Delayed lactose utilization among Shiga toxin-producing Escherichia coli of serogroup O121.

Two outbreaks of Shiga toxin-producing Escherichia coli O121:H19 associated with wheat flour, in the United States of America and Canada, involved strains with an unusual phenotype, delayed lactose utilization (DLU). These strains do not ferment lactose when initially cultured on MacConkey agar (MAC), but lactose fermentation occurs following subculture to a second plate of MAC. The prevalence of DLU was determined by examining the ß-galactosidase activity of 49 strains of E. coli O121, and of 37 other strains of E. coli. Twenty four of forty three O121:H19 and one O121:NM displayed DLU. Two strains (O121:NM and O145:H34) did not have detectable ß-galactosidase activity. ß-glucuronidase activity of O121 strains was also determined. All but six DLU strains had normal ß-glucuronidase activity. ß-glucuronidase activity was suppressed on MAC for 17 of 23 O121 non-DLU strains. Genomic analysis found that DLU strains possessed an insertion sequence, IS600 (1267 bp), between lacZ (ß-galactosidase) and lacY (ß-galactoside permease), that was not present in strains exhibiting normal lactose utilization. The insert might reduce the expression of ß-galactoside permease, delaying import of lactose, resulting in the DLU phenotype. The high probability of DLU should be considered when using lactose-containing media for the isolation of STEC O121.

A Clonal Shiga Toxin-Producing Escherichia coli O121:H19 Population Exhibits Diverse Carbon Utilization Patterns.

Shiga toxin-producing Escherichia coli (STEC) serotype O121:H19 is one of the major non-O157:H7 serotypes associated with severe human disease. Here we examined population structure, virulence potential, and metabolic profile of environmental STEC O121 strains recovered from a major produce production region in California and performed comparative analyses with STEC O121 clinical isolates. Multilocus sequence typing revealed that sequence type (ST)-655, a common ST in clinical strains, was the predominant genotype among the environmental strains. Phylotyping placed all STEC O121 strains in B1 group, a lineage containing other major non-O157 serogroups of STEC. Genes encoding different subtypes of Shiga toxin 1 and 2 were detected in O121, including stx1a, stx1d, stx2a, and stx2e. Furthermore, genes encoding intimin (eae) and enterohemolysin (ehxA) were detected in a majority of environmental strains (83.3%), suggesting that the majority of environmental STEC O121 strains are enterohemorrhagic E. coli. The STEC O121 strains with the same genotype were clustered together based on the carbon utilization pattern. Among the 122 carbon substrates that supported the growth of STEC O121 strains, 44 and 35 exhibited lineage (ST) and strain-specific metabolic profiles, respectively. Although clinical ST-655 strains displayed higher metabolic activity than environmental ST-655 strains for several carbon substrates, including l-alaninamide, 5-keto-d-gluconic acid, 3-O-ß-d-galactopyranosyl-d-arabinose, α-ketoglutaric acid, and lactulose, a few environmental strains with the enhanced metabolic potential for the above substrates were detected. Variations in curli biogenesis and swimming motility were also observed in ST-655 strains, suggesting that phenotypic variants are widespread in STEC. Considering the ecological niches that STEC colonizes, increased metabolic potential for plant-derived carbohydrates, mucus-derived substrates, or secondary metabolites produced by the indigenous microorganisms might have been selected. Such traits would confer STEC competitive advantages and facilitate survival and adaptation of STEC population to a given niche, including infected humans.

Escherichia coli O121:H19 infection identified on microagglutination assay and PCR.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are increasingly recognized as foodborne pathogens that trigger hemolytic uremic syndrome (HUS). The detection and isolation of these strains is important, but distinguishing their bacteriological profiles is difficult. A 2-year-old girl developed HUS with mild renal involvement 22 days after consuming barbecued meat. Clinical and laboratory findings gradually improved without specific treatment. Because neither enterohemorrhagic E. coli (EHEC) nor Shiga toxins were detected in stool cultures in a clinical laboratory and the patient tested negative for circulating antibodies to O157 lipopolysaccharide, the case was initially diagnosed as probable atypical HUS. Subsequent serodiagnostic microagglutination assay and polymerase chain reaction-based molecular testing, however, indicated the presence of the EHEC O121:H19 strain with stx2. Thus, to correctly diagnose and treat HUS, a system for detecting non-O157 STEC in a clinical setting is urgently needed.

Survivability and thermal resistance of Salmonella and Escherichia coli O121 in wheat flour during extended storage of 360 days.

Foodborne pathogens like Salmonella and Escherichia coli O121 can endure the harsh low water activity (aw) environment of wheat flour for elongated periods of time and can proliferate when hydrated for baking or other purposes. This study determined the survivability and thermal tolerance (D- and z-values) of Salmonella and Escherichia coli O121 in wheat flour and muffin batter (prepared from inoculated flour on the days of analyses) during the storage period of 360 days. The Salmonella and E. coli O121 studies were conducted as two independent experiments. Both studies were designed as randomized complete block with three replications as blocks. All experimental data were analyzed using one-way ANOVA and Tukey's test in Minitab® software, and P ≤ 0.05 was considered significant. The wheat flour was spray inoculated individually with 7-isolate Salmonella or 3-isolate E. coli O121 cocktail and then dried back to the original aw levels. On each analysis day, inoculated wheat flour (~5 g) or muffin batter (~2.5 g) was placed inside the TDT disks, heat treated at set temperatures in hot water baths, and sampled at predetermined time intervals for determining the survival microbial population. The population of E. coli O121 and Salmonella cocktails in wheat flour at day 1 were 7.6 ± 0.18 and 7.8 ± 0.07 log CFU/g, respectively, which decreased to 2.0 ± 0.40 and 2.8 ± 0.59 log CFU/g on day 360, respectively. The D-values of Salmonella and E. coli O121 cocktails in inoculated flour and muffin batter prepared from inoculated flour (on the day of analysis) were determined on days 1, 30, 90, 180, 270, and 360 [given enough surviving bacterial population (~3 to 4 log CFU/g) was present in the flour]. The population of Salmonella and E. coli O121 in wheat flour decreased by 5.0 and 5.6 log CFU/g, respectively, during the storage period of 360 days. The D70°C, D75°C, and D80°C values of Salmonella in wheat flour remained similar during the storage period. Whereas, for E. coli O157:H7 in wheat flour, the D70°C value decreased from 20.3 ± 2.82 to 7.1 ± 2.82 min, and D75°C decreased from 10.2 ± 2.14 to 2.7 ± 0.27 min, during the storage period of 180 days. The z-values of Salmonella or E. coli O157:H7 remained similar during the storage period. The D- and z-values from this research can be employed for validation of thermal process to ensure safety of wheat flour.

Comparative genomic and phenotypic analyses of the virulence potential in Shiga toxin-producing Escherichia coli O121:H7 and O121:H10.

Shiga toxin-producing Escherichia coli (STEC) O121 is among the top six non-O157 serogroups that are most frequently associated with severe disease in humans. While O121:H19 is predominant, other O121 serotypes have been frequently isolated from environmental samples, but their virulence repertoire is poorly characterized. Here, we sequenced the complete genomes of two animal isolates belonging to O121:H7 and O121:H10 and performed comparative genomic analysis with O121:H19 to assess their virulence potential. Both O121:H7 and O121:H10 strains carry a genome comparable in size with the O121:H19 genomes and belong to phylogroup B1. However, both strains appear to have evolved from a different lineage than the O121:H19 strains according to the core genes-based phylogeny and Multi Locus Sequence Typing. A systematic search of over 300 E. coli virulence genes listed in the Virulence Factor DataBase revealed a total of 73 and 71 in O121:H7 and O121:H10 strains, respectively, in comparison with an average of 135 in the O121:H19 strains. This variation in the virulence genes repertoire was mainly attributed to the reduction in the number of genes related to the Type III Secretion System in the O121:H7 and O121:H10 strains. Compared to the O121:H19 strains, the O121:H7 strain carries more adherence and toxin genes while the O121:H10 strain carries more genes related to the Type VI Secretion System. Although both O121:H7 and O121:H10 strains carry the large virulence plasmid pEHEC, they do not harbor all pEHEC virulence genes in O121:H19. Furthermore, unlike the O121:H19 strains, neither the O121:H7 nor O121:H10 strain carried the Locus of Enterocyte Effacement, OI-122, nor the tellurite resistance island. Although an incomplete Locus of Adhesion and Autoaggregation (LAA) was identified in the O121:H7 and O121:H10 strains, a limited number of virulence genes were present. Consistently, both O121:H7 and O121:H10 strains displayed significant reduced cytotoxicity than either the O157:H7 strain EDL933 or the O121:H19 strain RM8352. In fact, the O121:H7 strain RM8082 appeared to cause minimal cytotoxicity to Vero cells. Our study demonstrated distinct evolutionary lineages among the strains of serotypes O121:H19, O121:H10, and O121:H7 and suggested reduced virulence potentials in STEC strains of O121:H10 and O121:H7.

Escherichia coli O121 outbreak associated with raw milk Gouda-like cheese in British Columbia, Canada, 2018.

BACKGROUND: In 2018, a Shiga toxin-producing Escherichia coli O121 outbreak that affected seven individuals was associated with raw milk Gouda-like cheese produced in British Columbia, Canada. OBJECTIVES: To describe the E. coli O121 outbreak investigation and recommend greater control measures for raw milk Gouda-like cheese. METHODS: Cases of E. coli O121 were identified through laboratory testing results and epidemiologic surveillance data. The cases were interviewed on exposures of interest, which were analyzed against Foodbook Report values for British Columbia. Environmental inspection of the dairy plant and the cheese products was conducted to ascertain a source of contamination. Whole genome multi-locus sequence typing (wgMLST) was performed on all positive E. coli O121 clinical and food isolates at the provincial laboratory. RESULTS: Four out of the seven cases consumed the same raw milk Gouda-like cheese between August and October 2018. The implicated cheese was aged longer than the required minimum of 60 days, and no production deficiencies were noted. One sample of the implicated cheese tested positive for E. coli O121. The seven clinical isolates and one cheese isolate matched by wgMLST within 6.5 alleles. CONCLUSION: Raw milk Gouda and Gouda-like cheese has been implicated in three previous Shiga toxin-producing E. coli outbreaks in North America. It was recommended product labelling to increase consumer awareness and thermization of milk to decrease the risk of illness associated with raw milk Gouda and Gouda-like cheese.

The global population structure and evolutionary history of the acquisition of major virulence factor-encoding genetic elements in Shiga toxin-producing Escherichia coli O121:H19.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are foodborne pathogens causing serious diseases, such as haemorrhagic colitis and haemolytic uraemic syndrome. Although O157:H7 STEC strains have been the most prevalent, incidences of STEC infections by several other serotypes have recently increased. O121:H19 STEC is one of these major non-O157 STECs, but systematic whole genome sequence (WGS) analyses have not yet been conducted on this STEC. Here, we performed a global WGS analysis of 638 O121:H19 strains, including 143 sequenced in this study, and a detailed comparison of 11 complete genomes, including four obtained in this study. By serotype-wide WGS analysis, we found that O121:H19 strains were divided into four lineages, including major and second major lineages (named L1 and L3, respectively), and that the locus of enterocyte effacement (LEE) encoding a type III secretion system (T3SS) was acquired by the common ancestor of O121:H19. Analyses of 11 complete genomes belonging to L1 or L3 revealed remarkable interlineage differences in the prophage pool and prophage-encoded T3SS effector repertoire, independent acquisition of virulence plasmids by the two lineages, and high conservation in the prophage repertoire, including that for Stx2a phages in lineage L1. Further sequence determination of complete Stx2a phage genomes of 49 strains confirmed that Stx2a phages in lineage L1 are highly conserved short-tailed phages, while those in lineage L3 are long-tailed lambda-like phages with notable genomic diversity, suggesting that an Stx2a phage was acquired by the common ancestor of L1 and has been stably maintained. Consistent with these genomic features of Stx2a phages, most lineage L1 strains produced much higher levels of Stx2a than lineage L3 strains. Altogether, this study provides a global phylogenetic overview of O121:H19 STEC and shows the interlineage genomic differences and the highly conserved genomic features of the major lineage within this serotype of STEC.

Effect of Ph On Survival of Escherichia coli O157, Escherichia coli O121, and Salmonella enterica During Desiccation and Short-term Storage.

ABSTRACT: One intrinsic characteristic of low-moisture foods that is frequently overlooked is pH. Although pH affects the survival of microorganisms in high-moisture foods, its influence in low-moisture foods with less available moisture has not been examined. Escherichia coli O157:H7, E. coli O121, Salmonella enterica Anatum, and S. enterica Agona were grown on solid media with and without added glucose, harvested, and then suspended in buffer at pH 4, 5, and 7 for 10 min. All cultures were spotted individually onto cellulose filters and dried in a biohazard cabinet (23 ± 2°C) overnight (24 ± 2 h) and then stored in a 25°C incubator at 33% relative humidity. Populations were examined at regular intervals up to 26 (E. coli) or 29 (Salmonella) days. Additional controls for pH consisted of cultures held in buffer at pH 4, 5, and 7 at 25°C for the same time periods as the desiccated cells. For all strains tested, pH had an effect on survival whether stored dried or in liquid buffer (P < 0.05). However, when grown on solid media, acid adaptation (grown with glucose) before acid treatment did not appear beneficial to Salmonella during desiccation. Instead, both acid-adapted Salmonella serovars appeared less resistant during drying than did non-acid-adapted cells. Once dried, the rates of decline for Salmonella were not significantly different for acid-adapted and nonadapted cells (P > 0.05), indicating similar persistence following desiccation. A reverse trend was observed for E. coli O121; acid adaptation on solid media improved survival during desiccation and subsequent storage at low pH (P < 0.05). E. coli O157:H7 survival was significantly lower than that of either Salmonella or E. coli O121 under all conditions tested. Results indicate that the response to desiccation and pH stress differs between the microorganisms and under different growth conditions.

Desiccation and Thermal Resistance of Escherichia coli O121 in Wheat Flour.

Non-O157 Shiga toxin-producing Escherichia coli infections have recently been associated with wheat flour on two separate accounts in the United States and Canada. However, there is little information regarding the thermal resistance and longevity of non-O157 Shiga toxin-producing Escherichia coli during storage in low-moisture environments. The objectives of this study were to determine the thermal inactivation kinetics of E. coli O121 in wheat flour and to compare the thermal inactivation rates with those of other pathogens. Wheat flour, inoculated with E. coli O121, was equilibrated at 25°C to a water activity of 0.45 in a humidity-controlled conditioning chamber. Inoculated samples were treated isothermally at 70, 75, and 80°C, and posttreatment population survivor ratios were determined by plate counting. D- and z-values calculated with a log-linear model, were compared with those obtained in other studies. At 70, 75, and 80°C, the D-values for E. coli O121 were 18.16 ± 0.96, 6.47 ± 0.50, and 4.58 ± 0.40 min, respectively, and the z-value was 14.57 ± 2.21°C. Overall, E. coli O121 was observed to be slightly less thermally resistant than what has been previously reported for Salmonella Enteritidis PT30 in wheat flour as measured under the same conditions with the same methods.

Infecciones por Escherichia coli productora de toxina Shiga O121: H19 en pacientes atendidos en Mar del Plata / Infections caused by Escherichia coli producing Shiga O121: H19 toxin in patients treated in Mar del Plata

Escherichia coli productora de toxina Shiga (STEC) O157:H7 es el serotipo más frecuentemente identificado como agente causal de colitis hemorrágica y síndrome urémico hemolítico (SUH), aunque se han descripto más de 100 serotipos con potencial patogénico similar. El objetivo del trabajo fue describir casos de enfermedad humana asociados a la infección por STEC O121:H19, atendidos en la ciudad de Mar del Plata y establecer la relación genética de los aislamientos mediante técnicas de epidemiología molecular. Se observó un amplio espectro en la severidad clínica de los ocho casos estudiados: dos fueron asintomáticos (contactos de SUH), un paciente tuvo diarrea sanguinolenta, y cinco presentaron SUH. Uno de los pacientes con SUH falleció. Las cepas O121:H19 portadoras del genotipo stx2a/eae/ehxA fueron sensibles a los antibióticos ensayados y presentaron por electroforesis en gel de campo pulsado (Xbal-PFGE) distintos patrones de macrorrestricción, con similitud del 84,25%. El patrón AREXKX01.0072, detectado en un SUH y en su contacto, es nuevo en la Base de Datos Nacional de STEC no-O157 de la Argentina. La utilización de métodos estandarizados de detección y tipificación de STEC permite a los laboratorios de referencia monitorear la frecuencia temporal y la distribución geográfica de las cepas circulantes para la prevención y control de estos patógenos asociados a enfermedad humana.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is the most frequent serotype identified as causative agent of sporadic cases and outbreaks of diarrhea with or without blood, hemorrhagic colitis and hemolytic uremic syndrome (HUS), although more than 100 serotypes have been described of similar pathogenic potential. The aim of the study was to describe cases of human disease associated with STEC O121:H19 infections, assisted in Mar del Plata City, and to establish the genetic relationship of the isolates by molecular epidemiology techniques. A wide spectrum was observed in the clinical severity of the eight cases studied: two were asymptomatic (contacts of HUS), one patient had bloody diarrhea, and five cases presented HUS. One HUS case died. All STEC O121:H19 strains carried the stx2a/eae/ehxA genotype, were sensitive to all antibiotics tested and showed different macrorestriction patterns by pulsed-field gel electrophoresis (Xbal-PFGE), with 84.25% similarity. The pattern AREXKX01.0072, detected in a HUS case and in his contact, is new in the Argentine National Database of non-O157 STEC. The use of standardized methods for detection and typing of STEC allows reference laboratories to monitor the temporal frequency and geographical distribution of circulating strains for the prevention and control of these pathogens associated with human diseases.

Escherichia coli produtora de toxina Shiga (STEC) O157:H7 é o sorotipo mais frequentemente identificado como o agente causador de colite hemorrágica e síndrome hemolítica urêmica (SHU), embora tenham sido descritas mais de 100 sorotipos com potencial patogênico semelhantes. O objectivo foi o de descrever os casos de doença humana associadas com a infecção por STEC O121:H19, assistido, na cidade de Mar del Plata e estabelecer relação genética de isolados utilizando epidemiologia molecular. Um amplo espectro foi observado na severidade clínica dos oito casos estudados, dois eram assintomáticos (contacto SHU), uma paciente teve diarreia com sangue, e cinco tiveram SHU. Um caso de SHU faleceu. As cepas O121:H19 portaram o genótipo stx2a/eae/ehxA, foram sensíveis aos antibióticos testados e apresentaram, por eletroforese em gel de campo pulsado (Xbal-PFGE), diferentes padrões de macrorestrição, com similaridade de 84,25%. O padrão AREXKX01.0072 detectado em SHU e em seu contato, é novo para a Base de Dados Nacional de STEC não-O157 na Argentina. O uso de métodos padrão de detecção e tipagem de STEC permite os laboratórios de referência monitorar frequência temporal e distribuição geográfica de estirpes circulantes para a prevenção e controlo destes agentes patogénicos associados com a doença humana.