monoculture and coculture conditions. We have established the coculture as a human renal proximal tubule model to study water absorption and cytotoxicity in the presence of Stx2 and SubAB. We obtained and characterized cocultures of HGEC and HK-2. Under basal conditions, HGEC monolayers exhibited the lowest electrical resistance (TEER) and the highest water permeability, while the HGEC/HK-2 bilayers showed the highest TEER and the lowest water permeability. In addition, at times as short as 20–30 minutes, Stx2 and SubAB caused the inhibition of water absorption across HK-2 and HGEC monolayers and this effect was not related to a decrease in cell viability. However, toxins did not have inhibitory effects on water movement across HGEC/HK-2 bilayers. After 72 h, Stx2 inhibited the cell viability of HGEC and HK-2 monolayers, but these effects were attenuated in HGEC/HK-2 bilayers. On the other hand, SubAB cytotoxicity shows a tendency to be attenuated by the bilayers. Our data provide evidence about the different effects of these toxins on the bilayers respect to the monolayers. This in vitro model of communication between human renal microvascular endothelial cells and human proximal tubular epithelial cells is a representative model of the human proximal tubule to study the effects of Stx2 and SubAB related to the development of HUS.

Incidence of Top 6 shiga toxigenic <em>Escherichia coli</em> within two Ontario beef processing facilities: Challenges in screening and confirmation testing

The incidence of the Top 6 STEC serotypes was determined in two beef slaughter houses. In total, 328 samples were taken of hides, de-hided carcasses and the plant environment. Samples were enriched in Tryptic Soy Broth containing novobiocin then screened using RT-PCR GeneDiskO system that targeted stx, eae and wzx genes. It was found that 92.5% (172 of 186) of the hide samples. 72.5% (29 of 40) de-hided samples and 84.3% (86 of 102) of the environmental samples returned presumptive positive results. Serotypes O103, O45 and O121 were most commonly encountered although all the Top 6 serotypes were represented within individual samples. However, attempts to recover the Top 6 serotypes by culturing proved unsuccessful despite screening up to 20 colonies per CHROMAgar® plate of enriched sample. The reasons for the discrepancy between the RT-PCR and culture methods were found to be due to low levels of the target in enriched samples, presence of virulence factors in different cells and also the transient retention of stx. With regards the latter it was found that strains harboring a full set of virulence factors (eae, stx) were more common in grown cultures held post-incubation at 4 °C for 14 days. Moreover, no stx gene was recovered when isolates were sub-cultured on TSA but was present in the same strains grown on CHROMAgar®. In total 39 STEC isolates were recovered with the majority harboring stx1, stx2, eae and hylA. Only 3 of the isolates had stable complement of virulence factors and were identified as O172:H28, O76:H7 and O187:H52. Although no Top 6 STEC were isolated the presence of virulent strains on carcasses with the potential to cause Hemolytic Uremic Syndrome is of concern. The significance of those STEC that transiently harbor virulence factors is unclear although clearly impacts on diagnostic performance robustness when screening for the Top 6 non-O157 STEC.

Simultaneous Presence of Insertion Sequence Excision Enhancer and Insertion Sequence IS629 Correlates with Increased Diversity and Virulence in Shiga Toxin-Producing Escherichia coli.

Although new serotypes of enterohemorrhagic Escherichia coli (EHEC) emerge constantly, the mechanisms by which these new pathogens arise and the reasons emerging serotypes tend to carry more virulence genes than other E. coli are not understood. An insertion sequence (IS) excision enhancer (IEE) was discovered in EHEC O157:H7 that promoted the excision of IS3 family members and generating various genomic deletions. One IS3 family member, IS629, actively transposes and proliferates in EHEC O157:H7 and enterotoxigenic E. coli (ETEC) O139 and O149. The simultaneous presence of the IEE and IS629 (and other IS3 family members) may be part of a system promoting not only adaptation and genome diversification in E. coli O157:H7 but also contributing to the development of pathogenicity among predominant serotypes. Prevalence comparisons of these elements in 461 strains, representing 72 different serotypes and 5 preassigned seropathotypes (SPT) A to E, showed that the presence of these two elements simultaneously was serotype specific and associated with highly pathogenic serotypes (O157 and top non-O157 Shiga toxin-producing Escherichia coli [STEC]) implicated in outbreaks and sporadic cases of human illness (SPT A and B). Serotypes lacking one or both elements were less likely to have been isolated from clinical cases. Our comparisons of IEE sequences showed sequence variations that could be divided into at least three clusters. Interestingly, the IEE sequences from O157 and the top 10 non-O157 STEC serotypes fell into clusters I and II, while less commonly isolated serotypes O5 and O174 fell into cluster III. These results suggest that IS629 and IEE elements may be acting synergistically to promote genome plasticity and genetic diversity among STEC strains, enhancing their abilities to adapt to hostile environments and rapidly take up virulence factors.

Survival of Shiga toxin-producing Escherichia coli in broth as influenced by pH, water activity and temperature.

This study examined the effects of and interactions between pH, aw and temperature on the survival of the top six non-O157 STECs and Escherichia coli O157:H7. All variables significantly affected the survival of all STEC serotypes. However, aw bore the most significant effect, followed by temperature and then pH. Examination of the effect of the interaction between these variables revealed that the interaction between aw and temperature was the most significant followed by the interaction between pH and temperature and then aw and pH. Decrease in aw resulted in population reduction of all serotypes studied. This reduction in population was significantly increased with the increase in temperature and was further significantly enhanced with decreasing pH. Examination of the differences in the survival among the individual serotypes revealed that the response of each serotype to aw or temperature changes was significantly different, while their response to pH changes was similar. Analysis of the relative survival of individual non-O157 STECs to O157:H7 revealed that the survival of O121 and O45 was not significantly different to O157:H7 while O103, O111, O145 and O26 showed less tolerance to the combined treatments, and their survival was significantly different from O157:H7. Results of this study estimate the interaction between pH, aw and temperature on the survival of the top six non-O157 STECs relative to Escherichia coli O157:H7 and provide important growth and no-growth condition which will offer risk assessors a means of estimating the likelihood of these pathogens, if present, would grow in response to the interaction between the three variables assessed.

Intimin Gene ( eae ) Subtype-Based Real-Time PCR Strategy for Specific Detection of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 in Cattle Feces

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely, γ1, β1, ε, θ, and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae, and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx plus eae plus O genetic combinations were detected 143 times. However, taking into consideration the association between eae subtypes and O group markers, the resulting stx plus eae subtype plus O combinations were detected only 46 times. The 46 isolation assays performed allowed recovery of 22 E. coli strains belonging to one of the five targeted STEC serogroups. In contrast, only 2 of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but that were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11, and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtype-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces.

Occurrence, serotypes and virulence genes of non-O157 Shiga toxin-producing Escherichia coli in fresh beef, ground beef, and beef burger

This study determined the prevalence, serotypes and virulence genes distribution of non-O157 Shiga toxin-producing Escherichia coli in meat products collected from butchers shops and supermarkets in Mansoura city, Egypt. We have characterized 18 non-O157 STEC strains among the identified 100 E. coli isolates recovered from the examined 87 meat product samples. The prevalence of non-O157 STEC strains in fresh beef, ground beef and beef burger samples were 11.1% (3/27), 16.7% (5/30), and 33.3% (10/30), respectively. The eighteen non-O157 STEC isolated strains were serotyped into seven (38.9%) O111:H8, six (33.3%) O26:H11, two (11.1%) O111:H–, and one (5.56%) for each of O55:H7, O126:H5 and O128:H2. PCR assays for different virulence genes showed that nine (50%), eleven (61.1%), and nine (50%) strains carry stx1, stx2, and eae genes, respectively. The distribution of shiga toxin genes among the isolated strains indicated that seven (38.9%) strains harbored stx1 only, nine (50%) strains harbored stx2 only, and two (11.1%) strains harbored both stx1 and stx2. The eae gene was present in association with five (27.8%), three (16.7%), and one (5.6%) strains that harbored stx1 only, stx2 only, and both stx1 and stx2, respectively. This study concluded that the examined meat products, particularly beef burger, consumed in Egypt are considerably contaminated with a variety of non-O157 STEC serotypes, and hence consumption of such products may constitute a potential health risk for consumers.

Prevalences of Shiga Toxin Subtypes and Selected Other Virulence Factors among Shiga-Toxigenic Escherichia coli Strains Isolated from Fresh Produce

Shiga-toxigenic Escherichia coli (STEC) strains were isolated from a variety of fresh produce, but mostly from spinach, with an estimated prevalence rate of 0.5%. A panel of 132 produce STEC strains were characterized for the presence of virulence and putative virulence factor genes and for Shiga toxin subtypes. About 9% of the isolates were found to have the eae gene, which encodes the intimin binding protein, and most of these belonged to known pathogenic STEC serotypes, such as O157:H7 and O26:H11, or to serotypes that reportedly have caused human illness. Among the eae-negative strains, there were three O113:H21 strains and one O91:H21 strain, which historically have been implicated in illness and therefore may be of concern as well. The ehxA gene, which encodes enterohemolysin, was found in ∼60% of the isolates, and the saa and subAB genes, which encode STEC agglutinating adhesin and subtilase cytotoxin, respectively, were found in ∼30% of the isolates. However, the precise roles of these three putative virulence factors in STEC pathogenesis have not yet been fully established. The stx1a and stx2a subtypes were present in 22% and 56%, respectively, of the strains overall and were the most common subtypes among produce STEC strains. The stx2d subtype was the second most common subtype (28% overall), followed by stx2c (7.5%), and only 2 to 3% of the produce STEC strains had the stx2e and stx2g subtypes. Almost half of the produce STEC strains had only partial serotypes or were untyped, and most of those that were identified belonged to unremarkable serotypes. Considering the uncertainties of some of these Stx subtypes and putative virulence factors in causing human illness, it is difficult to determine the health risk of many of these produce STEC strains.

Discrimination of Enterohemorrhagic Escherichia coli (EHEC) from Non-EHEC Strains Based on Detection of Various Combinations of Type III Effector Genes

Enterohemorrhagic Escherichia coli (EHEC) strains comprise a subgroup of Shiga-toxin (Stx)-producing E. coli (STEC) and are characterized by a few serotypes. Among these, seven priority STEC serotypes (O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7) are most frequently implicated in severe clinical illness worldwide. Currently, standard methods using stx, eae, and O-serogroup-specific gene sequences for detecting the top 7 EHEC serotypes bear the disadvantage that these genes can be found in non-EHEC strains as well. Here, we explored the suitability of ureD, espV, espK, espN, Z2098, and espM1 genes and combinations thereof as candidates for a more targeted EHEC screening assay. For a very large panel of E. coli strains (n = 1,100), which comprised EHEC (n = 340), enteropathogenic E. coli (EPEC) (n = 392), STEC (n = 193), and apathogenic strains (n = 175), we showed that these genetic markers were more prevalent in EHEC (67.1% to 92.4%) than in EPEC (13.3% to 45.2%), STEC (0.5% to 3.6%), and apathogenic E. coli strains (0 to 2.9%). It is noteworthy that 38.5% of the EPEC strains that tested positive for at least one of these genetic markers belonged to the top 7 EHEC serotypes, suggesting that such isolates might be Stx-negative derivatives of EHEC. The associations of espK with either espV, ureD, or Z2098 were the best combinations for more specific and sensitive detection of the top 7 EHEC strains, allowing detection of 99.3% to 100% of these strains. In addition, detection of 93.7% of the EHEC strains belonging to other serotypes than the top 7 offers a possibility for identifying new emerging EHEC strains.

Serotypes and virulotypes of non-O157 shiga-toxin producing Escherichia coli (STEC) on bovine hides and carcasses

Four hundred and fifty beef animal hides and a similar number of carcasses were screened for STEC in 3 beef abattoirs over a 12 month period using PCR and culture based methods. 67% (301/450) of hides and 27% (122/450) of carcasses were STEC PCR positive. Forty isolates representing 12 STEC serotypes (O5:H-, O13:H2, O26:H11, O33:H11, O55:H11, O113:H4, O128:H8, O136:H12, O138:H48, O150:H2, O168:H8 and ONT:H11) and 15 serotype–virulotype combinations were identified.This study provides much needed non-O157 STEC surveillance data and also provides further evidence of bovines as a source of clinically significant STEC as well as identifying 3 emerging serotypes O5:H- (eae-β1), O13:H2 (eae-ζ), and O150:H2 (eae-ζ) that should be considered when developing beef testing procedures for non-O157 STEC.