Non-O157 Shiga Toxin Producing Research Articles

Comparison of Three Preharvest Sampling Strategies to Monitor Pathogens in Cattle Lairage Areas

The objective of this study was to compare preharvest monitoring strategies by evaluating three different sampling methods in the lairage area to determine pathogen recovery for each sampling method and incoming pathogen prevalence from the cattle to inform in-plant decision making. Samples were gathered over a 5-month period, from February to June 2022, at a harvesting and processing facility located in Eastern Nebraska. Sampling methods included (i) fecal pats, (ii) boot swabs, and (iii) MicroTally swab. A total of 329 samples were collected over the study period (fecal pats: n = 105, boot swabs: n = 104, and MicroTally swabs: n = 120). Specific media combinations, an incubation temperature of 42°C, and incubation timepoints (18–24 h) were utilized for each matrix and the prevalence of Salmonella, Escherichia coli O157:H7, and six non-O157 Shiga-toxin producing E. coli (STEC) was evaluated using the BAX system Real-Time PCR assay. Overall, results from the study concluded that boot swabs were an effective sampling method for pathogen detection in the cattle lairage area. Boot swabs (97.1%) were statistically more likely to detect for Salmonella (p < 0.05) when compared to fecal pats (67.6%) and MicroTally swab (77.5%) methods. For E. coli O157:H7 and STEC – O26, O121, O45, and O103 prevalence, boot swabs were significantly better at detecting for these pathogens (p < 0.05) than MicroTally swabs (OR = 3.16 – 11.95) and a comparable sampling method to fecal pats (OR = 0.93 – 2.01, p > 0.05). Lastly, all three sampling methods detected a very low prevalence for E. coli O111 and O145; therefore, no further analysis was conducted. The boot swab sampling method was strongly favored because they require little training to implement, are inexpensive, and they do not require much sampling labor; therefore, would be a simple and effective sampling method to implement within the industry to evaluate pathogen prevalence preharvest.

Risk Factors for Non-O157 Shiga Toxin–Producing Escherichia coli Infections, United States

Shiga toxin-producing Escherichia coli (STEC) causes acute diarrheal illness. To determine risk factors for non-O157 STEC infection, we enrolled 939 patients and 2,464 healthy controls in a case-control study conducted in 10 US sites. The highest population-attributable fractions for domestically acquired infections were for eating lettuce (39%), tomatoes (21%), or at a fast-food restaurant (23%). Exposures with 10%-19% population attributable fractions included eating at a table service restaurant, eating watermelon, eating chicken, pork, beef, or iceberg lettuce prepared in a restaurant, eating exotic fruit, taking acid-reducing medication, and living or working on or visiting a farm. Significant exposures with high individual-level risk (odds ratio >10) among those >1 year of age who did not travel internationally were all from farm animal environments. To markedly decrease the number of STEC-related illnesses, prevention measures should focus on decreasing contamination of produce and improving the safety of foods prepared in restaurants.

Isolation and Characterization of Non-O157 Shiga Toxin–Producing Escherichia coli in Foods Sold at Retail Markets in China

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are significant foodborne pathogens that can cause acute diarrhea in humans. This study was conducted to investigate the contamination by non-O157 STEC in different types of food sold at retail markets in the People's Republic of China and to characterize non-O157 STEC strains. From May 2012 to April 2014, 1,200 retail food samples were collected from markets in 24 cities in China. Forty-four non-O157 isolates were recovered from 43 STEC-positive samples. Of the isolates, 22 and 19 carried the stx1 and stx2 genes, respectively, and 3 harbored both stx1 and stx2. stx1a and stx2a were the most prevalent stx subtypes. Other virulence genes, ent, hlyA, astA, eae, espB, iha, subAB, and tia, were commonly detected. Diverse O serogroups were identified among these isolates. Multilocus sequence typing indicated the high genetic diversity of the isolates. Thirty-two sequence types (STs) were identified among the 44 isolates, with ST383 (9.09%), ST134 (6.82%), and ST91 (6.82%) the most prevalent. Nine new STs were found. The isolates had a high prevalence of resistance to cephalothin, ampicillin, tetracycline, trimethoprim-sulfamethoxazole, nalidixic acid, streptomycin, and chloramphenicol. Twenty isolates (45.45%) were resistant to at least three antibiotics. This study provides updated surveillance data for non-O157 STEC isolates from foods sold at retail markets. Virulent and multidrug-resistant non-O57 STEC strains were isolated from all types of food. Our findings highlight the need for increased monitoring of non-O157 STEC in retail foods.

Persistence of Non-O157 Shiga Toxin–Producing Escherichia coli in Dairy Compost during Storage

Dairy compost with 20, 30, or 40% moisture content (MC) was inoculated with a mixture of six non-O157 Shiga toxin-producing Escherichia coli (STEC) serovars at a final concentration of 5.1 log CFU/g and then stored at 22 and 4°C for 125 days. Six storage conditions-4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC-were investigated for the persistence of non-O157 STEC in the dairy compost. During the entire storage, fluctuations in indigenous mesophilic bacterial levels were observed within the first 28 days of storage. After inoculation, the non-O157 STEC population increased 0.69 and 0.79 log CFU/g in the dairy compost with 30 and 40% MC at 22°C within the first day, respectively; for all other storage conditions, the pathogen population decreased rapidly. After the 125-day storage, the reductions of non-O157 STEC for 4°C and 20% MC, 4°C and 30% MC, 4°C and 40% MC, 22°C and 20% MC, 22°C and 30% MC, and 22°C and 40% MC storage conditions were >4.52, >4.55, 3.89, >4.61, 3.60, and 3.17 log CFU/g, respectively. All the survival curves showed an extensive tail, indicating non-O157 STEC can survive at least for 125 days in the dairy compost. The survival data were analyzed with log-linear with tailing and Weibull models. Compared with the log-linear with tailing model, the Weibull model was found to be a better choice for predicting the survival of non-O157 STEC in dairy compost owing to a high overall R2 value (0.8738 to 0.9909). The decay rate of non-O157 STEC was higher in dairy compost stored at 4°C compared with at 22°C, and the same trend was found for the compost with 40% MC versus 20% MC. In addition, two non-O157 STEC serotypes (STEC O145 and O45) were detected on the last day of the longitudinal study and may deserve special attention in the Big 6 STEC group. Our results have provided scientific data for risk assessment of the microbiological safety of dairy compost to control non-O157 STEC during subsequent storage of dairy compost.

Molecular Characterization of Escherichia coli O157:H7 and non-O157 Shiga Toxin Producing E. coli from Retail Meat and Humans

Molecular Characterization of Escherichia coli O157:H7 and non-O157 Shiga Toxin Producing E. coli from Retail Meat and Humans

Antimicrobial Drug-Resistant Shiga Toxin-Producing Escherichia coli Infections, Michigan, USA.

High frequencies of antimicrobial drug resistance were observed in O157 and non-O157 Shiga toxin–producing E. coli strains recovered from patients in Michigan during 2010–2014. Resistance was more common in non-O157 strains and independently associated with hospitalization, indicating that resistance could contribute to more severe disease outcomes.

Molecular Characterization of Escherichia coli O157:H7 and non-O157 Shiga Toxin Producing E. coli from Retail Meat and Humans

A total of 550 meat samples (300 minced beef and 250 chicken meat) marketed in Zagazig City, Sharkia Governorate, Egypt, as well as 150 human stool samples were examined for Shiga toxin producing E. coli . Results revealed that the isolation rates of E. coli O157:H7 versus non- O157 were 1.7% versus 2.3% in minced beef, 0.8% versus 2% in chicken meat and 0.7% versus 2.7% in human stools. Other identified serotypes were including O111:H8 (25%), O26:H11 (20.8%), O55:H7 (16.7%) and O113:H21 (4.2%). Virulence associated genes were identified in E. coli serotypes, stx 1 and stx 2 were characterized in 16.7% and 62.5% of the isolates, while, eae A and hly A genes were identified in 50% and 70.8% of the examined serotypes, respectively. Genotyping of E. coli O157:H7 serotype from different sources using Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) fingerprinting revealed heterogenicity of the isolates, however, human and minced beef isolates were grouped in the same cluster indicating potential transmission of infection from contaminated beef to human consumers. In conclusion, ERIC-PCR is a highly discriminatory, reliable and cost-effective tool for tracing sources of infection with bacteria. Public health education and application of strict hygienic measures during slaughtering, transportation and preparation of meat are essential to minimize the risk of contamination and transmission of infection to consumers.

Growth of Escherichia coli O157:H7, Non-O157 Shiga Toxin–Producing Escherichia coli, and Salmonella in Water and Hydroponic Fertilizer Solutions

The desire for local, fresh produce year round is driving the growth of hydroponic growing systems in the United States. Many food crops, such as leafy greens and culinary herbs, grown within hydroponics systems have their root systems submerged in recirculating nutrient-dense fertilizer solutions from planting through harvest. If a foodborne pathogen were introduced into this water system, the risk of contamination to the entire crop would be high. Hence, this study was designed to determine whether Escherichia coli O157:H7, non-O157 Shiga toxin-producing E. coli , and Salmonella were able to survive and reproduce in two common hydroponic fertilizer solutions and in water or whether the bacteria would be killed or suppressed by the fertilizer solutions. All the pathogens grew by 1 to 6 log CFU/ml over a 24-h period, depending on the solution. E. coli O157:H7 reached higher levels in the fertilizer solution with plants (3.12 log CFU/ml), whereas non-O157 Shiga toxin-producing E. coli and Salmonella reached higher levels in the fertilizer solution without plants (1.36 to 3.77 log CFU/ml). The foodborne pathogens evaluated here survived for 24 h in the fertilizer solution, and populations grew more rapidly in these solutions than in plain water. Therefore, human pathogens entering the fertilizer solution tanks in hydroponic systems would be expected to rapidly propagate and spread throughout the system and potentially contaminate the entire crop.

Disinfectant and Antimicrobial Susceptibility Profiles of the Big Six Non-O157 Shiga Toxin–Producing Escherichia coli Strains from Food Animals and Humans

The disinfectant and antimicrobial susceptibility profiles of 138 non-O157 Shiga toxin-producing Escherichia coli strains (STECs) from food animals and humans were determined. Antimicrobial resistance (AMR) was moderate (39.1% of strains) in response to 15 antimicrobial agents. Animal strains had a lower AMR prevalence (35.6%) than did human strains (43.9%) but a higher prevalence of the resistance profile GEN-KAN-TET. A decreasing prevalence of AMR was found among animal strains from serogroups O45 > O145 > O121 > O111 > O26 > O103 and among human strains from serogroups O145 > O103 > O26 > O111 > O121 > O45. One animal strain from serogroups O121 and O145 and one human strain from serogroup O26 had extensive drug resistance. A high prevalence of AMR in animal O45 and O121 strains and no resistance or a low prevalence of resistance in human strains from these serogroups suggests a source other than food animals for human exposure to these strains. Among the 24 disinfectants evaluated, all strains were susceptible to triclosan. Animal strains had a higher prevalence of resistance to chlorhexidine than did human strains. Both animal and human strains had a similar low prevalence of low-level benzalkonium chloride resistance, and animal and human strains had similar susceptibility profiles for most other disinfectants. Benzyldimethylammonium chlorides and C10AC were the primary active components in disinfectants DC&R and P-128, respectively, against non-O157 STECs. A disinfectant FS512 MIC ≥ 8 μg/ml was more prevalent among animal O121 strains (61.5%) than among human O121 strains (25%), which may also suggest a source of human exposure to STEC O121 other than food animals. Bacterial inhibition was not dependent solely on pH but was correlated with the presence of dissociated organic acid species and some undissociated acids.

위합성용액에서 과일주스에 노출한 Non-O157 Shiga Toxin-Producing Escherichia coli의 산 저항성 평가

다양한 환경에서 분리된 시가독소 생산성 대장균(Shiga toxin-producing E. coli, STEC, n=18)을 초산혼합용액(AAS; 400 mM, pH 3.2, 30°C)에 노출한 후 산 저항성을 측정하였다. 또한, 선정된 4종류의 non-O157:H7 STEC균을 사과주스, 파인애플주스, 오렌지주스, 딸기주스(pH 3.8)에 정봉하여 4°C와 20°C에서 24시간 산 적응시킨 후 위합성용액(SGF, pH 1.5)에서 2시간 동안 생존능력을 평가하였다. Non-O157:H7 STEC를 AAS에 노출했을 때 O111 혈청형의 STEC는 평균 0.12 log CFU/mL 감소하여 다른 혈청형에 비하여 가장 강한 산 저항성을 나타냈고 O157:H7 STEC와 유의적 차이가 없었으며(P0.05), O26 혈청형의 STEC는 가장 민감한 것으로 나타났다. 반면, AAS에 glutamic acid를 첨가하였을 경우 모든 STEC는 혈청형과 관계없이 초산에 매우 강한 저항성을 나타내었다(P0.05). SGF에서 생존능력을 측정한 결과, 06E0218(O157:H7)은 다른 non-O157:H7 STEC 균들보다 생존능력이 낮았고 03-4669(O145:NM)가 가장 강한 생존능력을 나타내었다. 한편, 과일주스 중에서는 파인애플주스에 산 적응된 STEC가 SGF에 가장 강한 생존능력을 나타내었다. 4°C의 과일주스에 STEC를 산 적응시켰을 경우 20°C보다 SGF에 대한 생존능력이 현저하게 높았다(P0.05). 따라서 과일주스에 의한 non-O157:H7 STEC의 산 적응력 증가는 위장관 내 생존율 및 식중독 발생을 높일 수 있으므로 이에 대한 적절한 연구와 안전관리 옵션을 제공할 필요가 있을 것으로 판단된다.

Biofilm Formation, Virulence Gene Profiles, and Antimicrobial Resistance of Nine Serogroups of Non-O157 Shiga Toxin-Producing Escherichia coli.

The objectives of this study were to characterize the phenotype and genotype of 36 non-O157 Shiga toxin-producing Escherichia coli (STEC) strains isolated from humans, ovines, or bovines, including the top 6 (O26, O45, O103, O111, O121, and O145) and three other serogroups implicated in serious illness (O91, O113, and O128). Biofilms were formed by all strains with intermediate to strong biofilm producers (n = 24) more common at 22°C than at 37°C (p < 0.001) and 48 and 72 h (p < 0.001) than 24 h of incubation time. Biofilm-forming potential differed by serogroup and origin with O113 and human strains exhibiting the highest potential (p < 0.001). Biofilm-associated genes, csgA/csgD/crl/fimH (100%), flu (94%), rpoS (92%), ehaA(α) (89%), and cah (72%), were most prevalent, while mlrA (22%) and ehaA(β) (14%) were least prevalent, although there was no clear compliment of genes associated with strains exhibiting the greatest biofilm-forming capacity. Among 12 virulence genes screened, iha and ehxA were present in 92% of the strains. The occurrence of stx1 in the top 6 serogroups (8/12, 67%) did not differ (p = 0.8) from other serogroups (17/24, 71%), but stx2 was less likely (confidence interval [CI] = 0.14-1.12; p = 0.04) to be in the former (9/24, 38%) than the latter (9/12, 75%). Excluding serogroups, O91 and O121, at least one strain per serogroup was resistant to between three and six antimicrobials. Streptomycin (31%), sulfisoxazole (31%), and tetracycline (25%) resistance was most common and was 35-50% less likely (p < 0.05) in human than animal strains. All non-O157 STEC strains were able to form biofilms on an abiotic surface, with some exhibiting resistance to multiple antimicrobials. Potential as a reservoir of antimicrobial resistance genes may be another hazard of biofilms in food-processing plants. As a result, future strategies to control these pathogens may include measures to prevent biofilms.

Improving the Enrichment and Plating Methods for Rapid Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in Dairy Compost

A culture method to detect non-O157 Shiga toxin-producing Escherichia coli (STEC) was optimized in this study. The finished dairy compost with 30% moisture content was inoculated with a cocktail of six non-O157 STEC serovars at initial concentrations of 1 to 100 CFU/g. Afterward, non-O157 STEC cells in the inoculated dairy compost were enriched by four methods, followed by plating onto cefixime-tellurite sorbitol MacConkey agar supplemented with 5 mg/liter novobiocin (CTNSMAC) and modified Rainbow agar containing 5 mg/liter novobiocin, 0.05 mg/liter cefixime trihydrate, and 0.15 mg/liter potassium tellurite (mRBA). Immunomagnetic bead separation (IMS) was used to compare the cell concentration of individual non-O157 STEC serotypes after enrichment. There was no significant difference (P > 0.05) between CTN-SMAC and mRBA for non-O157 STEC enumeration. The single-step selective enrichment recovered ca. 0.54 log CFU/g more cells (ca. 0.41 log CFU/g for compost-adapted cells) (P < 0.05) compared with the two-step enrichment. Furthermore, the duration of the process to detect non-O157 STEC from dairy compost by selective enrichment, followed by IMS, was optimized. Among six non-O157 STEC serotypes, serotypes O111, O45, and O145 reached the highest cell density after enrichment in dairy compost, and the cell populations reached 7.3, 7.4, and 7.8 log CFU/g within 16 h of incubation, respectively. In contrast, without an enrichment step, the IMS detection limit of individual non-O157 STEC serovars ranged from 3.15 to 4.15 log CFU/g in dairy compost. These results demonstrate that low levels of non-O157 STEC can be detected within 2 days from dairy compost by using a culture method with an optimized enrichment procedure followed by IMS.

Escherichia coli O157:H7 strains harbor at least three distinct sequence types of Shiga toxin 2a-converting phages

BackgroundShiga toxin-producing Escherichia coli O157:H7 is a foodborne pathogen that causes severe human diseases including hemolytic uremic syndrome (HUS). The virulence factor that mediates HUS, Shiga toxin (Stx), is encoded within the genome of a lambdoid prophage. Although draft sequences are publicly available for a large number of E. coli O157:H7 strains, the high sequence similarity of stx-converting bacteriophages with other lambdoid prophages poses challenges to accurately assess the organization and plasticity among stx-converting phages due to assembly difficulties.MethodsTo further explore genome plasticity of stx-converting prophages, we enriched phage DNA from 45 ciprofloxacin-induced cultures for subsequent 454 pyrosequencing to facilitate assembly of the complete phage genomes. In total, 22 stx2a-converting phage genomes were closed.ResultsComparison of the genomes distinguished nine distinct phage sequence types (PSTs) delineated by variation in obtained sequences, such as single nucleotide polymorphisms (SNPs) and insertion sequence element prevalence and location. These nine PSTs formed three distinct clusters, designated as PST1, PST2 and PST3. The PST2 cluster, identified in two clade 8 strains, was related to stx2a-converting phages previously identified in non-O157 Shiga-toxin producing E. coli (STEC) strains associated with a high incidence of HUS. The PST1 cluster contained phages related to those from E. coli O157:H7 strain Sakai (lineage I, clade 1), and PST3 contained a single phage that was distinct from the rest but most related to the phage from E. coli O157:H7 strain EC4115 (lineage I/II, clade 8). Five strains carried identical stx2a-converting phages (PST1-1) integrated at the same chromosomal locus, but these strains produced different levels of Stx2.ConclusionThe stx2a-converting phages of E. coli O157:H7 can be categorized into at least three phage types. Diversification within a phage type is mainly driven by IS629 and by a small number of SNPs. Polymorphisms between phage genomes may help explain differences in Stx2a production between strains, however our data indicates that genes encoded external to the phage affect toxin production as well.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1934-1) contains supplementary material, which is available to authorized users.

Detection of Non-O157 Shiga Toxin–Producing Escherichia coli in 375 Grams of Beef Trim Enrichments across Multiple Commercial PCR Detection Platforms

Although serotype O157:H7 remains the pathogenic Shiga toxin–producing Escherichia coli (STEC) of primary concern worldwide, some focus in the United States has shifted to six particular non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145). Some of these serogroups have also emerged as concerns elsewhere around the world, including Europe. The objective of this work was to compare commercial detection methods with the U.S. Department of Agriculture (USDA) reference method for detection of non-O157 STEC in 375 g of beef trim using a limit of detection study design. Overall, the commercial platforms performed well, showing similar levels of sensitivity for detection of presumptive positives for O45, O26, O103, and O121 (PCR screen results only). For O111, one method that utilizes an integrated immunomagnetic separation and PCR approach was more sensitive than a PCR-only screen approach. Additionally, one commercial method showed more presumptive and confirmed positives overall. Use of an immunomagnetic separation tool, such as antibody-coated beads, aided considerably with the confirmation procedures and is an important step when confirming suspect samples. A secondary goal of this study was to evaluate isolation and International Organization for Standardization confirmation protocols used in Europe compared with strategies provided by the USDA Microbiology Laboratory Guidebook (MLG). Generally, results from the USDA confirmation plates (modified Rainbow agar) were better than the European Union confirmation plates (MacConkey agar with or without rhamnose). In summary, detection of non-O157 STEC in 375 g of beef trim can be performed by any of the three methods on the market evaluated in the study.

Growth of Strains of the Major Non-O157 Shiga Toxin–Producing Escherichia coli Serogroups Is Not Different from Growth of Escherichia coli O157:H7 in Neutral Broth (pH 7.4) and Acidified Broth (pH 5.6) at 10°C

Understanding the survival and growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) strains under cold temperatures may be important for protecting public health. The aim of this study was to compare the growth of three strains of each of the major non-O157 STEC serogroups (O26, O45, O103, O111, O121, and O145) with the growth of six O157:H7 STEC strains in broth at 10°C. Brain heart infusion broth (BHIB; pH 7.4) was inoculated with a single strain of stationary-phase STEC culture to produce a starting inoculum of ∼10(6) CFU/ml and stored at 10°C for up to 96 h (three trials per strain). Populations over time were fitted to the Baranyi and Roberts model, and lag-phase duration (LPD) and growth rate were calculated for each strain per trial. Average LPD ranged from 9.2 to 32.8 h for non-O157 STEC and from 10.5 to 17.2 h for O157 STEC. One strain of O26 STEC had a significantly longer LPD (P < 0.05) than did the other strains (32.8 h); otherwise, no significant differences were noted (P > 0.05). Growth rate ranged from 0.031 to 0.060 log CFU/ml/h for non-O157 STEC strains and from 0.034 to 0.046 log CFU/ml/h for O157 STEC strains. No significant difference in growth rate was noted among strains in BHIB at pH 7.4 and 10°C. In subsequent trials, growth of a single strain of each of the non-O157 STEC serogroups was compared with growth of four acid-tolerant O157 STEC strains in BHIB acidified to pH 5.6 with lactic acid. Acidification generally increased LPD and decreased the growth rate for strains, although the effect was variable and not significant. These findings suggest that growth patterns for strains of non-O157 STEC are similar to those for strains of O157 STEC in neutral and pH 5.6 BHIB at 10°C. Further research is needed to determine whether strains behave similarly in meat systems.

Pathogenicity Islands in Shiga Toxin–ProducingEscherichia coliO26, O103, and O111 Isolates from Humans and Animals

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are increasingly recognized as foodborne pathogens worldwide. Serogroups O26, O111, and O103 cause most known outbreaks related to non-O157 STEC. Pathogenicity islands (PAIs) play a major role in the evolution of STEC pathogenicity. To determine the distribution of PAIs often associated with highly virulent STECs (OI-122, OI-43/48, OI-57, and high pathogenicity islands) among STEC O26, O103, and O111, a collection of STEC O26 (n=45), O103 (n=29), and O111 (n=52) from humans and animals were included in this study. Pulsed-field gel electrophoresis (PFGE) with XbaI digestion was used to characterize the clonal relationship of the strains. In addition, a polymerase chain reaction-restriction fragment length polymorphism assay was used to determine eae subtypes. Additional virulence genes on PAIs were identified using specific PCR assays, including OI-122: pagC, sen, efa-1, efa-2, and nleB; OI-43/48: terC, ureC, iha, and aidA-1; OI-57: nleG2-3, nleG5-2, and nleG6-2; and HPI: fyuA and irp2. A PFGE dendrogram demonstrated that instead of clustering together with strains from the same O type (O111:H8), the O111:H11 (n=14) strains clustered together with strains of the same H type (O26:H11, n=45). In addition, O26:H11 and O111:H11 strains carried eae subtype β, whereas O111:H8 strains had eae γ2/θ. The O26:H11 and O111:H11 stains contained an incomplete OI-122 lacking pagC and a complete HPI. However, a complete OI-122 but no HPI was found in the O111:H8 strains. Additionally, aidA-1 of OI-43/48 and nleG6-2 of OI-57 were significantly associated with O26:H11 and O111:H11 strains but were almost missing in O111:H8 strains (p<0.001). This study demonstrated that H11 (O111:H11 and O26:H11) strains were closely related and may have come from the same ancestor.

Interactions of O157 and Non-O157 Shiga Toxin–ProducingEscherichia coli(STEC) Recovered from Bovine Hide and Carcass with Human Cells and Abiotic Surfaces

Different structures related to biofilm formation by Shiga toxin-producing Escherichia coli (STEC), particularly O157 strains, have been described, but there are few data regarding their involvement in non-O157 strains. The aim of this study was to determine the ability of 14 O157 and 8 non-O157 strains isolated from bovine hide and carcass to interact with biotic and abiotic surfaces and also to evaluate the role of different adhesins. Biofilm formation assays showed that four O157 and two non-O157 strains were able to adhere to glass, and that only one O157 strain adhered to polystyrene. Reverse transcriptase-polymerase chain reaction was carried out using biofilm-forming strains to determine the expression of antigen 43 (Ag43), curli, type 1 fimbriae, STEC autotransporter contributing to biofilm formation (Sab), calcium-binding antigen 43 homologue (Cah), and autotransporter protein of enterohemorrhagic E. coli (EhaA). Most of these structures were expressed under biofilm conditions. However, the lack of Ag43 in one non-O157 strain, as well as Cah and EhaA in two O157 strains, suggests that other adhesins are involved in biofilm formation in these strains. Despite the fact that adherence to HeLa cells was detected in 20 strains (91%), it was not possible to correlate biofilm formation with adherence patterns. Invasiveness in T84 and Caco-2 cells was observed in four and three O157 strains, respectively. Altogether, we showed that there are different sets of genes involved in the interactions of STEC with biotic and abiotic surfaces. Interestingly, one O157 strain that was able to form biofilm on both glass and polystyrene also adhered to and invaded human cells, indicating an important route for its persistence in the environment and interaction with the host. Additionally, the ability of non-O157 strains not carrying the LEE pathogenicity island to form biofilm highlights an industrial and health problem that cannot be neglected.

Evidence of Non-O157 Shiga Toxin—Producing Escherichia coli in the Feces of Meat Goats at a U.S. Slaughter Plant

Shiga toxin-producing Escherichia coli (STEC) are important human pathogens, and attention to non-O157 serogroups has increased in recent years. Although cattle are normally considered the primary reservoir for STEC, recent illnesses associated with goat contact have indicated that these animals are important potential reservoirs for the organisms. The prevalence of STEC, particularly non-O157 serogroups, in U.S. goats has not been well described. Our objective was to determine the prevalence of six major non-O157 STEC serogroups in the feces of meat goats. Rectal contents from 296 goats were collected postevisceration at a slaughter plant in the southeastern United States over 9 days during a 12-week period from August through October 2012. Samples were enriched in E. coli broth, and DNA was extracted and used as template in an 11-gene multiplex PCR that detected six non-O157 serogroups (O26, O45, O103, O121, O111, and O145) and virulence genes. Samples were considered positive when at least one non-O157 STEC serotype was present with either stx₁ or stx₂. All six non-O157 serogroups were detected by PCR in our samples, and 14.5% of samples were positive for at least one serogroup. Prevalence of O26 was highest, with 6.4% of goat fecal samples positive. The prevalence of O45 was 3.4%, O103 was 4.4%, O111 was 4.1%, O121 was 1.4%, and O145 was 3.0%. Twenty-two (7.4%) of 296 fecal samples had more than one non-O157 serogroup detected in the feces. Two samples had evidence of three non-O157 STEC serogroups. Goats appear to be an important reservoir for non-O157 STEC, and further work to understand the characteristics, epidemiology, and ecology of STEC in these animals is warranted.

Behavior of Non-O157 Shiga Toxin–Producing Escherichia coli, Enteroinvasive E. coli, Enteropathogenic E. coli, and Enterotoxigenic E. coli Strains on Alfalfa Sprouts

Data about the behavior of non-O157 Shiga toxin-producing Escherichia coli (non-O157 STEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), and enteropathogenic E. coli (EPEC) on seeds and alfalfa sprouts are not available. The behavior of STEC, EIEC, ETEC, and EPEC was determined during germination and sprouting of alfalfa seeds at 20 ± 2°C and 30 ± 2°C and on alfalfa sprouts at 3 ± 2°C. When alfalfa seeds were inoculated with STEC, EIEC, ETEC, or EPEC strains, all these diarrheagenic E. coli pathotypes (DEPs) grew during germination and sprouting of seeds, reaching counts of approximately 5 and 6 log CFU/g after 1 day at 20 ± 2°C and 30 ± 2°C, respectively. However, when the sprouts were inoculated after 1 day of seed germination and stored at 20 ± 2°C or 30 ± 2°C, no growth was observed for any DEP during sprouting at 20 ± 2°C or 30 ± 2°C for 9 days. Refrigeration reduced significantly (P < 0.0.5) the number of viable DEPs on sprouts after 20 days in storage; nevertheless, these decreases have no practical significance for the safety of the sprouts.

Rapid Identification and Differentiation of Non-O157 Shiga Toxin–ProducingEscherichia coliUsing Polymerase Chain Reaction Coupled to Electrospray Ionization Mass Spectrometry

A polymerase chain reaction (PCR)-mass spectroscopy assay was developed to identify non-O157 Shiga toxin-producing Escherichia coli (STEC) with Plex-ID biosensor system, a platform identifying short PCR amplicons by specific base compositions. This assay simultaneously amplifies five fragments of two housekeeping genes, two subunits of stx2 gene, and four other virulence genes of STEC. A total of 164 well-characterized STEC isolates were examined with the assay to build a DNA base composition database. Another panel of 108 diverse STEC isolates was tested with the established database to evaluate the assay's identification capability. Among the 108 isolates, the assay specificity was 100% for three (stx1, eae, and aggA) out of five tested virulence genes, but 99% for stx2 and 96% for hlyA, respectively. Main stx1/stx2 subtypes and multiple alleles of stx1/stx2 could be differentiated. The assay successfully identified several clinically significant serotypes, including O91:H14, O103:H25, O145:H28/NM, O113:H21, and O104:H4. Meanwhile, it was able to group isolates with different levels of pathogenic potential. The results suggest that this high-throughput method may be useful in clinical and regulatory laboratories for STEC identification, particularly strains with increased pathogenic potential.