RpoS acts as a global repressor of virulence gene expression in E. coli O104:H4 and enteroaggregative E. coli.

In 2011, in Germany, Escherichia coli O104:H4 caused the enterohemorrhagic E. coli (EHEC) outbreak with the highest incidence rate of hemolytic uremic syndrome. This pathogen carries an exceptionally potent combination of EHEC- and enteroaggregative E. coli (EAEC)-specific virulence factors. Here, we identified an E. coli O104:H4 isolate that carried a single nucleotide polymorphism (SNP) in the start codon (ATG > ATA) of rpoS, encoding the alternative sigma factor S. The rpoS ATG > ATA SNP was associated with enhanced EAEC-specific virulence gene expression. Deletion of rpoS in E. coli O104:H4 Δstx2 and typical EAEC resulted in a similar effect. Both rpoS ATG > ATA and ΔrpoS strains exhibited stronger virulence-related phenotypes in comparison to wild type. Using promoter-reporter gene fusions, we demonstrated that wild-type RpoS repressed aggR, encoding the main regulator of EAEC virulence. In summary, our work demonstrates that RpoS acts as a global repressor of E. coli O104:H4 virulence, primarily through an AggR-dependent mechanism.

Sporadic Detection of Escherichia coli O104:H4 Strain C227/11Φcu in the Edible Parts of Lamb's Lettuce Cultured in Contaminated Agricultural Soil Samples.

In the current study, we demonstrate that E. coli O104:H4 strain C227/11Φcu, a derivative of the 2011 enterohemorrhagic/enteroaggregative (EHEC/EAEC) E. coli outbreak strain, migrated into the edible portion of lamb's lettuce plants upon contamination of the surrounding soil. Seeds were surface-sterilized and cultivated on Murashige-Skoog agar or in autoclaved agricultural soil. Migration into the edible portions was investigated by inoculating the agar or soil close to the plants with 108 colony-forming units (CFU). The edible parts, which did not come into contact with the contaminated medium or soil, were quantitatively analyzed for the presence of bacteria after 2, 4 and 8 weeks. Strain C227/11Φcu could colonize lamb's lettuce when contamination of medium or soil occurs. The highest recovery rate (27%) was found for lettuce cultivated in agar, and up to 1.6 × 103 CFU/g lettuce was detected. The recovery rate was lower for the soil samples (9% and 13.5%). Although the used contamination levels were high, migration of C227/11Φcu from the soil into the edible parts was demonstrated. This study further highlights the risk of crop plant contamination with pathogenic E. coli upon soil contamination.

Shiga-Toxin-Producing Strains of Escherichia coli O104:H4 and a Strain of O157:H7, Which Can Cause Human Hemolytic Uremic Syndrome, Differ in Biofilm Formation in the Presence of CO2 and in Their Ability to Grow in a Novel Cell Culture Medium.

One pathogen that commonly causes gastrointestinal illnesses from the consumption of contaminated food is Escherichia coli O157:H7. In 2011 in Germany, however, there was a prominent outbreak of bloody diarrhea with a high incidence of hemolytic uremic syndrome (HUS) caused by an atypical, more virulent E. coli O104:H4 strain. To facilitate the identification of this lesser-known, atypical E. coli O104:H4 strain, we wanted to identify phenotypic differences between it and a strain of O157:H7 in different media and culture conditions. We found that E. coli O104:H4 strains produced considerably more biofilm than the strain of O157:H7 at 37 °C (p = 0.0470-0.0182) Biofilm production was significantly enhanced by the presence of 5% CO2 (p = 0.0348-0.0320). In our study on the innate immune response to the E. coli strains, we used HEK293 cells that express Toll-like receptors (TLRs) 2 or 4. We found that E. coli O104:H4 strains had the ability to grow in a novel HEK293 cell culture medium, while the E. coli O157:H7 strain could not. Thus, we uncovered previously unknown phenotypic properties of E. coli O104:H4 to further differentiate this pathogen from E. coli O157:H7.

O-Antigen Diversification Masks Identification of Highly Pathogenic Shiga Toxin-Producing Escherichia coli O104:H4-Like Strains.

Shiga toxin-producing Escherichia coli (STEC) can give rise to a range of clinical outcomes from diarrhea to the life-threatening systemic condition hemolytic-uremic syndrome (HUS). Although STEC O157:H7 is the serotype most frequently associated with HUS, a major outbreak of HUS occurred in 2011 in Germany and was caused by a rare serotype, STEC O104:H4. Prior to 2011 and since the outbreak, STEC O104:H4 strains have only rarely been associated with human infections. From 2012 to 2020, intensified STEC surveillance was performed in Germany where the subtyping of ~8,000 clinical isolates by molecular methods, including whole-genome sequencing, was carried out. A rare STEC serotype, O181:H4, associated with HUS was identified, and like the STEC O104:H4 outbreak strain, this strain belongs to sequence type 678 (ST678). Genomic and virulence comparisons revealed that the two strains are phylogenetically related and differ principally in the gene cluster encoding their respective lipopolysaccharide O-antigens but exhibit similar virulence phenotypes. In addition, five other serotypes belonging to ST678 from human clinical infection, such as OX13:H4, O127:H4, OgN-RKI9:H4, O131:H4, and O69:H4, were identified from diverse locations worldwide. IMPORTANCE Our data suggest that the high-virulence ensemble of the STEC O104:H4 outbreak strain remains a global threat because genomically similar strains cause disease worldwide but that the horizontal acquisition of O-antigen gene clusters has diversified the O-antigens of strains belonging to ST678. Thus, the identification of these highly pathogenic strains is masked by diverse and rare O-antigens, thereby confounding the interpretation of their potential risk.

The conjugative transfer of plasmid-mediated mobile colistin resistance gene, mcr-1, to Escherichia coli O157:H7 and Escherichia coli O104:H4 in nutrient broth and in mung bean sprouts.

The emergence of mobile colistin resistant gene (mcr-1) in Enterobacteriaceae has become a global public health concern. Dissemination of the mcr-1 gene through conjugation of bacteria associated with food may occur. This research investigated the transfer frequency of the mcr-1 gene among Escherichia coli in liquid media and during growth of mung bean sprouts. The donor strain E. coli NCTC 13846 (mcr-1 positive) and recipient strains of E. coli O157:H7 and E. coli O104:H4 were used. Mating experiments in vitro were conducted at 4, 25, and 37 °C for up to 36 h. The in vivo mating experiments (growing sprouts) were conducted in a sprout growth chamber with irrigation of 1 min/h over 6 days following inoculation of mung bean seeds with the donor and a recipient. The highest transfer frequencies in TSB media, 2.86E-07 and 3.24E-07, occurred at 37 °C after mating for 24 h for E. coli O104:H4 and E. coli O157:H7, respectively. Transconjugants were not detected in liquid media at 4 °C. Moreover, transfer frequency (5.68E-05 per recipient) of mcr-1 was greater during mung bean sprout growth for E. coli O104:H4 compared to E. coli O157:H7 (1.02E-05 per recipient) Day 3 to Day 6. This study indicates that the transfer of antibiotic resistant gene(s) among bacteria during mung bean sprout production may facilitate the spread of antibiotic resistant bacteria in the environment and to humans.

Transduction of stx2a mediated by phage (Φ11-3088) from Escherichia coli O104:H4 in vitro and in situ during sprouting of mung beans.

Escherichia coli O104:H4 strain 11-3088 encoding Stx2a is epidemiologically related to the foodborne outbreak associated with sprouts in Germany, 2011. Sprouting provides suitable conditions for bacterial growth and may lead to transduction of non-pathogenic strains of E. coli with Stx phages. Although transduction of E. coli by Stx phages in food has been documented, data on the phages from E. coli O104:H4 is limited. This study determined the host range of the bacteriophage Φ11-3088 from E. coli O104:H4 using E. coli O104:H4 ∆stx2::gfp::ampr and demonstrated phage transduction during sprouting. The Φ11-3088∆stx transduced 5/45 strains, including generic E. coli, pap-positive E. coli O103:H2, ETEC, and S. sonnei. The expression level of Φ11-3088∆stx differed among lysogens upon induction. Of the 3 highly induced lysogens, the lytic cycle was induced in E. coli O104:H4∆stx2::gfp::ampr and O103:H2 but not in S. sonnei. E. coli DH5α was the only strain susceptible to lytic infection by Φ11-3088∆stx. To explore the effect of drying and rehydration during seed storage and sprouting on phage induction and transduction, mung beans inoculated with the phage donor E. coli O104:H4∆stx2::gfp::ampr (8 log CFU/g) were dried, rehydrated, and incubated with the phage recipient E. coli DH5α (7 log CFU/g) for 96 h. Sprouted seeds harbored about 3 log CFU/g of putative lysogens that acquired ampicillin resistance. At the end of sprouting, 71 % of putative lysogens encoded gfp, confirming phage transduction. Overall, stx transfer by phages may increase the cell counts of STEC during sprouting by converting generic E. coli to STEC.

Sporadic Occurrence of Enteroaggregative Shiga Toxin-Producing Escherichia coli O104:H4 Similar to 2011 Outbreak Strain.

We describe the recent detection of 3 Shiga toxin-producing enteroaggregative Escherichia coli O104:H4 isolates from patients and 1 from pork in the Netherlands that were genetically highly similar to isolates from the 2011 large-scale outbreak in Europe. Our findings stress the importance of safeguarding food supply production chains to prevent future outbreaks.

Effect of Citral on the Thermal Inactivation of Escherichia coli O104:H4 in Ground Beef.

ABSTRACT: The objective of the present study was to analyze the combined effect of heat treatment (55 to 62.5°C) and citral (0 to 3%) on the heat resistance of Escherichia coli O104:H4 inoculated into ground beef. Inoculated meat packages were immersed in a circulating water bath stabilized at 55, 57.5, 60, or 62.5°C for various times. The surviving microbial cells were counted after culture on tryptic soy agar. A factorial design (4 × 4) was used to analyze the effects and interaction of heat treatment and citral. Heat and citral promoted E. coli O104:H4 thermal inactivation, suggesting a synergistic effect. At 55°C, the incorporation of citral at 1, 2, and 3% decreased D-values (control = 42.75 min) by 85, 89, and 91%, respectively (P < 0.05). A citral concentration-dependent effect (P < 0.05) also was noted at other evaluated temperatures. These findings could be of value to the food industry for designing a safe thermal process for inactivating E. coli O104:H4 in ground beef under similar thermal inactivation conditions.

Diversity of Potentially Pathogenic Escherichia coli O104 and O9 Serogroups Isolated before 2011 from Fecal Samples from Children from Different Geographic Regions.

In 2011, an outbreak of hemorrhagic colitis and hemolytic uremic syndrome (HUS) was reported in Europe that was related to a hybrid STEAEC of Escherichia coli (E. coli) O104:H4 strain. The current study aimed to analyze strains of E. coli O104 and O9 isolated before 2011. The study included 47 strains isolated from children with and without diarrhea between 1986 and 2009 from different geographic regions, as well as seven reference strains. Serotyping was carried out on 188 anti-O and 53 anti-H sera. PCR was used to identify DEC genes and phylogenetic groups. Resistance profiles to antimicrobials were determined by diffusion in agar, while PFGE was used to analyze genomic similarity. Five serotypes of E. coli O104 and nine of O9 were identified, as well as an antigenic cross-reaction with one anti-E. coli O9 serum. E. coli O104 and O9 presented diarrheagenic E. coli (DEC) genes in different combinations and were located in commensal phylogenetic groups with different antimicrobial resistance. PFGE showed that O104:H4 and O9:(H4, NM) strains from SSI, Bangladesh and México belong to a diverse group located in the same subgroup. E. coli O104 and O9 were classified as commensal strains containing DEC genes. The groups were genetically diverse with pathogenic potential making continued epidemiologic surveillance important.

Survival of Enterohemorrhagic Escherichia coli O104:H4 Strain C227/11Φcu in Agricultural Soils Depends on rpoS and Environmental Factors.

The consumption of contaminated fresh produce caused outbreaks of enterohemorrhagic (EHEC) Escherichia coli. Agricultural soil might be a reservoir for EHEC strains and represent a contamination source for edible plants. Furthermore, the application of manure as fertilizer is an important contamination route. Thus, the German fertilizer ordinance prohibits the use of manure 12 weeks before crop harvest to avoid pathogen transmission into the food chain. In this study, the survival of E. coli O104:H4 strain C227/11Φcu in soil microenvironments with either diluvial sand or alluvial loam at two temperatures was investigated for more than 12 weeks. It was analyzed whether the addition of cattle manure extends EHEC survival in these microenvironments. The experiments were additionally performed with isogenic ΔrpoS and ΔfliC deletion mutants of C227/11Φcu. The survival of C227/11Φcu was highest at 4 °C, whereas the soil type had a minor influence. The addition of cattle manure increased the survival at 22 °C. Deletion of rpoS significantly decreased the survival period under all cultivation conditions, whereas fliC deletion did not have any influence. The results of our study demonstrate that EHEC C227/11Φcu is able to survive for more than 12 weeks in soil microenvironments and that RpoS is an important determinant for survival.

Resveratrol suppresses NLRP3 inflammasome activation in colonic epithelial cells triggered by Escherichia coli O104: H4 / 中国临床药理学与治疗学

AIM: To study the effects of resveratrol (RES) on the mitochondrial damage, oxidative stress injury and NLRP3 inflammasome activation in colonic epithelial cells (Caco-2) induced by Escherichia coli O104: H4. METHODS: Caco-2 cells pre-treated with 200 μmol/L RES for 12 h, then cells were infected with 10

The Role of Pea (Pisum sativum) Seeds in Transmission of Entero-Aggregative Escherichia coli to Growing Plants.

Crop plants can become contaminated with human pathogenic bacteria in agro-production systems. Some of the transmission routes of human pathogens to growing plants are well explored such as water, manure and soil, whereas others are less explored such as seeds. Fenugreek seeds contaminated with the entero-hemorrhagic Escherichia coli O104:H4 were suspected to be the principle vectors for transmission of the pathogen to sprouts at the food-borne disease outbreak in Hamburg and surrounding area in 2011. In this study we raised the questions of whether cells of the entero-aggregative E. coli O104:H4 strain 55989 is capable of colonizing developing plants from seeds and if it would be possible that, via plant internalization, these cells can reach the developing embryonic tissue of the next generation of seeds. To address these questions, we followed the fate of strain 55989 and of two other E. coli strains from artificially contaminated seeds to growing plants, and from developing flower tissue to mature seeds upon proximate introductions to the plant reproductive organs. Escherichia coli strains differing in origin, adherence properties to epithelial cells, and virulence profile were used in our experimentation to relate eventual differences in seed and plant colonization to typical E. coli properties. Experiments were conducted under realistic growth circumstances in greenhouse and open field settings. Entero-aggregative E. coli strain 55989 and the two other E. coli strains were able to colonize the root compartment of pea plants from inoculated seeds. In roots and rhizosphere soil, the strains could persist until the senescent stage of plant growth, when seeds had ripened. Colonization of the above-soil parts was only temporary at the start of plant growth for all three E. coli strains and, therefore, the conclusion was drawn that translocation of E. coli cells via the vascular tissue of the stems to developing pea seeds seems unlikely under circumstances realistic for agricultural practices. Proximate introductions of cells of E. coli strains to developing flowers also did not result in internal seed contamination, indicating that internal seed contamination with E. coli is an unlikely event. The fact that all three E. coli strains showed stronger preference for the root-soil zones of growing pea plants than for the above soil plant compartments, in spite of their differences in clinical behaviour and origin, indicate that E. coli in general will colonize root compartments of crop plants in production systems.

Antimicrobial Activity of Silver-Treated Bacteria against other Multi-Drug Resistant Pathogens in Their Environment.

Silver is a potent antimicrobial agent against a variety of microorganisms and once the element has entered the bacterial cell, it accumulates as silver nanoparticles with large surface area causing cell death. At the same time, the bacterial cell becomes a reservoir for silver. This study aims to test the microcidal effect of silver-killed E. coli O104: H4 and its supernatant against fresh viable cells of the same bacterium and some other species, including E. coli O157: H7, Multidrug Resistant (MDR) Pseudomonas aeruginosa and Methicillin Resistant Staphylococcus aureus (MRSA). Silver-killed bacteria were examined by Transmission Electron Microscopy (TEM). Agar well diffusion assay was used to test the antimicrobial efficacy and durability of both pellet suspension and supernatant of silver-killed E. coli O104:H4 against other bacteria. Both silver-killed bacteria and supernatant showed prolonged antimicrobial activity against the tested strains that extended to 40 days. The presence of adsorbed silver nanoparticles on the bacterial cell and inside the cells was verified by TEM. Silver-killed bacteria serve as an efficient sustained release reservoir for exporting the lethal silver cations. This promotes its use as a powerful disinfectant for polluted water and as an effective antibacterial which can be included in wound and burn dressings to overcome the problem of wound contamination.

The Locus of Heat Resistance Confers Resistance to Chlorine and Other Oxidizing Chemicals in Escherichia coli.

Some chlorine-resistant Escherichia coli isolates harbor the locus of heat resistance (LHR), a genomic island conferring heat resistance. In this study, the protective effect of the LHR for cells challenged by chlorine and oxidative stress was quantified. Cloning of the LHR protected against NaClO (32 mM; 5 min), H2O2 (120 mM; 5 min), and peroxyacetic acid (105 mg/liter; 5 min) but not against 5.8 mM KIO4, 10 mM acrolein, or 75 mg/liter allyl isothiocyanate. The lethality of oxidizing treatments for LHR-negative strains of E. coli was about 2 log10 CFU/ml higher than that for LHR-positive strains of E. coli The oxidation of cytoplasmic proteins and membrane lipids was quantified with the fusion probe roGFP2-Orp1 and the fluorescent probe BODIPY581/591, respectively. The fragment of the LHR coding for heat shock proteins protected cytoplasmic proteins but not membrane lipids against oxidation. The middle fragment of the LHR protected against the oxidation of membrane lipids but not of cytoplasmic proteins. The addition of H2O2, NaClO, and peroxyacetic acid also induced green fluorescent protein (GFP) expression in the oxidation-sensitive reporter strain E. coli O104:H4 Δstx2::gfp::amp Cloning of pLHR reduced phage induction in E. coli O104:H4 Δstx2::gfp::amp after treatment with oxidizing chemicals. Screening of 160 strains of Shiga toxin-producing E. coli (STEC) revealed that none of them harbors the LHR, additionally suggesting that the LHR and Stx prophages are mutually exclusive. Taking our findings together, the contribution of the LHR to resistance to chlorine and oxidative stress is based on the protection of multiple cellular targets by different proteins encoded by the genetic island.IMPORTANCE Chlorine treatments are used in water and wastewater sanitation; the resistance of Escherichia coli to chlorine is thus of concern to public health. We show that a genetic island termed the locus of heat resistance (LHR) protects E. coli not only against heat but also against chlorine and other oxidizing chemicals, adding to our knowledge of the tools used by E. coli to resist stress. Specific detection of the oxidation of different cellular targets in combination with the cloning of fragments of the LHR provided insight into mechanisms of protection and demonstrated that different fragments of the LHR protect different cellular targets. In E. coli, the presence of the LHR virtually always excluded other virulence factors. It is tempting to speculate that the LHR is maintained by strains of E. coli with an environmental lifestyle but is excluded by pathogenic strains that adapted to interact with vertebrate hosts.

Plant variety and soil type influence Escherichia coli O104:H4 strain C227/11ϕcu adherence to and internalization into the roots of lettuce plants.

Human disease outbreaks caused by pathogenic Escherichia coli are increasingly associated with the consumption of contaminated fresh produce. Internalization of enteroaggregative/enterohemorrhagic E. coli (EAEC/EHEC) strains into plant tissues may present a serious threat to public health. In the current study, the ability of the fluorescing Shiga toxin-negative E. coli O104:H4 strain C227/11ϕcu/pKEC2 to adhere to and to internalize into the roots of Lactuca sativa and Valerianella locusta grown in diluvial sand (DS) and alluvial loam (AL) was investigated. In parallel, the soil microbiota was analyzed by partial 16S rRNA gene sequencing. The experiments were performed in a safety level 3 greenhouse to simulate agricultural practice. The adherence of C227/11ϕcu/pKEC2 to the roots of both plant varieties was increased by at least a factor three after incubation in DS compared to AL. Compared to V. locusta, internalization into the roots of L. sativa was increased 12-fold in DS and 108-fold in AL. This demonstrates that the plant variety had an impact on the internalization ability, whereas for a given plant variety the soil type also affected bacterial internalization. In addition, microbiota analysis detected the inoculated strain and showed large differences in the bacterial composition between the soil types.

Differential transcriptome analysis of enterohemorrhagic Escherichia coli strains reveals differences in response to plant-derived compounds.

BACKGROUND: Several serious vegetable-associated outbreaks of enterohemorrhagic Escherichia coli (EHEC) infections have occurred during the last decades. In this context, vegetables have been suggested to function as secondary reservoirs for EHEC strains. Increased knowledge about the interaction of EHEC with plants including gene expression patterns in response to plant-derived compounds is required. In the current study, EHEC O157:H7 strain Sakai, EHEC O157:H- strain 3072/96, and the EHEC/enteroaggregative E. coli (EAEC) hybrid O104:H4 strain C227-11φcu were grown in lamb's lettuce medium and in M9 minimal medium to study the differential transcriptional response of these strains to plant-derived compounds with RNA-Seq technology. RESULTS: Many genes involved in carbohydrate degradation and peptide utilization were similarly upregulated in all three strains, suggesting that the lamb's lettuce medium provides sufficient nutrients for proliferation. In particular, the genes galET and rbsAC involved in galactose metabolism and D-ribose catabolism, respectively, were uniformly upregulated in the investigated strains. The most prominent differences in shared genome transcript levels were observed for genes involved in the expression of flagella. Transcripts of all three classes of the flagellar hierarchy were highly abundant in strain C227-11φcu. Strain Sakai expressed only genes encoding the basal flagellar structure. In addition, both strains showed increased motility in presence of lamb's lettuce extract. Moreover, strain 3072/96 showed increased transcription activity for genes encoding the type III secretion system (T3SS) including effectors, and was identified as a powerful biofilm-producer in M9 minimal medium. CONCLUSION: The current study provides clear evidence that EHEC and EHEC/EAEC strains are able to adjust their gene expression patterns towards metabolization of plant-derived compounds, demonstrating that they may proliferate well in a plant-associated environment. Moreover, we propose that flagella and other surface structures play a fundamental role in the interaction of EHEC and EHEC/EAEC with plants.

The Role of the AggR Regulon in the Virulence of the Shiga Toxin-Producing Enteroaggregative Escherichia coli Epidemic O104:H4 Strain in Mice.

An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a large outbreak of bloody diarrhea and the hemolytic uremic syndrome in 2011. We previously developed an ampicillin (Amp)-treated C57BL/6 mouse model to measure morbidity (weight loss) and mortality of mice orally infected with the prototype Stx-EAEC strain C227-11. Here, we hypothesized that mice fed C227-11 cured of the pAA plasmid or deleted for individual genes on that plasmid would display reduced virulence compared to animals given the wild-type (wt) strain. C227-11 cured of the pAA plasmid or deleted for the known pAA-encoded virulence genes aggR, aggA, sepA, or aar were fed to Amp-treated C57BL/6 mice at doses of 1010-1011CFU. Infected animals were then either monitored for morbidity and lethality for 28 days or euthanized to determine intestinal pathology and colonization levels at selected times. The pAA-cured, aggR, and aggA mutants of strain C227-11 all showed reduced colonization at various intestinal sites. However, the aggR mutant was the only mutant attenuated for virulence as it showed both reduced morbidity and mortality. The aar mutant showed increased expression of the aggregative adherence fimbriae (AAF) and caused greater systemic effects in infected mice when compared to the C227-11 wt strain. However, unexpectedly, both the aggA and aar mutants displayed increased weight loss compared to wt. The sepA mutant did not exhibit altered morbidity or mortality in the Amp-treated mouse model compared to wt. Our data suggest that the increased morbidity due to the aar mutant could possibly be via an effect on expression of an as yet unknown virulence-associated factor under AggR control.

Estandarización de una técnica de PCR múltiple para la detección de los serogrupos O157, O104 y "big six" de Escherichia coli productora de la toxina Shiga (STEC) / Standardization of a multiplex PCR technique for the detection of Shiga toxin-producing Escherichia coli (STEC) serogroups O157, O104 and "big six"

Los serogrupos O26, O45, O103, O104, O111, O121, O145 y O157 de STEC se relacionan con un elevado número de casos de SUH a nivel mundial, por lo que están incluidos dentro de las categorías de mayor riesgo para los humanos, según los criterios de autoridades alimentarias de Estados Unidos y Europa. El método convencional de identificación de antígenos O y H se realiza por aglutinación con antisueros de conejo. Este método además de ser muy costoso y laborioso, no se encuentra disponible en el país para empleo masivo. En este contexto, el objetivo de este estudio observacional descriptivo de corte transverso ha sido la estandarización de una técnica de PCR múltiple para la detección de estos 8 serogrupos, a fin de contar con un sistema de detección eficiente, sensible y con potencial de aplicación en la industria alimentaria. Se estandarizaron reacciones de PCR empleando como controles positivos cepas E. coli de referencia correspondientes a la totalidad de los serogrupos citados. Se obtuvieron productos de tamaños esperados para cada serogrupo, no se observaron amplificaciones cruzadas o falsos positivos. Esta técnica estandarizada podría representar una herramienta rápida y menos costosa que la técnica serológica, con la capacidad de ser aplicada a diferentes matrices, permitiendo la detección de estos serogrupos en aislados STEC de ganado en pie, fuentes de agua de consumo, alimentos e incluso en aislamientos clínicos asociados a enfermedades humanas(AU)

STEC serogroups O26, O45, O103, O104, O111, O121, O145, and O157, are related to a high number of cases of HUS worldwide, so they are included in the categories of greatest risk for humans, according to the food administration criteria of the United States and Europe. The conventional method of identifying antigens O and H is carried out by agglutination with rabbit antisera. This method is very expensive and laborious and is not available in the country for massive-scale use. In this context, the objective of this cross-sectional descriptive observational study has been the standardization of a multiplex PCR technique for the detection of these 8 serogroups, in order to have an efficient and sensitive detection system with the potential for application in the food industry. PCR reactions were standardized using as positive controls reference E. coli strains to correspond to all the mentioned serogroups. Products of expected sizes were obtained for each serogroup; no cross-amplification or false positives were observed. This standardized technique could represent a quick and less expensive tool than the serological technique, with the possibility to be applied to different kind of samples, allowing the detection of these serogroups in STEC isolates of live cattle, sources of drinking water, food and even in clinical isolates associated with human diseases(AU)

A non-beta-lactam antibiotic inhibitor for enterohemorrhagic Escherichia coli O104:H4.

The overuse of antibiotics has caused an increased prevalence of drug-resistant bacteria. Bacterial resistance in E. coli is regulated via production of ß-lactam-hydrolyzing ß-lactamases enzymes. Escherichia coli O104: H4 is a multi-drug resistant strain known to resist ß-lactam as well as several other antibiotics. Here, we report a molecular dynamic simulation-combined docking approach to identify, screen, and verify active pharmacophores against enterohemorrhagic Escherichia coli (EHEC). Experimental studies revealed a boronic acid cyclic monomer (BACM), a non-ß-lactam compound, to inhibit the growth of E. coli O104: H4. In vitro Kirby Bauer disk diffusion susceptibility testing coupled interaction analysis suggests BACM inhibits E. coli O104:H4 growth by not only inhibiting the ß-lactamase pathway but also via direct inhibition of the penicillin-binding protein. These results suggest that BACM could be used as a lead compound to develop potent drugs targeting beta-lactam resistant Gram-negative bacterial strains. KEY MESSAGES: • An in silico approach was reported to identify pharmacophores against E. coli O104: H4. • In vitro studies revealed a non-ß-lactam compound to inhibit the growth of E. coli O104: H4. • This non-ß-lactam compound could be used as a lead compound for targeting beta-lactam strains.

Shiga toxin-induced haemolytic uraemic syndrome and the role of antibiotics: a global overview.

OBJECTIVES: The administration of antibiotics in infections caused by Shiga toxin producing E. coli (STEC) strains, such as O157:H7, was and remains controversial, as it has been associated with the development of haemolytic uraemic syndrome (HUS). We conducted a literature review to better examine this association. METHODS: We searched the PubMed and Google Scholar databases for relevant articles, using the key words: ``haemolytic uraemic syndrome'', ``Shiga toxin'', ``E. coli O157:H7'', ``E. coli O104:H4'', ``STEC colitis'', ``STEC antibiotics'', ``STEC fosfomycin'', ``STEC trimethoprim sulfamethoxazole'', ``STEC fluoroquinolones'', ``STEC ciprofloxacin'', ``STEC rifaximin'', ``STEC gentamycin'', ``STEC colistin'', "Shiga toxin binding agent", "Shiga toxin monoclonal antibody" and ``STEC Japan epidemic''. RESULTS: Numerous studies report that antibiotics increase the risk of HUS development, while others report that antibiotics do not have any effect or can even reduce the rate of HUS development in STEC infections. The infecting STEC strain, the type of antibiotic as well as the timing of its administration appear to significantly affect the development of HUS in a STEC infected patient. CONCLUSIONS: It appears that, while some antibiotics such as b-lactams and TMP/SMX may be detrimental, others appear to be safe and can prevent the development of HUS. Of note, fosfomycin appears to be the antibiotic with the most positive results from clinical studies, and may be able to avert HUS development, especially if administered within the first two or three days from diarrhoea onset. Fluoroquinolones have also shown positive outcomes in clinical studies, despite demonstrating unfavourable results in in vitro studies. Other agents, such as colistin, gentamycin and rifamycins, have shown promising results in in vitro studies and require further evaluation.