Shiga Toxin Producing Research Articles

LAMBEDA PHAGE

Background: Recognition of bacteriophages in many aspects plays an important role such as controlling the number and variation of bacteria and participation in horizontal gene transfer, which is an important process in bacterial evolution. Bacteriophages use small proteins to take over the host molecular machinery and thus interfere with central metabolic processes in infected bacteria. In general, phages inhibit or reverse host transcription to transcribe their genome. Mechanical and structural studies of phage host transcription may lead to the development of new antibacterial agents. Result: The result shows that phage vB_Eco4M-7 must be a lytic virus. This was confirmed by monitoring faglitic development by a one-step growth test. In addition, phage of relatively small uniform plaques (1 mm in diameter) occur without lysogenesis. Electron microscopic analysis showed that vB_Eco4M-7 belongs to the family Myoviridae. Based on mass spectrometric analysis, including the fragmentation pattern of unique peptides, 33 vB_Eco4M-7 phage proteins are assigned to the annotated reading frames. The results indicate that the phage studied is a potential candidate for phage treatment and / or food protection against E. coli, which produce Shiga toxin, as most of these strains belong to the O157 serotype. Conclusion: In general, phages inhibit or reverse host transcription to transcribe their genome. Mechanical and structural studies of phage host transcription may lead to the development of new antibacterial agents. The result shows that phage vB_Eco4M-7 must be a lytic virus. This was confirmed by monitoring faglitic development by a one-step growth test.

Occurrence of Virulence Genes and Antimicrobial Resistance of E. coli O157:H7 Isolated from the Beef Carcass of Bahir Dar City, Ethiopia.

E. coli O157:H7 is one of the most virulent foodborne pathogens. The aim of this study was to isolate E. coli O157:H7, determine virulence genes carried by the organism, and assess the antimicrobial susceptibility pattern of the isolates from beef carcass samples at Bahir Dar city. Swab samples (n = 280) were collected from the carcass of cattle slaughtered at the abattoir and processed using sorbitol MacConkey agar supplemented with cefixime telluride and confirmed with latex agglutination test. A polymerase chain reaction was performed on isolates for the detection of virulence genes stx1, stx2, hlyA, and eae. Antimicrobial susceptibility testing was performed using the disk diffusion method. Of 280 samples processed, 25 (8.9%) isolates were positive. Out of 25 isolates subjected for molecular detection, 8 (32%) and 14 (56%) isolates possessed stx1 and stx2 genes, respectively; from those, 5 (20%) isolates had both genes for the production of Shiga toxins. Compared from other virulent genes relatively higher proportion of 18 (72%) isolates carried the hlyA gene. Only 5 (2%) isolates were positive for eae. Resistance was detected in all 25 (100%) isolates and 3 (12%) against clindamycin and trimethoprim, respectively. This study result highlights the potential threat to public health. The abattoir workers need to be aware about the pathogen and should follow appropriate practices to prevent contamination of meat intended for human consumption.

The effect of two ribonucleases on the production of Shiga toxin and stx-bearing bacteriophages in Enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.

Characterization of Shiga Toxin 2a Encoding Bacteriophages Isolated From High-Virulent O145:H25 Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) may cause severe disease mainly due to the ability to produce Shiga toxins (Stx) encoded on bacteriophages. In Norway, more than 30% of the reported cases with STEC O145:H25 develop hemolytic uremic syndrome (HUS), and most cases, with known travel history, acquired the infection domestically. To describe phage characteristics associated with high virulence, we extracted the Stx2a phage sequences from eight clinical Norwegian O145:H25 STEC to conduct in-depth molecular characterization using long and short read sequencing. The Stx2a phages were annotated, characterized, and compared with previously published Stx2a phages isolated from STEC of different serotypes. The Norwegian O145:H25 Stx2a phages showed high sequence identity (>99%) with 100% coverage. The Stx2a phages were located at the integration site yciD, were approximately 45 kbp long, and harbored several virulence-associated genes, in addition to stx2a, such as nanS and nleC. We observed high sequence identity (>98%) and coverage (≥94%) between Norwegian O145:H25 Stx2a phages and publicly available Stx2a phages from O145:H25 and O145:H28 STEC, isolated from HUS cases in the USA and a hemorrhagic diarrhea case from Japan, respectively. However, low similarity was seen when comparing the Norwegian O145:H25 Stx2a phage to Stx2a phages from STEC of other serotypes. In all the Norwegian O145:H25 STEC, we identified a second phage or remnants of a phage (a shadow phage, 61 kbp) inserted at the same integration site as the Stx2a phage. The shadow phage shared similarity with the Stx2a phage, but lacked stx2a and harbored effector genes not present in the Stx2a phage. We identified a conserved Stx2a phage among the Norwegian O145:H25 STEC that shared integration site with a shadow phage in all isolates. Both phage and shadow phage harbored several virulence-associated genes that may contribute to the increased pathogenicity of O145:H25 STEC.

Characterizing the Cattle Gut Microbiome in Farms with a High and Low Prevalence of Shiga Toxin Producing Escherichia coli.

Cattle are the main reservoirs of Shiga toxin producing Escherichia coli (STEC), a major foodborne pathogen associated with acute enteric disease and hemolytic–uremic syndrome in humans. A total of 397 beef and dairy cattle from 5 farms were included in this study, of which 660 samples were collected for 16S rRNA gene sequencing. The microbiota of farms with a high-STEC prevalence (HSP) had greater richness compared to those of farms with a low-STEC prevalence (LSP). Longitudinal analyses showed STEC-shedders from LSP farms had higher microbiome diversity; meanwhile, changes in the microbiome composition in HSP farms were independent of the STEC shedding status. Most of the bacterial genera associated with STEC shedding in dairy farms were also correlated with differences in the percentage of forage in diet and risk factors of STEC carriage such as days in milk, number of lactations, and warm temperatures. Identifying factors that alter the gut microbiota and enable STEC colonization in livestock could lead to novel strategies to prevent fecal shedding and the subsequent transmission to humans.

Isolation and Characterization of Shiga Toxin-Producing Escherichia coli from Retail Beef Samples from Eight Provinces in China.

While Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen worldwide, data on the molecular and phylogenetic properties of STEC isolates from retail beef samples in China remain scant. Fresh retail beef samples (n = 1062) were collected from eight provinces, and STEC isolates were recovered and characterized. PCR data showed that more than 50% of the samples were stx positive, and 82 STEC isolates were recovered from 14.8% (79/535) stx-positive enriched broths. In contrast, all ciprofloxacin resistant isolates (n = 19) and 13 cefotaxime (CTX) resistant isolates were eae positive and belonged to three serotypes: O111:H8, O26:H11, or O157:H7. Point mutations in quinolone resistance-determining regions and plasmid-mediated quinolone resistance determinants were identified in 16 and 20 isolates, respectively. BlaCTX-M and a point mutation (C-42T) in ampC promoter were detected in 15 and 8 of the CTX resistant isolates, respectively. In addition, macrolide resistance gene mphA was identified in eight azithromycin resistant O111:H8 isolates and one O26:H11 isolate. Single nucleotide polymorphism analysis demonstrated that the O26 and O157 isolates had multiple origins, but the O111 isolates were closely related. Taken together, our data demonstrated that several sequence types associated with hemolytic uremic syndrome from the retail beef samples in China had developed into dangerous multidrug resistant pathogens. The resistant phenotype can facilitate their transmission among the farm animals and human beings when there is an antimicrobial selective pressure.

Survival of Salmonella and Shiga Toxin–Producing Escherichia coli and Changes in Indigenous Microbiota during Fermentation of Home-Brewed Kombucha

Survival and growth of Salmonella and Shiga toxin-producing Escherichia coli (STEC) were investigated in kombucha prepared from four brands of commercially available kombucha kits intended for use by home brewers. Changes in populations of the indigenous microbiota responsible for fermentation of kombucha were also determined. An initial population of Salmonella (6.77 log CFU/mL) decreased to below the detection limit (0.30 log CFU/mL) within 10 days in kombucha prepared from two of the test brands. Populations of 1.85 and 1.20 log CFU/mL were detected in two brands fermented for 14 days. An initial STEC population of 7.02 log CFU/mL decreased to <0.30 log CFU/mL in two brands within 14 days; 0.77 and 0.87 log CFU/mL were detected in kombucha prepared from the other two brands. Salmonella and STEC increased within 1 day in three brands of base tea used to prepare kombucha and were stable throughout 14 days of incubation. Both pathogens steadily declined in base tea prepared from one brand of kombucha kit. Inactivation of the pathogens occurred as the pH of the kombucha decreased, but a clear correlation between rates of inactivation among different brands of kits and decrease in pH was not evident. Growth and peak populations of mesophilic aerobic microorganisms, yeasts, lactic acid bacteria, and acetic acid bacteria varied depending on the kombucha kit brand. No strong evidence was found of a correlation between the behavior of Salmonella or STEC and that of any of these groups of indigenous microbiota. Results of this study show that survival of Salmonella and STEC in kombucha and base tea used to prepare kombucha is dependent on inherent differences in commercially available kombucha kits intended for use in home settings. Strict application of hygienic practices is essential for preventing contamination with Salmonella or STEC and reducing the risk of illness associated the consumption of kombucha.

Impacts of Microbial Food Safety in China and Beyond.

Impacts of Microbial Food Safety in China and Beyond.

The interactions of bacteriophage Ace and Shiga toxin-producing Escherichia coli during biocontrol.

Strictly lytic phages are considered powerful tools for biocontrol of foodborne pathogens. Safety issues needed to be addressed for the biocontrol of Shiga toxin-producing Escherichia coli (STEC) include: lysogenic conversion, Shiga toxin production through phage induction, and emergence/proliferation of bacteriophage insensitive mutants (BIMs). To address these issues, two new lytic phages, vB_EcoS_Ace (Ace) and vB_EcoM_Shy (Shy), were isolated and characterized for life cycle, genome sequence and annotation, pH stability and efficacy at controlling STEC growth. Ace was efficient in controlling host planktonic cells and did not stimulate the production of the Stx prophage or Shiga toxin. A single dose of phage did not lead to the selection of BIMs. However, when reintroduced, BIMs were detected after 24 h of incubation. The gain of resistance was associated with lower virulence, as a subset of BIMs failed to agglutinate with O157-specific antibody and were more sensitive to human serum complement. BIM's biofilm formation capacity and susceptibility to disinfectants was equal to that of the wild-type strain. Overall, this work demonstrated that phage Ace is a safe biocontrol agent against STEC contamination and that the burden of BIM emergence did not represent a greater risk in environmental persistence and human pathogenicity.

Zoonotic Potential of Escherichia Coli in Poultry Intestinal Contents in Ismailia City, Egypt with Special Reference to Shiga Toxin-Producing (STEC) Strains

Avian colibacillosis can affect birds of all ages. It is now one of the major causes of economic losses in the poultry industry, as well as a public health risk all over the world.This study aimed to determine the occurrence of E. coli in market-age poultry and the risk of its zoonotic infections in Ismailia city, Egypt. A total of 350 samples were collected from the intestinal contents of the slaughtered poultry (100 farm chickens, 100 backyard chickens, 50 ducks, 50 geese, and 50 turkeys) in Ismailia City. On the other hand, a total of 200 samples were collected from humans at outpatient clinics (100 stool samples and 100 urine samples) from the same investigated area. Samples were examined using bacteriological and molecular examination for genus-specific and virulence gene detection. Results revealed that the isolation rates of E. coli were 70% in farm chickens and turkeys, 88% in backyard chickens, 86% in domestic ducks, 72% in geese. Of avian E. coli isolates, the rates of Shiga toxin-producing E. coli (STEC) strains were 91.4%, 96.6%, 90.9%, 41.7%, and 51.4%% in farm chickens, backyard chickens, ducks, geese, and turkey respectively. In humans, the isolation rate of E. coli was 42% in urine samples and 66% in stool samples. The human STEC isolates were higher in stool samples (26%) than in urine samples (6%). The isolation rates of E. coli were significantly higher in persons who were in contact with poultry than in persons who were not in contact with poultry (p ≤ 0.01). In conclusion, the high isolation rates of STEC and detection with similarities of some E. coli virulence genes (It, St, eaeA, Stx1, and Stx2 genes) from poultry intestinal contents and human samples indicated a significant risk of zoonotic transmission of E. coli via food chain in the investigated area.

Validation of Commonly Used Antimicrobial Interventions on Bob Veal Carcasses for Reducing Shiga Toxin–Producing Escherichia coli Surrogate Populations

Ruminants are natural reservoirs of Shiga toxin-producing Escherichia coli (STEC), and the STEC can be easily transferred to carcasses during the conversion of animals to meat. Three experiments were conducted to validate the efficacy of lactic acid (LA; 4%), peroxyacetic acid (PAA; 300 ppm), and hot water (HW; 80°C) for their individual or combined abilities to reduce STEC surrogates on bob veal carcasses pre- and postchill and through fabrication. In experiment 1, hot carcasses (n = 9) were inoculated with a five-strain cocktail (ca. 8 log CFU/mL) containing rifampin-resistant surrogate E. coli (BAA-1427, BAA-1428, BAA-1429, BAA-1430, and BAA-1431) and then treated with HW, LA, or PAA. Carcasses were then chilled (0 ± 1°C; 24 h) and split in half, and each side was treated with either LA or PAA. In experiment 2, hot carcasses (n = 3) were inoculated and chilled (24 h). After 24 h, the carcasses were split, and each side was treated with either LA or PAA. For experiment 3, carcasses (n = 3) were chilled for 24 h, split, inoculated, and treated with either LA or PAA. After chilling, carcasses from all three experiments were fabricated to subprimals and the cut surfaces were sampled to determine the translocation of bacteria. Experiment 1 showed that LA+LA was the most effective (P ≤ 0.05) treatment for reducing surrogate E. coli on veal. In experiments 2 and 3, LA and PAA were similar (P > 0.05) in their abilities to reduce E. coli on chilled veal carcasses. In experiments 1 and 2, all antimicrobial treatments resulted in undetectable levels (<0.2 log CFU/cm2) of surrogate E. coli on cut surfaces after fabrication, whereas low levels (1.7 and 1.0 log CFU/cm2 for LA and PAA, respectively) were observed in experiment 3. Of the antimicrobial interventions utilized, LA was more effective for reducing STEC surrogate populations on veal carcasses, pre- and/or postchill.

Bloody Diarrhea and Shiga Toxin–Producing Escherichia coli Hemolytic Uremic Syndrome in Children: Data from the ItalKid-HUS Network

To analyze the results of an enhanced laboratory-surveillance protocol for bloody diarrhea aimed at identifying children with Shiga toxin-producing Escherichia coli (STEC) infection early in the course of the disease toward the early identification and management of patients with hemolytic uremic syndrome (HUS). The study (2010-2019) involved a referral population of 2.3 million children. Stool samples of patients with bloody diarrhea were screened for Shiga toxin (Stx) genes. Positive patients were rehydrated and monitored for hemoglobinuria until diarrhea resolved or STEC-HUS was diagnosed. A total of 4767 children were screened; 214 (4.5%) were positive for either Stx1 (29.0%) or Stx2 (45.3%) or both Stx1+2 (25.7%); 34 patients (15.9%) developed STEC-HUS (0.71% of bloody diarrheas). Hemoglobinuria was present in all patients with HUS. Patients with Stx2 alone showed a greater risk of STEC-HUS (23.7% vs 12.7%) and none of the patients with Stx1 alone developed HUS. During the same period of time, 95 other patients were diagnosed STEC-HUS but were not captured by the screening program (26 had nonbloody diarrhea, 11 came from areas not covered by the screening program, and 58 had not been referred to the screening program, although they did meet the inclusion criteria). At HUS presentation, serum creatinine of patients identified by screening was significantly lower compared with that of the remaining patients (median 0.9 vs 1.51mg/dL). Nearly 1% of children with bloody diarrhea developed STEC-HUS, and its diagnosis was anticipated by the screening program for Stx. The screening of bloody diarrhea for Stx is recommended, and monitoring patients carrying Stx2 with urine dipstick for hemoglobinuria is suggested to identify the renal complication as early as possible.

Psychoactive Drugs Induce the SOS Response and Shiga Toxin Production in Escherichia coli.

Several classes of non-antibiotic drugs, including psychoactive drugs, proton-pump inhibitors (PPIs), non-steroidal anti-inflammatory drugs (NSAIDs), and others, appear to have strong antimicrobial properties. We considered whether psychoactive drugs induce the SOS response in E. coli bacteria and, consequently, induce Shiga toxins in Shiga-toxigenic E. coli (STEC). We measured the induction of an SOS response using a recA-lacZ E. coli reporter strain, as RecA is an early, reliable, and quantifiable marker for activation of the SOS stress response pathway. We also measured the production and release of Shiga toxin 2 (Stx2) from a classic E. coli O157:H7 strain, derived from a food-borne outbreak due to spinach. Some, but not all, serotonin selective reuptake inhibitors (SSRIs) and antipsychotic drugs induced an SOS response. The use of SSRIs is widespread and increasing; thus, the use of these antidepressants could account for some cases of hemolytic-uremic syndrome due to STEC and is not attributable to antibiotic administration. SSRIs could have detrimental effects on the normal intestinal microbiome in humans. In addition, as SSRIs are resistant to environmental breakdown, they could have effects on microbial communities, including aquatic ecosystems, long after they have left the human body.

Variability in the Occupancy of Escherichia coli O157 Integration Sites by Shiga Toxin-Encoding Prophages.

Escherichia coli O157:H7 strains often produce Shiga toxins encoded by genes on lambdoid bacteriophages that insert into multiple loci as prophages. O157 strains were classified into distinct clades that vary in virulence. Herein, we used PCR assays to examine Shiga toxin (Stx) prophage occupancy in yehV, argW, wrbA, and sbcB among 346 O157 strains representing nine clades. Overall, yehV was occupied in most strains (n = 334, 96.5%), followed by wrbA (n = 213, 61.6%), argW (n = 103, 29.8%), and sbcB (n = 93, 26.9%). Twelve occupancy profiles were identified that varied in frequency and differed across clades. Strains belonging to clade 8 were more likely to have occupied sbcB and argW sites compared to other clades (p < 0.0001), while clade 2 strains were more likely to have occupied wrbA sites (p < 0.0001). Clade 8 strains also had more than the expected number of occupied sites based on the presence of stx variants (p < 0.0001). Deletion of a 20 kb non-Stx prophage occupying yehV in a clade 8 strain resulted in an ~18-fold decrease in stx2 expression. These data highlight the complexity of Stx prophage integration and demonstrate that clade 8 strains, which were previously linked to hemolytic uremic syndrome, have unique Stx prophage occupancy profiles that can impact stx2 expression.

Escherichia coli Shiga Toxins and Gut Microbiota Interactions.

Escherichia coli (EHEC) and Shigella dysenteriae serotype 1 are enterohemorrhagic bacteria that induce hemorrhagic colitis. This, in turn, may result in potentially lethal complications, such as hemolytic uremic syndrome (HUS), which is characterized by thrombocytopenia, acute renal failure, and neurological abnormalities. Both species of bacteria produce Shiga toxins (Stxs), a phage-encoded exotoxin inhibiting protein synthesis in host cells that are primarily responsible for bacterial virulence. Although most studies have focused on the pathogenic roles of Stxs as harmful substances capable of inducing cell death and as proinflammatory factors that sensitize the host target organs to damage, less is known about the interface between the commensalism of bacterial communities and the pathogenicity of the toxins. The gut contains more species of bacteria than any other organ, providing pathogenic bacteria that colonize the gut with a greater number of opportunities to encounter other bacterial species. Notably, the presence in the intestines of pathogenic EHEC producing Stxs associated with severe illness may have compounding effects on the diversity of the indigenous bacteria and bacterial communities in the gut. The present review focuses on studies describing the roles of Stxs in the complex interactions between pathogenic Shiga toxin-producing E. coli, the resident microbiome, and host tissues. The determination of these interactions may provide insights into the unresolved issues regarding these pathogens.

Pediatric Enteric Diagnostic Stewardship: The Right Test in the Right Context.

In this issue of Pediatrics , Cotter et al1 offer valuable analysis of the utility of molecular (nucleic acid amplification) enteric pathogen diagnostic technology in hospitalized children. These tests return highly worthwhile information for some infections, but many of their results are prone to misinterpretation. However, diagnostic guidelines based on classic microbiologic techniques do not perform well either.2 Molecular enteric microbiology is, therefore, a model opportunity for diagnostic stewardship to maximize worth and minimize wasteful expense. Cotter et al1 find that only 3.7% of tested hospitalized children benefitted from molecular diagnosis in terms of decreased length of stay and/or initiation of appropriate treatment. These data reinforce previous findings: enteric diagnostic use increases briskly after molecular testing becomes available and the tests find many unexpected signals reflecting difficult-to-interpret and/or nonactionable microbes. The authors candidly acknowledge the study’s limitations: nongeneralizability (their findings do not apply to outpatients) and reliance on historic comparison data. The benefit they cite applies to hospitalized children, and such populations are probably enriched for severe bacterial infections for which rapid and accurate molecular diagnosis has the greatest value; one would expect different yields in emergency facilities and ambulatory settings. … Address correspondence to Gillian A. M. Tarr, PhD, School of Public Health, University of Minnesota, MMC 807, 420 Delaware St SE, Minneapolis, MN 55455. E-mail: gtarr{at}umn.edu

Tissue-resident macrophages mediate neutrophil recruitment and kidney injury in shiga toxin-induced hemolytic uremic syndrome

Enterohaemorrhagic E.coli cause major epidemics worldwide with significant organ damage and very high percentages of death. Due to the ability of enterohaemorrhagic E.coli to produce shiga toxin these bacteria damage the kidney leading to the hemolytic uremic syndrome. A therapy against this serious kidney disease has not been developed yet and the impact and mechanism of leukocyte activation and recruitment are unclear. Tissue-resident macrophages represent the main leukocyte population in the healthy kidney, but the role of this important cell population in shiga toxin-producing E.coli-hemolytic uremic syndrome is incompletely understood. Using state of the art microscopy and mass spectrometry imaging, our preclinical study demonstrated a phenotypic and functional switch of tissue-resident macrophages after disease induction in mice. Kidney macrophages produced the inflammatory molecule TNFα and depletion of tissue-resident macrophages via the CSF1 receptor abolished TNFα levels in the kidney and significantly diminished disease severity. Furthermore, macrophage depletion did not only attenuate endothelial damage and thrombocytopenia, but also activation of thrombocytes and neutrophils. Moreover, we observed that neutrophils infiltrated the kidney cortex and depletion of macrophages significantly reduced the recruitment of neutrophils and expression of the neutrophil-attracting chemokines CXCL1 and CXCL2. Intravital microscopy revealed that inhibition of CXCR2, the receptor for CXCL1 and CXCL2, significantly reduced the infiltration of neutrophils and reduced kidney injury. Thus, our study shows activation of tissue-resident macrophages during shiga toxin-producing E.coli-hemolytic uremic syndrome leading to the production of disease-promoting TNFα and CXCR2-dependent recruitment of neutrophils.

Standardized Shiga-Toxin Encoding Genes Real-Time PCR Screening Methods Comparison and Development of an Internally Controlled Assay for Pan-stx2 Detection.

Various primer and probe sets have been developed and standardized, but certain sets may have low efficiency or miss some stx-subtypes. To compare the efficiency of the recommended stx screening primers and probe sets in four standardized methods and develop a new primers and probe system with an internal amplification control (IAC) for all known stx2 subtypes. The inclusivity and specificity of recommended screening primers and probe sets in four standardized methods were compared. A new pan-stx2 primer and probe set was adapted from the International Organization for Standardization (ISO) method for all known stx2 subtypes. The robustness of the new method was assessed in seven laboratories and also assessed in ground beef and bean sprout samples. None of the recommended screening primers and probe sets in the four standardized methods could efficiently amplify all the stx2 subtypes because of various mismatches in the primers or the probe sequences. A new primers and probe system adapted from the ISO method, through introducing degenerate bases in primers and probe sequences with an IAC, showed high amplification efficiency and specificity for all known stx2 subtypes in ground beef and bean sprouts samples. The specificity of the new method was assessed in seven laboratories and showed robust and consistent results. This study provided evidence for Shiga-toxin producing Escherichia coli (STEC) screening method development, and the newly developed primers and probes system should be considered in the revision of the standardized methods. None of the recommended screening primer and probe set in the four official methods could efficiently amplify all the stx2 subtypes. A new developed primer and probe set showed high amplification efficiency and specificity for all known stx2 subtypes in fresh ground beef and bean sprouts samples. The newly developed stx2 screening system showed robustness and consistency during interlaboratory study.

Characterization of Shiga-toxin producing Escherichia coli (STEC) isolated from retail raw meats in Southeast China

Shiga toxin-producing Escherichia coli (STEC) is an important zoonotic foodborne pathogen that can induce acute gastrointestinal symptoms related to human infection. In this study, we characterized 49 STEC strains isolated from 816 retail raw meats from 2010 to 2016 in Southeast China for their serotype, virulence-associated genes, phylogenetic group, antibiotic resistance, and biofilm-forming ability. These isolates belonged to 33 divergent O: H serotypes, among which O157: H7 (12.24%, 6/49) was the most common serotype. Moreover, 22.45% (11/49) isolates belonged to the “top six” serotypes most frequently implicated in human infections. Among the 11 virulence-associated genes studied, stx2 (71.43%, 35/49) was the most prevalent virulence factor, with 87.76% (43/49) of isolates carrying two or more types of virulence factors. The B1 (55.10%, 27/49) and D (28.57%, 14/49) phylogenetic groups were predominant among isolates, whereas no strain belonged to group B2. A total of 59.18% (29/49) of isolates were resistant to 13 out of 16 tested antimicrobials, with resistance rate ranging from 4.08% (2/49) for both ceftazidime and chloramphenicol to 46.94% (23/49) for streptomycin. We detected eight out of 30 antibiotic-resistance genes (tetA, tetB, strA, strB, gyrA, floR, sulI, and sulII), with obvious mismatch between the phenotypes and genotypes of these isolates. Most strains (87.76%, 43/49) produced biofilms and 23.26% (10/43) were categorized as high producers. In conclusion, raw meats retailed in Southeast China contain STEC with multifarious serotypes, virulence-associated genes, antimicrobial resistance and biofilm-forming ability, some of which pose potential health risks to consumers.

Detection of stx1 and stx2 and subtyping of Shiga toxin-producing Escherichia coli using asymmetric PCR combined with lateral flow immunoassay

Shiga toxin-producing Escherichia coli (STEC) is known as a kind of foodborne pathogens that can produce Shiga toxin type1, type 2, or both, encoded by stx1 and stx2 genes, respectively.Rapid and accurate detection of STEC and stx1/stx2 subtyping assay in food and environment is essential for foodborne outbreak investigation. In this study, a method for the accurate and rapid subtyping of STEC single colony using asymmetric polymerase chain reaction (asPCR) combined with lateral flow immunoassay (LFIA) was established. AsPCR was conducted using biotinylated stx1 and digoxin-labeled stx2 primers to obtain several single stranded DNA amplicons labeled with biotin or/and digoxin. Subsequently, these amplicons were tagged with specific probes labeled with carboxylate-modified red polystyrene microspheres using sequential hybridization. Finally, STEC with different Shiga toxin gene types could be distinguished depending on the appearance of different test lines on an LFIA strip. Thus, the STEC single colony could be identified and subtyped accurately and conveniently using this method. This method showed high specificity. Twenty-four strains of STEC in milk were detected and subtyped using our method, which combines asPCR with LFIA.