Application of multilocus variable number tandem repeat analysis to monitor Verocytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales: emergence of a profile associated with a national outbreak.

AIMS: Evaluation of multilocus variable number tandem repeat analysis (MLVA) to subtype all isolates of Vero cytotoxin-producing Escherichia coli O157 phage type 8 in England and Wales. METHODS AND RESULTS: Over a 13 month period from December 2010, 483 isolates of VTEC O157 PT8 were tested by MLVA; 39% were received in the first 4 months of 2011, when infections are generally low. One profile, or single locus variants of it, was present in 249 (52%) isolates but was not common previously. These cases represented a national increase in PT8, associated epidemiologically with soil-contaminated vegetables. Most of the 177 other MLVA profiles were unique to a single isolate. Profiles shared by >1 isolate included cases from two small community, food-borne outbreaks and 11 households. Several shared profiles were found among 23 isolates without known links. Apart from one group, isolates linked to travel abroad had very diverse profiles. CONCLUSIONS: Multilocus variable number tandem repeat analysis discriminated apparent sporadic isolates of the same PT and assisted in detection of cases in an emerging national outbreak. SIGNIFICANCE AND IMPACT OF THE STUDY: Multilocus variable number tandem repeat analysis is an epidemiologically valid complement to surveillance and applicable as a rapid, practical test for large numbers of isolates.

Ongoing outbreak of Shigella flexneri serotype 3a in men who have sex with men in England and Wales, data from 2009-2011.

Diagnoses of Shigella flexneri in the United Kingdom (UK) are usually travel-related. However, since 2009, there has been an overall increase in UK-acquired cases. The Health Protection Agency has been investigating a national outbreak of S. flexneri detected in 2011 and which is still ongoing. Cases occurred mostly in men who have sex with men and were of serotype 3a. The investigation aimed at obtaining epidemiological data to inform targeted outbreak management and control.

Shiga toxin-producing Escherichia coli O157 associated with human infections in Switzerland, 2000-2009.

Shiga toxin-producing Escherichia coli (STEC), an important foodborne pathogen, can cause mild to severe bloody diarrhoea (BD), sometimes followed by life-threatening complications such as haemolytic uraemic syndrome (HUS). A total of 44 O157 strains isolated from different patients from 2000 through 2009 in Switzerland were further characterized and linked to medical history data. Non-bloody diarrhoea was experienced by 15.9%, BD by 61.4% of the patients, and 29.5% developed HUS. All strains belonged to MLST type 11, were positive for stx2 variants (stx2 and/or stx2c), eae and ehxA, and only two strains showed antibiotic resistance. Of the 44 strains, nine phage types (PTs) were detected the most frequent being PT32 (43.2%) and PT8 (18.2%). By PFGE, 39 different patterns were found. This high genetic diversity within the strains leads to the conclusion that STEC O157 infections in Switzerland most often occur as sporadic cases.

Verocytotoxigenic Escherichia coli O157 in animals on public amenity premises in England and Wales, 1997 to 2007.

At the request of the public health authorities, 31 public amenity premises in England and Wales containing animals of various species were investigated for the presence of verocytotoxigenic Escherichia coli (VTEC) O157 between 1997 and 2007, because of putative associations with human cases. VTEC O157 was confirmed in one or more species on 19 (61.3 per cent) of the premises. There were significant associations between the presence of VTEC O157 and the number of species sampled, the size of the enterprise, the presence of young cattle and the presence of adult pigs. E coli O157 was isolated from 305 (17.8 per cent) of 1715 samples taken from all the premises, and verocytotoxin genes were detected by PCR in 184 (98.4 per cent) of 187 representative isolates. On positive premises, the highest mean proportion of positive samples (29.0 per cent) was in cattle, followed by sheep (24.4 per cent), donkeys (14.6 per cent), pigs (14.3 per cent), horses (12.3 per cent) and goats (9.9 per cent). A high proportion of positive samples was obtained from camelid species sampled on three of the premises. The main phage types (PT) were 2 and 21/28, which were those most commonly isolated from human cases during the same period. A single PT was detected on 14 of the 19 positive premises, with up to six different species having the same PT.

Incidence of K1 antigen in Escherichia coli isolated from blood and cerebrospinal fluid of patients in the United Kingdom.

Altogether 411 cultures of Escherichia coli isolated from blood and 60 from cerebrospinal fluid (CSF) of patients in the United Kingdom were identified biochemically and serologically. They were tested for the presence of K1 antigen by an antiserum-agar method using horse meningococcus group B antiserum and by slide agglutination using E. coli 07.K1.H-antiserum. In total 71 cultures from blood (17%) and 29 from CSF (48%) gave positive results by both methods and were considered to possess the K1 antigen. Among the cultures from patients less than 3 years of age the K1 antigen was found significantly more often in those isolated from CSF (53%) than in those from blood (29%). The K1 antigen was found significantly more frequently in cultures isolated from the blood of patients less than 3 years old (29%) than in those from the blood of older patients (13%). Cultures which gave negative results in the slide agglutination test also gave negative results by the antiserum-agar method but positive results obtained by slide agglutination were not always confirmed using the antiserum-agar technique. Slide agglutination was considered to be valuable for the elimination of K1 negative cultures, but positive results required confirmation using the antiserum-agar method.

Strains of Escherichia coli O157:H8 from human diarrhoea belong to attaching and effacing class of E coli.

AIMS: To determine whether 17 Escherichia coli O157:H8 strains isolated from patients with diarrhoea in the United Kingdom were putative pathogens. METHODS: The strains had been isolated by the use of O157 antiserum, available for the detection of Vero cytotoxin (VT) producing strains of E coli O157 that are usually of flagellar (H) type 7, but may also be non-motile. The strains were examined for VT production, for their ability to adhere to HEp-2 cells, and for hybridisation with several DNA probes that recognise pathogenic properties of E coli. Their ability to ferment sorbitol and to produce beta-glucuronidase was also investigated, as these tests are used to discriminate VT positive O157 strains. RESULTS: The O157:H8 strains did not produce VT. All gave localised attachment to HEp-2 cells, associated with a positive fluorescence-actin staining test, and all hybridised with the E coli attaching and effacing (eae) probe. In addition to the difference in VT production, O157:H8 strains could be distinguished from VT positive O157 strains by their beta-glucuronidase activity, their failure to produce enterohaemolysin, and their lack of hybridisation with the CVD419 probe derived from a plasmid in an O157:H7 strain. CONCLUSIONS: The 0157:H8 strains had in vitro properties characteristic of the class of E coli that causes attaching and effacing lesions in epithelial intestinal cells. They may therefore be considered a putative cause of diarrhoea but their prevalence remains to be established. Several O157:H8 strains failed to ferment sorbitol in agar plates and therefore could be misidentified as VT positive O157 strains. Confirmatory tests for VT production are needed when O157 strains are isolated from faeces.

Resistance to ciprofloxacin in pathogenic Enterobacteriaceae in England and Wales in 1996.

In 1996, 6% of Escherichia coli from extraintestinal infections were resistant to ciprofloxacin with minimum inhibitory concentrations (MICs) > or = 2 mg/l (high level resistance). Low level resistance (MIC 0.125-1 mg/l) was also identified in 7% of Salmonella typhi, 4% of S paratyphi A, and 4% of non-typhoidal salmonellas. However, resistance to ciprofloxacin was rarely identified in shigellas. For E coli, physicians should be aware that treatment failures may occur when patients with invasive illness are treated with ciprofloxacin before the results of laboratory sensitivity tests are available. For salmonellas an increasing number of treatment failures have been recorded for patients infected with strains with low level resistance. Because of the increasing incidence of Enterobacteriaceae with low level resistance to ciprofloxacin, it is recommended that for this group of organisms a breakpoint of 0.125 mg/l should be included in laboratory sensitivity tests.

The use of serodiagnosis in the retrospective investigation of a nursery outbreak associated with Escherichia coli O157:H7.

AIMS: To use serology to investigate an outbreak of verocytotoxin (VT) producing Escherichia coli O157 in a hospital nursery, following the detection of faecal E coli O157 (phage type 49) producing VT type 2. METHODS: ELISA and immunoblotting techniques, based on lipopolysaccharide (LPS) purified from E coli O157; diagnostic bacteriology; serotyping and phage typing; DNA probes for VT. RESULTS: 29 of 126 sera contained antibodies to the LPS of E coli O157: 10 were from children, three were from staff, and 11 were from hospital kitchen staff. Five parents of children attending the nursery were antibody positive. Sixty four sera from other hospital staff and controls did not contain antibodies to the LPS of E coli O157. CONCLUSIONS: Serology detected evidence of infection with E coli O157 in 23% of sera examined. By bacteriology alone, only a single case of infection with E coli O157 would have been detected. Serology is valuable in providing evidence of infection with E coli O157.

Increasing incidence of antibiotic resistance in shigellas from humans in England and Wales: recommendations for therapy.

Since 1983 the incidence of resistance to ampicillin in Shigella dysenteriae, Sh. flexneri, and Sh. boydii infections in England and Wales has increased from 42% to 65% and the incidence of resistance to trimethoprim, from 6% to 64%. Furthermore, of 1524 strains received in 1995-1996, 46% were resistant to both of these antimicrobials. For Sh. sonnei almost 50% of isolates were resistant to ampicillin or trimethoprim and 15% were resistant to both of these antimicrobials. These results demonstrate that if antibiotic therapy had been indicated for infections with Sh. dysenteriae, Sh. flexneri, and Sh. boydii, then treatment with either ampicillin or trimethoprim may have been ineffective in almost 50% of cases and for Sh. sonnei, in 15% of cases. It is concluded that if it is necessary to commence treatment before the results of laboratory-based sensitivity tests are available, the best options would be to use nalidixic acid for children and a fluoroquinolone antibiotic such as ciprofloxacin or ofloxacin, for adults.

Comparative study of five different techniques for epidemiological typing of Escherichia coli O157.

A set of 47 Austrian human, food, and veterinary Escherichia coli O157:H7 isolates was used to evaluate five different epidemiological typing methods. Ribotyping using an automated microbial characterization system (RiboPrinter) was not suitable for detection of epidemiological relatedness. All but one E. Coli strain were typeable by phage typing. Random amplified polymorphic DNA-PCR fingerprinting was performed using primer M13 containing the sequence 5'-GAG GGT GGC GGT TCT-3' and primer 1247 (5'-AAGAGCCCGT-3'). Although both methods recognized only two clusters, both dendrograms grouped most of the EHEC O157 isolates into epidemiologically related subgroups. Pulsed-field gel electrophoresis of XbaI digested total DNA was a valuable subtyping system. We found that major differences can exist between results of multiple subtyping methods. E. coli O157 isolates should not be classified as epidemiologically related or nonrelated on the basis of a single typing method alone.

Verocytotoxin-producing Escherichia coli (VTEC) O157 and other VTEC from human infections in England and Wales: 1995-1998.

A total of 3429 isolations of verocytotoxin-producing Escherichia coli O157 (VTEC O157) was confirmed from human sources in England and Wales during the period 1995-1998. The largest annual total was 1087 in 1997. Most infections occurred in the third quarter of each year. The overall rate of infection ranged from 1.28 to 2.10/100,000 population and showed regional variation. The highest incidence was in children aged 1-4 years. Annually, between 5% and 11% of strains were from patients who had travelled abroad. There were 67 general outbreaks of infection represented by 407 (11.9%) VTEC O157 isolates. Outbreaks involved transmission by contaminated food or water, person-to-person spread and direct or indirect animal contact, and five were associated with foreign travel. The majority (76%) of strains carried verocytotoxin (VT) 2 genes and 23.3% were VT1+VT2. Most strains had the flagellar antigen H7, but c. 14% were non-motile. Approximately 20% of isolates were resistant to antimicrobial agents, predominantly streptomycin, sulphonamides and tetracycline. In addition to VTEC O157, strains of serogroup O157 that did not possess VT genes were identified. These were either derivatives of VTEC O157 that had lost VT genes or were strains with H antigens other than H7 that have never been associated with VT production. Strains of VTEC other than O157 were characterised. Most were associated with diarrhoea, bloody diarrhoea or haemolytic uraemic syndrome and had virulence markers in addition to VT.

Identification of enteropathogenic Escherichia coli isolated in Britain as enteroaggregative or as members of a subclass of attaching-and-effacing E. coli not hybridising with the EPEC adherence-factor probe.

Strains of Escherichia coli from sporadic cases of diarrhoea and belonging to serotypes O44:H18, O55:H7, O111ab:H21, O111ab:H25 or O126:H27 were examined for virulence properties. With the exception of O111ab:H25 these are considered to be classical enteropathogenic E. coli (EPEC) serotypes. The strains had been isolated in Britain from the faeces of children less than 3 years old. Of the serotypes examined, 7 of 13 O44:H18 strains, all of 10 O111ab:H21 strains and 13 of 21 O126:H27 strains belonged to the enteroaggregative class of E. coli (EAggEC) that attached to HEp-2 cells in the characteristic aggregative pattern and hybridised with the EAggEC probe. They also caused mannose-resistant haemagglutination of rat erythrocytes, a property which may be a useful marker for their identification. Strains of O44:H18 with similar properties were also isolated from three small outbreaks in Britain, one of which involved elderly patients. EAggEC have not been considered previously as aetiological agents of diarrhoea in developed countries and have rarely been reported as belonging to EPEC serotypes. All 15 O55:H7 strains and seven of eight O111ab:H25 strains were also considered to be potentially diarrhoeagenic as they gave localised attachment (LA) to HEp-2 cells that resulted in a positive fluorescence actin-staining test. This test is considered to correlate with the attaching-and-effacing virulence mechanisms of EPEC in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Subtyping of virulence genes in verocytotoxin-producing Escherichia coli (VTEC) other than serogroup O157 associated with disease in the United Kingdom.

Verocytotoxin-producing Escherichia coli (VTEC) causes a wide spectrum of disease in humans, from mild diarrhoea to haemolytic uraemic syndrome (HUS). The verocytotoxin (vtx) and intimin (eae) genes of VTEC strains, other than those of serogroup O157, were subtyped to identify common properties that may be associated with increased pathogenicity. Strains were isolated from patients with HUS, those with diarrhoea or from asymptomatic individuals. Strains of VTEC that carried vtx(2) gene subtypes vtx(2) and vtx(2c) were most commonly associated with HUS, whereas strains from patients with less severe disease and from the healthy control group were more likely to have vtx(1c) or vtx(2d) genes. The eae gene was detected more frequently in strains isolated from HUS patients than in those associated with cases of diarrhoea; beta-intimin was the most common intimin subtype in strains isolated from both groups of patients. None of the strains from the healthy control group carried the eae gene.

Use of gene probes and adhesion tests to characterise Escherichia coli belonging to enteropathogenic serogroups isolated in the United Kingdom.

Nine hundred and twenty-five Escherichia coli isolates from cases of diarrhoea in the United Kingdom and belonging to enteropathogenic E. coli (EPEC) O serogroups were examined for virulence properties. The tests included adhesion to HEp-2 cells, the fluorescence actin staining (FAS) test (which correlates with the ability to cause attaching and effacing lesions) and DNA hybridisations with probes to detect sequences for eaeA (E. coli attaching and effacing factor), EAF (EPEC adherence factor), verocytotoxins VT1 and VT2, enteroaggregative E. coli and diffusely adherent E. coli. The O serogroups examined were 18, 26, 44, 55, 86, 111, 114, 119, 125, 126, 127, 128 and 142. Six hundred and sixty strains (71.4%) hybridised with at least one of the DNA probes. Over 80% of strains in O serogroups 26, 55, 119, 125, 127 and 142 and 41% of strains of serogroups 86, 111, 114, 126 and 128 hybridised with the eae probe and most showed localised attachment and were FAS-positive. However, <10% of these eae probe-positive strains hybridised with the EAF probe. Eighty-four of 232 strains in O serogroups 44, 86, 111, and 126 were enteroaggregative. VT genes were detected in 57 of 402 strains in O serogroups 26, 55, 111 and 128. Identification of EPEC by serogrouping was shown to be an effective method of identifying strains with pathogenic potential, although the organisms were diverse in their properties.

Increasing incidence of resistance to nalidixic acid in shigellas from humans in England and Wales: implications for therapy.

Among shigellas isolated from patients in England and Wales in 2002, 10% of subgroups A, B and C, and 13% of subgroup D (Shigella sonnei), were resistant to nalidixic acid. As a consequence, should antimicrobial therapy be indicated, the efficacy of nalidixic acid as the preferred treatment for children with bacillary dysentery has been jeopardised.

Vero cytotoxin-producing Escherichia coli in a study of infectious intestinal disease in England.

An investigation of infectious intestinal disease in England included examination of feces for Vero cytotoxin-producing Escherichia coli (VTEC). Using DNA probe hybridization 27 VTEC strains were identified, 12 were from cases, and of these three belonged to serogroup O157. The remaining 15 strains were isolated from controls. The strains were confirmed biochemically as E. coli, they were serotyped and characterized according to their toxin production, the presence of sequences encoding intimin (eae) and enterohemolysin was determined and resistance to antimicrobial agents was determined. Six of the nine cases with non-O157 VTEC were less than 16 years old, only two of the 15 controls were under 16. Infection with more than one micro-organism was also considered.

Antibiotic resistance in Escherichia coli isolated from blood and cerebrospinal fluid: a 6-year study of isolates from patients in England and Wales.

A study of the incidence of resistance to antimicrobial drugs in Escherichia coli from blood and CSF made in England and Wales in the 6-year period 1991 1996 has demonstrated a significant increase in the incidence of strains resistant to ampicillin and ciprofloxacin, two antibiotics used for first-line therapy of invasive disease. In particular, there has been a dramatic change in the occurrence of isolates with low level or high level resistance to ciprofloxacin; over 90% of isolates in the high level group were also resistant to at least four other antimicrobials. Physicians in England and Wales should be aware that there is now an increasing possibility of treatment failures when ciprofloxacin is used for the treatment of invasive E. coli infections.

Molecular epidemiology of multi-resistant Escherichia coli.

In this case-control study multi-resistant Escherichia coli isolates were characterized on a molecular level and risk factors for their development were identified. Thirty-two multi-resistant E. coli strains were isolated from the urine of 13 patients attending a renal clinic for chronic urinary tract infection (UTI) and from different sites of 11 terminally ill patients with nosocomial infections hospitalized on five different wards. All 32 isolates were resistant to ciprofloxacin, cotrimoxazole and produced beta-lactamase. All strains contained plasmids of 2-110 MDa of which a 50 MDa and a 100 MDa plasmid were present in 81% of the strains. Pulse-field gel electrophoresis (PFGE) analysis demonstrated 17 genotypes among 32 strains which indicates a polyclonal outbreak with some geographic clustering. Monitoring of patients over the study period showed that either the resident genotype remained the same and that these retained strains underwent changes in their plasmid contents, or that they were replaced by a different genotype after several months of therapy for chronic UTI. Univariate analysis indicated that multi-resistant E. coli develop in the presence of long-term selective ciprofloxacin pressure at a dosing regimen of 250 mg bid for more than 20 days and that treatment with a broad spectrum antimicrobial for more than three days favours the selection of multi-resistant E. coli in the flora of terminally ill patients with multiple disorders.

Use of strain typing to provide evidence for specific interventions in the transmission of VTEC O157 infections.

Transmission of verocytotoxin (VT)-producing Escherichia coli (VTEC) occurs by three main routes. These comprise food- or water-borne infections, acquisition of disease by direct or indirect contact with animals and person-to-person spread. Phenotypic typing of VTEC belonging to serogroup O157 is achieved by phage typing and identification of VT type. These properties quickly provide evidence for the linkage of human cases and their association to potential sources. DNA-based subtyping methods such as pulsed field gel electrophoresis (PFGE) are generally required to increase discrimination of VTEC O157 strains so that the spread of specific strains can be monitored. Phenotypic and DNA-based methods were used in the investigation of 85 general outbreaks of VTEC O157 infection between 1995 and 1999. Results were used in conjunction with epidemiological data to provide direct or indirect evidence for the likely route of transmission. Detailed strain fingerprinting identified specific food vehicles and reservoirs of infection in animals. Typing supported the implementation of measures to control the spread of infection that included pasteurisation orders, product withdrawal, temporary closure of retail premises and open farms and the introduction of HACCP-based working practices. In outbreaks involving widely distributed foods, DNA-based examination of apparently sporadic isolates with the same phage and VT type as outbreak strains was performed to identify additional potential outbreak cases and estimate the spread of infection. Strain typing was applied in outbreaks in nurseries and other institutions to monitor person-to-person spread, including careers and their families and to assess the involvement of community cases occurring at the same time. Rapid exchange of epidemiological, microbiological and typing data will be increasingly important in investigation of VTEC O157 outbreaks.

Detection and characterization of Shiga toxin-producing Escherichia coli in feral pigeons.

Escherichia coli strains producing a variant of Shiga toxin 2 (Stx2), designated Stx2f, have been recently described in the stools of feral pigeons. During 1997-1998, 649 pigeons were trapped and examined in three different squares of Rome. Stool samples were collected from each bird and enrichment cultures were examined for the presence of Stx by the vero cell assay. Stx-producing E. coli (STEC) were isolated from the positive cultures and characterized by serotyping and PCR analysis of stx and other virulence-related genes. Stx was detected in 10.8% of the stool enrichment cultures. The percentage of positive birds did not differ significantly for the three flocks considered and the season of sample collection. Conversely, STEC carriage was significantly more frequent in young than in adult birds (17.9 versus 8.2%). None of the birds examined showed signs of disease. STEC strains were isolated from 30 of 42 Stx-positive cultures examined. All the strains produced Stx2f, and most of them possessed genes encoding for intimin and the cytolethal distending toxin (CLDT). Six serogroups were identified, but most of the isolates belonged to O45, O18ab, and O75. Molecular typing indicated that most of the isolates within a flock were clonally-related. This work confirms that pigeons represent a natural reservoir of STEC strains characterized by the production of the toxin variant Stx2f, and by the frequent presence of eae and cldt genes. Further work is needed to clarify whether these STEC may represent a cause of avian disease or even a potential health hazard for humans.