The prevalence and spread of Escherichia coli O157:H7 at a commercial beef abattoir.

AIMS: To investigate the prevalence and virulence characteristics of Escherichia coli O157:H7 after a number of beef process operations at a commercial Irish abattoir. METHODS AND RESULTS: Two 12-month studies were carried out. The first study (study 1) examined the prevalence of E. coli O157:H7 at up to six sites on carcasses at eight stages of the dressing, washing, chilling and boning process. The second study (study 2) examined the prevalence of E. coli O157:H7 in bovine faeces and rumen contents post-slaughter and on dressed, washed carcasses. Isolates from both studies were phage-typed and the presence of genes encoding verocytotoxin, enterohaemolysin and intimin production was determined. E. coli O157:H7 was isolated from four of 36 carcasses in study 1. E. coli O157:H7 was detected during hide removal and was detected at multiple carcass sites and multiple process stages, including boning. On two carcasses, contamination was first detected at the bung following its freeing and tying. All isolates from study 1 were phage type (PT) 2, eaeAO157 and ehlyA positive, but were verocytotoxin 1 (VT1) and verocytotoxin 2 (VT2) negative. In study 2, E. coli O157:H7 was isolated from 2.4% of faecal, 0.8% of rumen and 3.2% of carcass samples. In some cases, isolates recovered from the faeces of a particular animal, the resulting carcass and adjacent carcasses on the line had the same phage typing and virulence characteristic profile patterns. All isolates from study 2 were eaeAO157 and ehlyA positive and only one isolate was VT1 and VT2 negative. Most isolates were PT 32. A higher frequency of positive isolations was noted from samples taken during spring and late summer. CONCLUSION: These studies show that in a typical Irish beef abattoir, carcass contamination with E. coli O157:H7 can occur during hide removal and bung tying and this contamination can remain on the carcass during subsequent processing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data that is necessary for the understanding of how E. coli O157:H7 contamination of beef occurs.

Coagulase gemne variants associated with distinct populations of Staphylococcus aureus.

An identifying characteristic of Staphylococcus aureus is the production of staphylocoagulase (coagulase). The aim of this study was to determine the clonal distribution of coagulase gene (coa) variants within populations of S. aureus defined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and protein A variation. The N-terminal region of the coa gene from 43 methicillin-susceptible (MSSA) and 252 methicillin-resistant (MRSA) S. aureus human isolates and 9 animal S. aureus isolates was amplified and digested with HinfI. Twelve types were identified amongst the MSSA isolates and the majority (93%) of MRSA isolates were assigned to 5 of the 12 types. MLST and PFGE analysis identified epidemic populations of MRSA and each epidemic population was characterized by a different coagulase type. Nine of the 12 MLST-defined clonal complex ancestral genotypes recently described each carried a different coagulase type suggesting that coagulase evolution and the evolution of the clonal complexes are intimately related.

A foodborne outbreak of Vero cytotoxin-producing Escherichia coli O157:H-phage type 8 in hospital.

This paper describes the epidemiological and microbiological aspects of the largest outbreak of Vero cytotoxin-producing Escherichia coli O157 (VTEC O157) infection in a hospital setting in which the route of transmission was foodborne. The outbreak, which was caused by a relatively uncommon phage type of VTEC O157, occurred in four geriatric continuing care wards in May 1997. The total number of people found to be excreting the organism was 37, of whom 16 were inpatients and 11 were staff. Twelve people displayed enteric symptoms. In addition, all but two of 10 cases identified in the local community were thought to be associated with the outbreak. An epidemiological investigation amongst the hospital patients revealed a statistically significant association between VTEC O157 infection and attendance at a concert party on the continuing care wards on 17 May 1997 (relative risk = 3.22;P= 0.006). There was an even stronger relationship between consumption of home-baked cream-filled cakes brought to that party and evidence of infection (relative risk = 19.35;P= 0.00002). Further investigations in the local community, coupled with microbiological evidence, supported the epidemiological finding that homemade cream cakes brought into the hospital were the vehicle of infection for the outbreak. There was no secondary spread within the hospital. The outbreak serves as a reminder of the hazard posed by foodstuffs brought into a hospital from outside.

Differences in levels of secreted locus of enterocyte effacement proteins between human disease-associated and bovine Escherichia coli O157.

Ongoing extensive epidemiological studies of verotoxin-carrying Escherichia coli O157 (stx(+) eae(+)) have shown this bacterial pathogen to be common in cattle herds in the United States and the United Kingdom. However, the incidence of disease in humans due to this pathogen is still very low. This study set out to investigate if there is a difference between strains isolated from human disease cases and those isolated from asymptomatic cattle which would account for the low disease incidence of such a ubiquitous organism. The work presented here has compared human disease strains from both sporadic and outbreak cases with a cross-section, as defined by pulsed-field gel electrophoresis, of E. coli O157 strains from cattle. Human (n = 22) and bovine (n = 31) strains were genotyped for carriage of the genes for Shiga-like toxin types 1, 2, and 2c; E. coli secreted protein genes espA, espB, and espP; the enterohemolysin gene; eae (intimin); ast (enteroaggregative E. coli stable toxin [EAST]); and genes for common E. coli adhesins. Strains were also phenotyped for hemolysin, EspP, Tir, and EspD expression as well as production of actin and cytoskeletal rearrangement associated with attaching and effacing (A/E) lesions on HeLa cells. The genotyping confirmed that there was little difference between the two groups, including carriage of stx(2) and stx(2c), which was similar in both sets. ast alleles were confirmed to all contain mutations that would prevent EAST expression. espP mutations were found only in cattle strains (5 of 30). Clear differences were observed in the expression of locus of enterocyte effacement (LEE)-encoded factors between strains and in different media. EspD, as an indicator of LEE4 (espA, -B, and -D) expression, and Tir levels in supernatants were measured. Virtually all strains from both sources could produce EspD in Luria-Bertani broth, although at very different levels. Standard trichloroacetic acid precipitation of secreted proteins from tissue culture medium produced detectable levels of EspD from the majority of strains of human origin (15 of 20) compared with only a few (4 of 20) bovine strains (P < 0.001), which is indicative of much higher levels of protein secretion from the human strains. Addition of bovine serum albumin carrier protein before precipitation and enhanced detection techniques confirmed that EspD could be detected after growth in tissue culture medium for all strains, but levels from strains of human origin were on average 90-fold higher than those from strains of bovine origin. In general, levels of secretion also correlated with ability to form A/E lesions on HeLa cells, with only the high-level protein secretors in tissue culture medium exhibiting a localized adherence phenotype. This research shows significant differences between human- and bovine-derived E. coli O157 (stx(+) eae(+)) strains and their production of certain LEE-encoded virulence factors. These data support the recent finding of Kim et al. (J. Kim, J. Nietfeldt, and A. K. Benson, Proc. Natl. Acad. Sci. USA 96:13288-13293, 1999) proposing different E. coli O157 lineages in cattle and humans and extend the differential to the regulation of virulence factors. Potentially only a subset of E. coli O157 isolates (stx(+) eae(+)) in cattle may be capable of causing severe disease in humans.

Comparison of two methods for serotyping Campylobacter spp.

Two serotyping schemes (Penner and Laboratory of Enteric Pathogens [LEP]) based on soluble heat-stable antigens were used to analyze 3,788 Campylobacter sp. isolates. A significant percentage (36.6%) was untypeable using LEP serotyping; greater cross-reaction was also observed. The relative discrimination capabilities of the techniques were similar. Penner serotyping fulfils more of the requisite criteria for typing methods.

Characterization of a recurrent clonal type of Escherichia coli O157:H7 causing major outbreaks of infection in Scotland.

A particular recurrent clonal type of Escherichia coli O157 has been isolated from multiple clinical, veterinary, food, and environmental sources throughout Scotland since 1989. Significant genotypic variation was detected among isolates from distinct outbreaks, with the presence or absence of single fragments being sufficient to delineate outbreak groups within the clonal type.

Molecular epidemiological investigation of an outbreak of Campylobacter jejuni identifies a dominant clonal line within Scottish serotype HS55 populations.

Three molecular typing methods, pulsed-field gel electrophoresis (PFGE), ribotyping, and flagellin (flaA) gene typing, were used to discriminate within a group of 28 Campylobacter jejuni, heat-stable serotype 55 (HS55) isolates derived from cases of campylobacter enteritis occurring throughout Scotland, including 9 isolates associated with an outbreak. PFGE was found to be most discriminatory, identifying 6 distinct profiles, followed by ribotyping (5 profiles), and then flagellin gene typing (4 profiles). The coincidence of all three genotypic markers identified a dominant clonal line within the HS55 group, accounting for each of the outbreak strains, and for 9 of the 19 sporadic strains. A second, closely related, clonal line accounted for a further 5 of the sporadic strains, and also included the HS55 reference strain. Identification and monitoring of such clonal lines should facilitate more effective future epidemiological surveillance of C. jejuni.

Augmentation of killing of Escherichia coli O157 by combinations of lactate, ethanol, and low-pH conditions.

The acid tolerance of Escherichia coli O157:H7 strains can be overcome by addition of lactate, ethanol, or a combination of the two agents. Killing can be increased by as much as 4 log units in the first 5 min of incubation at pH 3 even for the most acid-tolerant isolates. Exponential-phase, habituated, and stationary-phase cells are all sensitive to incubation with lactate and ethanol. Killing correlates with disruption of the capacity for pH homeostasis. Habituated and stationary-phase cells can partially offset the effects of the lowering of cytoplasmic pH.

Genetic heterogeneity of Escherichia coli O157:H7 in Scotland and its utility in strain subtyping.

From April 1994 to March 1995, seven outbreaks of Escherichia coli O157:H7 infection occurred throughout Scotland, including the largest milk-borne outbreak to date worldwide. Various vehicles of infection were identified, and there were 144 confirmed cases in total. All isolates associated with the outbreaks were subjected to detailed subtyping: phage typing, testing for carriage of verotoxin genes (VT), and pulsed-field gel electrophoresis. The outbreak strains were of three different phage types (2, 4, and 28). Those of phage type 2 and 28 were VT1-/VT2+, those of phage type 4 were VT1+/VT2+. To discriminate outbreak-associate isolates from the high sporadic background, real-time pulsed-field gel electrophoresis analyses were performed. The results demonstrated that, within each of the seven outbreak groups, the macrorestriction profiles observed were indistinguishable, whereas profiles for sporadic isolates were not. The consistent genetic heterogeneity observed within the Scottish Escherichia coli O157 population can be exploited in epidemiological investigations.

Evidence for recombination in the flagellin locus of Campylobacter jejuni: implications for the flagellin gene typing scheme.

The flagellin subunit of the flagellar filament in Campylobacter jejuni is encoded by two highly homologous tandem genes, flaA and flaB. The flaA gene was sequenced in 18 strains of C. jejuni, including isolates from three outbreak groups. Sequences obtained were compared with flaA sequences available in the GenBank database, and all were analyzed for mosaic gene structure by using recently described statistical tests for detecting gene conversion among aligned sets of sequences. Strong evidence was found supporting recombination between flaA genes of different strains (i.e., intergenomic recombination). Intragenomic recombination between the flaA and flaB genes of C. jejuni TGH9011 was also demonstrated. Both mechanisms of recombination may act as a potential means by which pathogenic strains can generate increased antigenic diversity, so allowing them to escape the immunological responses of the host. Furthermore, demonstration of recombination within and between flagellin loci of natural strains suggests that flagellin gene typing (restriction fragment length polymorphism analysis of PCR-amplified flagellin genes) cannot be considered a stable method for long-term monitoring of pathogenic Campylobacter populations.

Molecular epidemiology of Escherichia coli O157:H7 by pulsed-field gel electrophoresis and comparison with that by bacteriophage typing.

One hundred twenty-four Escherichia coli O157:H7 isolates were characterized by pulse-field gel electrophoresis, bacteriophage typing, and PCR of verotoxin genes. Diversity indices obtained--0.786 for phage types and 0.987 for pulsed-field gel electrophoresis types--demonstrated that phage typing falls below the critical value (0.9) required for confident interpretation of results.

A comparison of immunomagnetic separation, direct culture and polymerase chain reaction for the detection of verocytotoxin-producing Escherichia coli O157 in human faeces.

Verocytotoxin-producing Escherichia coli O157 (O157 VTEC) has become well recognized as an important enteric pathogen. The number of organisms present in environmental and clinical samples may be low and efforts have been made to increase the sensitivity of O157 VTEC detection. Immunomagnetic seperation (IMS) has been shown to improve O157 VTEC detection in bovine faeces and food samples. A milkborne outbreak of O157 VTEC infection allowed us to compare the isolation rates from human faeces by IMS, direct faecal culture on sorbitol-MacConkey agar and a PCR test for verotoxin gene carriage. Of 142 faecal samples examined, 20 were positive on both direct culture and IMS and a further 13 on IMS alone. Therefore, IMS increased the detection rate of individual cases of O157 VTEC infection and also compared well with PCR. We recommend IMS for use in routine diagnostic laboratories where a more sensitive method than direct faecal culture is required for O157 VTEC isolation.

Characterization of coagulase-negative staphylococci by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses.

Coagulase-negative staphylococci are important nosocomial pathogens. At present, no wholly satisfactory typing scheme exists for these organisms. Therefore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were assessed as characterization methods. A total of 100 type strains and nontyped isolates representing nine species of coagulase-negative staphylococci were analyzed. Each species had a reproducible, characteristic whole-cell banding pattern when analyzed by either method. These species-specific profiles were obtained for all isolates despite disparate geographical origins and clinical isolation sites. Intraspecies similarities, calculated by using the Dice coefficient, were significantly higher than interspecies similarities. Although some species were more heterogeneous than others, the allocation of isolates to any particular species was reinforced by the high degree of interspecies dissimilarity. Application of SDS-PAGE also distinguished discrete subspecies groups. These groups possessed the characteristic profile of their species but were distinguished by a group of variable polypeptides. Species-specific banding patterns were also obtained by immunoblotting of whole-cell polypeptides. Differences between immunoblot and SDS-PAGE profiles could be attributed to variations of antigenicity of particular polypeptides. However, both SDS-PAGE and immunoblotting provided reproducible and sensitive methods for characterization of coagulase-negative staphylococci. Standardization of these techniques could provide the basis for a primary typing scheme.

Differentiation of staphylococcal species and strains by ribosomal RNA gene restriction patterns.

Staphylococcal DNA was digested with endonucleases and probed with labelled ribosomal RNA (rRNA) from Escherichia coli. Reproducible restriction patterns containing between seven and 22 bands were obtained for seven different species of staphylococci. These profiles were species-specific with different strains of a particular species sharing an identical or similar restriction pattern. The results reported here indicate that rRNA gene restriction pattern analyses have an application in the taxonomy of staphylococci.

Characterisation of methicillin-resistant isolates of Staphylococcus aureus by analysis of whole-cell and exported proteins.

Thirty-four isolates of methicillin-resistant Staphylococcus aureus (MRSA) from patients in Glasgow Royal Infirmary were studied. Whole-cell protein profiles obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) were compared with banding patterns produced by immunoblots of exported proteins. Human plasma was used as a source of staphylococcal antibodies for the immunoblots. SDS-PAGE of whole-cell extracts did not usefully distinguish different isolates of MRSA. Reproducible banding patterns were obtained by immunoblots of exported proteins. Analyses of immunoblots by use of the Dice coefficient demonstrated that isolates of MRSA could be divided into two main groups. Immunoblots of exported proteins provided a rapid, reproducible and sensitive method for characterisation of MRSA.