Urease genes in non-O157 Shiga toxin-producing Escherichia coli: mostly silent but valuable markers for pathogenicity

The distribution of ureC was investigated among 294 Escherichia coli isolates, comprising 72 strains from the E. coli standard reference collection (ECOR), 62 strains from the diarrhoeagenic E. coli (DEC) collection, and 160 clinical isolates of Shiga toxin-producing E. coli (STEC). The ureC gene was more frequent among STEC isolates harbouring eae than among those lacking eae (p < 0.0001). All clinical STEC isolates of serogroups O111 and O145 contained ureC, but only two of 294 isolates expressed urease activity. The silencing of urease expression could not be linked to a stop codon in ureD. The frequent occurrence of ure genes in eae-positive STEC isolates makes them valuable markers for virulence.     

Prevalence, characterisation and clinical profiles of Shiga toxin-producing Escherichia coli in The Netherlands.

Detection of Shiga toxin-producing Escherichia coli (STEC) in The Netherlands is traditionally limited to serogroup O157. To assess the relative importance of STEC, including non-O157 serogroups, stool samples submitted nationwide for investigation of enteric pathogens or diarrhoea were screened with real-time PCR for the presence of the Shiga toxin genes. Patients were selected if their stool contained blood upon macroscopic examination, if they had a history of bloody diarrhoea, were diagnosed with haemolytic uraemic syndrome, or were aged <6 years (irrespective of the bloody aspect of the stool). PCR-positive stools were forwarded to a central laboratory for STEC isolation and typing. In total, 4069 stools were examined, with 68 (1.7%) positive PCR results. The highest prevalence was for stools containing macroscopic blood (3.5%), followed by stools from patients with a history of bloody diarrhoea (2.4%). Among young children, the prevalence (1.0%) was not significantly higher than among random, non-bloody, stool samples from diarrhoeal patients (1.4%). STEC strains were isolated from 25 (38%) PCR-positive stools. Eleven O-serogroups were detected, including five STEC O157 strains. As serogroup O157 represented only 20% of the STEC isolates, laboratories should be encouraged to use techniques enabling them to detect non-O157 serogroups, in parallel with culture, for isolation and subsequent characterisation of STEC strains for public health surveillance and detection of outbreaks.     

非O157产志贺毒素大肠杆菌分离株亚碲酸钾抗性水平及抗性基因簇分析

目的 了解我国部分地区非O157产志贺毒素大肠杆菌(Shiga toxin-producing Escherichia coli,STEC)分离株的亚碲酸钾抗性水平、抗性基因簇及其关系.方法 使用平皿法检测亚碲酸钾抗性水平,采用PCR方法检测亚碲酸钾抗性基因簇.结果 在所检测的39株非O157 STEC中,仅有5株菌亚碲酸钾抗性水平介于128～512 μg/ml,同时携带亚碲酸钾抗性基因簇(terABCDE).另有2株菌的亚碲酸钾抗性水平为8 μg/ml,3株菌为2 μg/ml,其余29株菌均＜1 μg/ml,且这34株菌terABCDE均阴性.结论 大多数非O157 STEC分离株对亚碲酸钾敏感,在使用含亚碲酸钾的选择性培养基分离非O157 STEC时应慎重.     

Clonal diversity of Shiga toxin-producing Escherichia coli O157:H7/H- in Germany--a ten-year study

Two hundred and ten E. coli O157:H7/H- strains isolated from single cases and outbreaks of diarrhoea and haemolytic uraemic syndrome (HUS) in Germany between 1988 and 1998 were characterised by a range of molecular subtyping methods and phage typing in order to analyse their clonal nature. A high clonal heterogeneity, together with a considerable clonal stability, has been identified among the bacterial isolates and no single clonal type appeared to be geographically dominant. It is recommended to apply pulsed-field gel electrophoresis (PFGE) together with P gene profile determination (number and genomic positions of lambdoid bacteriophages) as laboratory tools for an extended epidemiological surveillance of E. coli OOFF phage typing will remain helpful as a first line of analysis, particularly in outbreak situations.     

A family outbreak of haemolytic uraemic syndrome and haemorrhagic colitis caused by verocytotoxigenic Escherichia coli O157 from unpasteurised cow''s milk in Slovakia

This report describes a family outbreak of verocytotoxigenic Escherichia coli O157 (VTEC) infection, involving nine persons from one extended family, which occurred in eastern Slovakia. Three children suffered from haemolytic uraemic syndrome, two children had bloody diarrhoea, and four adults were asymptomatic carriers. Fourteen sorbitol-non-fermenting E. coli O157 isolates harbouring the vtx2, eae and ehxA genes were obtained. Verocytotoxin 2 activity was demonstrated in all 14 isolates. After epidemiological surveillance, the source of infection was identified as unpasteurised cow's milk.     

Designation of O174 and O175 to temporary O groups OX3 and OX7, and six new E. coli O groups that include Verocytotoxin-producing E. coli (VTEC): O176, O177, O178, O179, O180 and O181

Two temporary Escherichia coli O group strains OX3 and OX7 are given permanent status as O174 and O175, respectively. Both these test strains were originally isolated from cases of human diarrhoea. Whereas the O174 strain is negative for known virulence genes, the O175 strain is positive with the probe derived from the CVD432 plasmid associated with the aggregative adherence phenotype, the Enteroaggregative heat-stable enterotoxin 1 gene (astA) and daaC (F1845 afimbrial adhesin) associated with the diffuse adherence (DA) phenotype. Additionally, six E. coli strains are established as antigenic test strains for six new O groups, designated O176, O177, O178, O179, O180 and O181. All six strains produced Verocytotoxin and were positive for vtx1, vtx2, or both genes. Additional virulence genes associated with diarrhoeal disease in humans were found in four of the strains. O176 and O177 strains were isolated from calves, O178 and O181 strains from meat, the O179 strain was from human bloody diarrhoea, and the O180 strain from swine. Preliminary data on the occurrence and epidemiology of these eight new O groups amongst groups of diarrhoeagenic E. coli are reviewed.     

Evidence of transmission of verocytotoxin-producing Escherichia coli O111 from a cattle stable to a child

Infections with verocytotoxigenic Escherichia coli (VTEC) other than O157 have been assumed to have the same epidemiology as those with VTEC O157, but the source of infection is rarely defined for sporadic cases. This report describes a child with VTEC O111:H- infection who was probably infected by playing in a cattle stable and/or by drinking raw milk from the cows in this stable. E. coli O111 isolates colonising the cattle were indistinguishable from the patient isolate by the use of serotyping, pulsed-field gel electrophoresis, and virulence profiling.     

Bacterial and epidemiologic study of the resistance to oxyiminocephalosporins in Escherichia coli in a French hospital

Objective: To understand the mechanisms and epidemiology of resistance to oxyiminocephalosporins in Escherichia coli over a 2-year period in a French hospital.
Methods: Forty-four strains, resistant or intermediately resistant to one of the oxyiminocephalosporins or aztreonam, were collected from 35 patients. MIC determinations were carried out for the 44 isolates using a panel of β-lactam antibiotics, and characterization of the β-lactamases they produced by isoelectric focusing and catalytic activity measurement. Extended-spectrum β-lactamase production was studied by use of the double disk diffusion test. Conjugation experiments were used to search for plasmidic cephalosporinase. An epidemiologic study was then performed, by use of molecular typing of the strains with an ERIC-PCR method and a case-control analysis.
Results: Less than 1% of all the E. coli isolates at our hospital showed decreased susceptibility to oxyiminocephalosporins. Only three of the 44 isolates showed synergy between clavulanate and a third-generation cephalosporin and produced an extended-spectrum β-lactamase. For the other strains, a β-lactamase with a highly basic isoelectric point was detected. Spectrophotometric measures confirmed that most of these isolates were AmpC hyper-producers. No plasmidic cephalosporinase could be detected by conjugation experiments. Molecular typing showed all isolates to be different, except for two strains isolated in two patients of the same hospital unit, and for the repeated isolates of some patients. When 20 case patients were compared to 40 randomly selected control patients, prior receipt of an antimicrobial and more specifically of a β-lactam agent was significantly associated with case patients.
Conclusions: Although it appears to be very rare, the resistance to broad-spectrum cephalosporins needs our attention, because of the high frequency of E. coli infections and β-lactam use in their treatment.     

Shiga toxin-producing Escherichia coli: incidence and clinical features in a setting with complete screening of patients with suspected infective diarrhoea

ObjectivesShiga toxin-producing Escherichia coli (STEC) causes diarrhoeal disease, bloody diarrhoea, and haemolytic uraemic syndrome. The aim of this study was to describe the incidence of STEC and the clinical features of STEC patients from a well-defined Danish population in which all fecal samples of patients with suspected infective gastroenteritis were analysed for STEC.MethodsIn this population-based cohort study, all stool samples referred to two clinical microbiology laboratories were screened for STEC by culture and/or PCR. Epidemiological (n=170) and clinical (n=209) characteristics were analysed using data from local and national registries.ResultsOverall, 75,132 samples from 30,073 patients were screened resulting in 217 unique STEC-isolates. The epidemiological analysis showed an incidence of 10.1 cases per 100,000 person-years, which was more than twofold higher than the incidence in the rest of Denmark (3.4 cases per 100,000 person-years, p <0.001). Three groups were associated with a higher incidence: age <5 years (n=28, p <0.001), age ≥65 years (n=38, p 0.045), and foreign ethnicity (n=27, p 0.003). In the clinical analysis, patients with STEC harbouring only the Shiga toxin 1 gene (stx₁-only isolates) showed a lower frequency of acute (n=11, p <0.05) and bloody diarrhoea (n=5, p <0.05) and a higher frequency of gastrointestinal symptoms for ≥3 months (n=8, p <0.05) than the other STEC patients.ConclusionsWe report a more than twofold higher incidence in the project area compared with the rest of Denmark, indicating that patients remain undiagnosed when selective STEC screening is used. We found an association between patients with stx₁-only isolates and long-term gastrointestinal symptoms.     

Oral immunization of mice with Lactococcus lactis expressing Shiga toxin truncate confers enhanced protection against Shiga toxins of Escherichia coli O157:H7 and Shigella dysenteriae

Regardless of the communal impact of Shiga toxins, till today neither a specific treatment nor licensed vaccine is available. Lactococcus lactis (L. lactis), generally regarded as safe organism, is well known to provide a valuable approach regarding the oral delivery of vaccines. This study was undertaken to evaluate the protective efficacy of Stx2a1 expressed in nisin‐inducible L. lactis, against Shiga toxins (Stx1, Stx2) in mouse model. Oral immunization of BALB/c mice with LL‐Stx2a1 elicited significant serum antibody titer with elevated fecal and serum IgA, along with minimized intestinal and kidney damage resulting in survival of immunized animals at 84% and 100% when challenged with 10 × LD₅₀ of Escherichia coli O157 and Shigella dysenteriae toxins, respectively. HeLa cells incubated with immune sera and toxin mixture revealed high neutralizing capacity with 90% cell survivability against both the toxins. Mice immunized passively with both toxins and antibody mixture survived the observation period of 15 days, and the controls administered with sham sera and toxins were succumbed to death within 3 days. Our results revealed protective efficacy and toxin neutralization ability of LL‐Stx2a1, proposing it as an oral vaccine candidate against Shiga toxicity mediated by E. coli O157 and S. dysenteriae.     

How to: molecular investigation of a hospital outbreak

BackgroundStudying hospital outbreaks by using molecular tools, i.e. synthesizing the molecular epidemiology data to its appropriate clinical-epidemiologic context, is crucial in order to identify infection source, infer transmission dynamics, appropriately allocate prevention resources and implement control measures. Whole-genome sequencing (WGS) of pathogens has become the reference standard, as it is becoming more accessible and affordable. Consequently, sequencing of the full pathogen genome via WGS and major progress in fit-for-purpose genomic data analysis tools and interpretation is revolutionizing the field of outbreak investigations in hospitals. Metagenomics is an additional evolving field that might become commonly used in the future for outbreak investigations. Nevertheless, practitioners are frequently limited in terms of WGS or metagenomics, especially for local outbreak analyses, as a result of costs or logistical considerations, reduced or lack of locally available resources and/or expertise. As a result, traditional approaches, including pulsed-field gel electrophoresis, repetitive-element palindromic PCR and multilocus sequence typing, along with other typing methods, are still widely used.AimsTo provide practitioners with evidenced-based action plans for usage of the various typing techniques in order to investigate the molecular epidemiology of nosocomial outbreaks, of clinically significant pathogens in acute-care hospitals.SourcesPubMed search with relevant keywords along with personal collection of relevant publications.ContentRepresentative case scenarios and critical review of the relevant scientific literature.ImplicationsThe review provides practical action plans to manage molecular epidemiologic investigations of outbreaks caused by clinically significant nosocomial pathogens, while prioritizing the use and timely integration of the various methodologies.     

Shiga toxin 2 overexpression in Escherichia coli O157:H7 strains associated with severe human disease

Variation in disease severity among Escherichia coli O157:H7 infections may result from differential expression of Shiga toxin 2 (Stx2). Eleven strains belonging to four prominent phylogenetic clades, including clade 8 strains representative of the 2006 U.S. spinach outbreak, were examined for stx2 expression by real-time PCR and western blot analysis. Clade 8 strains were shown to overexpress stx2 basally, and following induction with ciprofloxacin when compared to strains from clades 1–3. Differences in stx2 expression generally correlated with Stx2 protein levels. Single-nucleotide polymorphisms identified in regions upstream of stx2AB in clade 8 strains were largely absent in non-clade 8 strains. This study concludes that stx2 overexpression is common to strains from clade 8 associated with hemolytic uremic syndrome, and describes SNPs which may affect stx2 expression and which could be useful in the genetic differentiation of highly-virulent strains.     

Development of a monoclonal antibody-based ELISA to detect Escherichia coli O157:H7

A pair of monoclonal antibodies (mAb) from 10 murine hybridomas secreting Escherichia coli O157:H7 (E. coli O157:H7)-specific mAbs were selected for the development of the sandwich ELISA to detect E. coli O157:H7. On the basis of pairwise interaction analysis, mAb-1 was selected as a capture antibody while mAb-6 was used as a detection antibody. The buffer system which provided the greatest difference between the specific E. coli O157:H7-positive antigen and the negative control was chosen. This sandwich ELISA showed good linearity when the concentration of E. coli O157:H7 was in the range of 10⁵–10⁸ cfu/mL, and the sensitivity was 1×10⁴ cfu/mL. With 8-h enrichment of bacteria, this ELISA was found to detect 0.4 cfu/g E. coli O157:H7 in artificially contaminated green tea samples.     

Molecular characterisation of enteroinvasive Escherichia coli O136:K78 isolates from patients of a diarrhoea outbreak in China

Purpose: A diarrhoea outbreak occurred in a kindergarten, which caused 21 relevant infected cases. Our object was to confirm the pathogens and their molecular characterisation. Materials and Methods: Faecal samples from 21 patients were collected on the 3^rd day after their symptom onset, and a regular epidemiological investigation was conducted. Bacterial isolation was performed in accordance with standard laboratory protocol, serological and molecular characterisations were determined by serum agglutination test and real-time polymerase chain reaction (PCR) method, respectively. The pulsed field gel electrophoresis (PFGE) and 16S rRNAs were conducted to determine the homology. Results: Eleven enteroinvasive Escherichia coli (EIEC) O136:K78 strains were isolated. The serum agglutination test showed that all strains’ serotypes were E. coli (EIEC) O136:K78. Real-time PCR showed that 10 (91%) strains carried the invasion plasmid antigen H gene (ipaH), carried by all four Shigella species and EIEC. The strain that didn’t carry the ipaH gene had different biochemical reactions of L-lizyna and L-rhamnose with the other strains. The complete 16S rRNA sequences showed 98.4% identity between ipaH-negative isolate and the others, and the PFGE indicated that the ipaH-negative isolate was not homological with other isolates in this diarrhoea outbreak. Conclusions: The diarrhoea outbreak was caused by E. coli (EIEC) O136:K78.     

Characteristics of Shiga toxin producing- and enteropathogenic Escherichia coli of the emerging serotype O80:H2 isolated from humans and diarrhoeic calves in Belgium

Objectives

Recently a highly virulent Escherichia coli O80:H2 pathotype carrying Shiga toxin genes, the intimin subtype eaeξ, and genes associated with the extraintestinal pathogenic E. coli (ExPEC) pS88 plasmid was described in France. In this study we examine the relatedness of Belgian E. coli O80:H2 isolated from humans and diarrhoeic calves as well their similarities with the French pathotype.

Epidemiological characterization of a nosocomial outbreak of extended spectrum β‐lactamase Escherichia coli ST‐131 confirms the clinical value of core genome multilocus sequence typing

Enhanced precision of epidemiological typing in clinically suspected nosocomial outbreaks is crucial. Our aim was to investigate whether single nucleotide polymorphism (SNP) analysis and core genome (cg) multilocus sequence typing (MLST) of whole genome sequencing (WGS) data would more reliably identify a nosocomial outbreak, compared to earlier molecular typing methods. Sixteen isolates from a nosocomial outbreak of ESBL E. coli ST‐131 in southeastern Sweden and three control strains were subjected to WGS. Sequences were explored by SNP analysis and cgMLST. cgMLST clearly differentiated between the outbreak isolates and the control isolates (>1400 differences). All clinically identified outbreak isolates showed close clustering (≥2 allele differences), except for two isolates (>50 allele differences). These data confirmed that the isolates with >50 differing genes did not belong to the nosocomial outbreak. The number of SNPs within the outbreak was ≤7, whereas the two discrepant isolates had >700 SNPs. Two of the ESBL E. coli ST‐131 isolates did not belong to the clinically identified outbreak. Our results illustrate the power of WGS in terms of resolution, which may avoid overestimation of patients belonging to outbreaks as judged from epidemiological data and previously employed molecular methods with lower discriminatory ability.     

Escherichia coli O157: Insights into the adaptive stress physiology and the influence of stressors on epidemiology and ecology of this human pathogen

Abstract

Escherichia coli O157, a foodborne pathogen of major concern for public health, has been associated with numerous outbreaks of haemorrhagic colitis and hemolytic uremic syndrome worldwide. Human infection with E. coli O157 has been primarily associated with the food-chain transmission route. This transmission route commonly elicits a multi-faceted adaptive stress response of E. coli O157 for an extended period of time prior to human infection. Several recent research articles have indicated that E. coli O157:H7 has evolved unique survival characteristics which can affect the epidemiology and ecology of this zoonotic pathogen. This review article summarizes the recent knowledge of the molecular responses of E. coli O157 to the most common stressors found within the human food chain, and further emphasizes the influence of these stressors on the epidemiology and ecology of E. coli O157.