Enterohaemorrhagic and other Shiga toxin-producing Escherichia coli (STEC): Where are we now regarding diagnostics and control strategies?

Escherichia coli comprises a highly diverse group of Gram-negative bacteria and is a common member of the intestinal microflora of humans and animals. Generally, such colonization is asymptomatic; however, some E. coli strains have evolved to become pathogenic and thus cause clinical disease in susceptible hosts. One pathotype, the Shiga toxigenic E. coli (STEC) comprising strains expressing a Shiga-like toxin is an important foodborne pathogen. A subset of STEC are the enterohaemorrhagic E. coli (EHEC), which can cause serious human disease, including haemolytic uraemic syndrome (HUS). The diagnosis of EHEC infections and the surveillance of STEC in the food chain and the environment require accurate, cost-effective and timely tests. In this review, we describe and evaluate tests now in routine use, as well as upcoming test technologies for pathogen detection, including loop-mediated isothermal amplification (LAMP) and whole-genome sequencing (WGS). We have considered the need for improved diagnostic tools in current strategies for the control and prevention of these pathogens in humans, the food chain and the environment. We conclude that although significant progress has been made, STEC still remains an important zoonotic issue worldwide. Substantial reductions in the public health burden due to this infection will require a multipronged approach, including ongoing surveillance with high-resolution diagnostic techniques currently being developed and integrated into the routine investigations of public health laboratories. However, additional research requirements may be needed before such high-resolution diagnostic tools can be used to enable the development of appropriate interventions, such as vaccines and decontamination strategies.

Biosecurity-based interventions and strategies to reduce Campylobacter spp. on poultry farms.

The prevention and control of Campylobacter colonization of poultry flocks are important public health strategies for the control of human campylobacteriosis. A critical review of the literature on interventions to control Campylobacter in poultry on farms was undertaken using a systematic approach. Although the focus of the review was on aspects appropriate to the United Kingdom poultry industry, the research reviewed was gathered from worldwide literature. Multiple electronic databases were employed to search the literature, in any language, from 1980 to September 2008. A primary set of 4,316 references was identified and scanned, using specific agreed-upon criteria, to select relevant references related to biosecurity-based interventions. The final library comprised 173 references. Identification of the sources of Campylobacter in poultry flocks was required to inform the development of targeted interventions to disrupt transmission routes. The approach used generally involved risk factor-based surveys related to culture-positive or -negative flocks, usually combined with a structured questionnaire. In addition, some studies, either in combination or independently, undertook intervention trials. Many of these studies were compromised by poor design, sampling, and statistical analysis. The evidence for each potential source and route of transmission on the poultry farm was reviewed critically, and the options for intervention were considered. The review concluded that, in most instances, biosecurity on conventional broiler farms can be enhanced and this should contribute to the reduction of flock colonization. However, complementary, non-biosecurity-based approaches will also be required in the future to maximize the reduction of Campylobacter-positive flocks at the farm level.

Influence of production system on the rate of onset of Campylobacter colonization in chicken flocks reared extensively in the United Kingdom.

1. Because thermophilic Campylobacter spp. are common in chicken flocks reared extensively, cross-sectional and longitudinal studies were carried out on organic and free-range farms to determine the onset of colonisation (lag phase) and likely sources of flock infection. 2. For 14 organic and 14 free range flocks, there was a difference in lag phases, with the former being colonized at a mean of 14·1 d in comparison with 31·6 d for the latter. Whereas most free-range flocks became colonized when released on to pasture, those reared organically were usually colonized at the housed brooding stage. 3. Further study of organic flocks on three farms over 7 successive crop cycles confirmed that colonisation was strongly influenced by the prevailing husbandry conditions and was not a consequence of the length of the rearing period. 4. Molecular epidemiological investigations on a farm showing the shortest lag phase, using PFGE typing with two different restriction enzymes (SmaI and KpnI) and flaA SVR sequence typing, revealed that potential sources of colonisation for organic chickens were already present on the farm at the time of chick placement. Such sources included the ante area of the brooding house, surrounding pasture and other livestock being kept on the farm. 5. Overall, the study demonstrated that, under UK conditions, the prevalence of colonisation was greater in extensive flocks (95-100%) than it was for conventional broilers (55%), similar to the situation in other countries, but all three management systems showed comparable levels of caecal carriage in positive birds (log(10)/g 6·2-6·7).

Selecting for development of fluoroquinolone resistance in a Campylobacter jejuni strain 81116 in chickens using various enrofloxacin treatment protocols.

AIMS: To determine the effect of various enrofloxacin dose regimes on the colonization and selection of resistance in Campylobacter jejuni strain 81116P in experimentally colonized chickens. METHODS AND RESULTS: Two experiments were undertaken, in which 14-day-old chickens were colonized with 1 × 10(7) -1 × 10(9 ) CFU g(-1) Camp. jejuni strain 81116P and then treated with enrofloxacin at 12-500 ppm in drinking water for various times. Caecal colonization levels were determined at various time-points after start-of-treatment, and the susceptibility of recovered isolates to ciprofloxacin was monitored. Resistance was indicated by growth on agar containing 4 µg ml(-1) ciprofloxacin, MICs of 16 µg ml(-1) and the Thr86Ile mutation in gyrA. Enrofloxacin at doses of 12-250 ppm reduced Camp. jejuni colonization over the first 48-72 h after start-of-treatment. The degree of reduction in colonization was dose, but not treatment time, dependent. In all cases, maximal colonization was re-established within 4-6 days. Fluoroquinolone-resistant organisms were recoverable within 48 h of start-of-treatment; after a further 24 h all recovered isolates were resistant. In contrast, a dose of 500 ppm enrofloxacin reduced colonization to undetectable levels within 48 h, and the treated birds remained Campylobacter negative throughout the remaining experimental period. By high pressure liquid chromatography, for all doses, the maximum concentrations of enrofloxacin and ciprofloxacin in the caecal contents were detected at the point of treatment completion. Thereafter, levels declined to undetectable by 7 days post-treatment withdrawal. CONCLUSIONS: In a model using chickens maximally colonized with Camp. jejuni 81116P, treatment with enrofloxacin, at doses of 12-250 ppm in drinking water, enables the selection, and clonal expansion, of fluoroquinolone-resistant organisms. However, this is preventable by treatment with 500 ppm of enrofloxacin. SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment of chickens with enrofloxacin selects for resistance in Camp. jejuni in highly pre-colonized birds. However, a dose of 500 ppm enrofloxacin prevented the selection of resistant campylobacters.

Investigation of the presence and protective effects of maternal antibodies against Campylobacter jejuni in chickens.

The role of maternal antibodies in the lag phase of Campylobacter positivity, widely observed in commercial broiler flocks, was investigated. The results indicate that 3-wk-old birds derived from a commercial flock are more susceptible to colonization with Campylobacter jejuni than 1-to-2-wk-old birds. This increasing susceptibility parallels the loss of maternally derived, circulating, anti-Campylobacter, immunoglobulin Y antibodies as detected by enzyme-linked immunosorbent assay. The role of these antibodies in resistance to colonization was further investigated using progeny from breeder flocks of known Campylobacter status. These results confirmed that maternal antibodies confer partial protection against Campylobacter colonization on young chickens (1-2 wk old). This protection was directed against challenge with both homologous and heterologous strains of C. jejuni and even against strains with a high colonization potential. However, evidence presented indicates that newly hatched chicks, with the highest levels of maternal antibodies, were as susceptible to Campylobacter challenge as 3-wk-old birds. This conundrum was investigated further, and an increase in resistance was detected from 1 to 3 days of age. The reasons for this are, as yet, unknown, but the observation validates the use of newly hatched chicks in models of Campylobacter colonization. Moreover, this high susceptibility in the first few days of life may explain the occasional early flock colonization observed, especially when environmental exposure to Campylobacter is high, for example, in free-range birds.

Factors related to the carriage of Verocytotoxigenic E. coli, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica in cattle, sheep and pigs at slaughter.

A 12-month abattoir study was undertaken from January 2003. We collected 7492 intestinal samples from cattle, sheep and pigs at slaughter. Rectal samples were taken from cattle and sheep and caecal samples from pigs. They were examined for verocytotoxigenic E. coli (VTEC) O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica. Data were collected on the animal from which the sample came and this information was analysed to look at potential risk factors for carriage of these organisms. Logistic regression models were run where an adequate number of positive results were available. This revealed that VTEC O157 carriage in cattle was associated with the summer period and that age was a protective factor. Salmonella carriage in pigs was associated with lairage times >12 h, the North East and not feeding when there was no bedding available. In cattle, carriage was associated with the summer period, the Eastern region of GB and dairy animals. In sheep a spring seasonal effect was seen, which coincided with the lambing period. The carriage of thermophilic Campylobacter in cattle was associated with single-species abattoirs, with age a protective factor. In sheep, winter was a risk period with lairage management influential. For pigs, lairage times of <12 h were found to be associated with carriage. A seasonal trend for carriage of Y. enterocolitica in all species was demonstrated with the period December-May a risk. For cattle, age was also a risk factor; for sheep feeding in the lairage and for pigs being held overnight were risk factors.

Genetic instability is associated with changes in the colonization potential of Campylobacter jejuni in the avian intestine.

AIMS: A panel of pulsed field gel electrophoresis (PFGE) type variants of Campylobacter jejuni, previously identified as of clonal origin, were investigated to determine whether genomic instability could be observed during competitive growth. METHODS AND RESULTS: Upon recovery from frozen storage, some variants had undergone alterations in PFGE profiles, but subsequent culture produced constant genotypes. Individual variants did not display differences in colonization potential when tested in orally challenged 1-day-old chickens. However, competitive colonization using mixtures of two or three PFGE types generally resulted, by 4 weeks postchallenge, in one predominant PFGE type in all birds. For some variant mixtures, a minor population of novel PFGE types was detected in individual birds. The creation of new variants appeared to be dependent on the extent of competition and of the individual host. Genomic rearrangements most likely explain this increase in genetic diversity, apparently without the involvement of natural transformation or plasmid acquisition. In vitro cultivation of mixed inoculations were again selected for particular variants; but genetic diversity was not generated, suggesting that the selection pressures in vitro differed from those active in vivo. CONCLUSION: These observations support the hypothesis that by generating genetic diversity, C. jejuni can improve its phenotypic fitness to survive and colonize subsequent hosts. SIGNIFICANCE AND IMPACT OF THE STUDY: The consequences of such observations for the development of campylobacter control strategies for poultry may be substantial.

Intestinal carriage of verocytotoxigenic Escherichia coli O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica, in cattle, sheep and pigs at slaughter in Great Britain during 2003.

An abattoir survey was undertaken to determine the prevalence of foodborne zoonotic organisms colonizing cattle, sheep and pigs at slaughter in Great Britain. The study ran for 12 months from January 2003, involved 93 abattoirs and collected 7703 intestinal samples. The design was similar to two previous abattoir surveys undertaken in 1999-2000 allowing comparisons. Samples were examined for VTEC O157, Salmonella, thermophilic Campylobacter and Yersinia enterocolitica. The prevalence of VTEC O157 faecal carriage was 4.7% in cattle, 0.7% in sheep and 0.3% in pigs. A significant decrease in sheep was detected from the previous survey (1.7%). Salmonella carriage was 1.4% in cattle, a significant increase from the previous survey of 0.2%. In sheep, faecal carriage was 1.1% a significant increase from the previous survey (0.1%). In pigs, carriage was 23.4%, consistent with the previous study. Thermophilic Campylobacter spp. were isolated from 54.6% of cattle, 43.8% of sheep and 69.3% of pigs. Y. enterocolitica was isolated from 4.5% of cattle, 8.0% of sheep and 10.2% of pigs.

Use of multilocus sequence typing to investigate the association between the presence of Campylobacter spp. in broiler drinking water and Campylobacter colonization in broilers.

The presence of campylobacters in broiler chickens and throughout the broiler water delivery systems of 12 farms in northeastern Scotland was investigated by sensitive enrichment methods and large-volume filtration. Campylobacter presence was independent of the water source and whether the water was treated. The genotypes of Campylobacter jejuni isolates recovered from chickens and various locations within the water delivery systems were compared by multilocus sequence typing. Matching strains in shed header tanks and birds were found at 1 of the 12 farms investigated. However, the sequence of contamination or whether the source was within or outside the shed was not determined. Nevertheless, these data provide evidence that drinking water could be associated with broiler infection by campylobacters.

Crossing the boundaries.

In the past, research on zoonotic diseases has been fragmented. Teresa Belcher and Diane Newell discuss the formation of Med-Vet-Net, a "virtual institute", which aims to integrate activity between researchers in human and veterinary medicine across the European Union.

A multiplex polymerase chain reaction to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus -species venerealis: use on UK isolates of C. fetus and other Campylobacter spp.

AIMS: Subspeciation of Campylobacter fetus subsp. fetus (CFF) and Campylobacter fetus subsp. venerealis (CFV) is important for international animal import regulations. Phenotyping can be unreliable, and genotyping by techniques like pulsed field gel electrophoresis is difficult in routine diagnostic laboratories. A PCR subspeciation technique has been reported [Aust Vet J (1997) 75, 827]; we aimed to develop this PCR and investigate its use on UK C. fetus isolates. METHODS AND RESULTS: We augmented the PCR with further primers, and tested 76 isolates of C. fetus and 16 isolates of other Campylobacter spp. PCR failed to correlate well with phenotyping, especially for CFV. We characterized the amplicon of the CFV-specific primers (reported as plasmid derived, but unavailable on the public databases); and predicted a parA gene sequence, anticipated to be plasmid-associated. However, although plasmid isolations from selected CFV isolates demonstrated the presence of several plasmids, there was no correlation between plasmid profile and PCR result. Further, the parA sequence was not detected by PCR in any of the plasmid bands. CONCLUSIONS: This PCR is not suitable for subspeciation of C. fetus in the UK. The results suggest that this is a reflection of the presence of an unusual clone of CFV currently present in cattle in this country. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR cannot substitute for phenotyping of C. fetus isolates in the UK. The reasons for failure of PCR genotyping may reflect local strains and/or plasmid profiles. Further study is required to better elucidate molecular sub-speciation of C. fetus.

Comparison of the biotypes of Yersinia enterocolitica isolated from pigs, cattle and sheep at slaughter and from humans with yersiniosis in Great Britain during 1999-2000.

AIMS: To investigate the relationship between livestock carriage of Yersinia enterocolitica and human disease. The biotypes/serotypes of strains recovered from the faeces of pigs, cattle and sheep at slaughter during a national survey in Great Britain in 1999-2000, were compared with those of strains isolated from human cases of yersiniosis during the same period. METHODS AND RESULTS: The faecal carriage of Y. enterocolitica by cattle, sheep and pigs at slaughter was 6.3, 10.7 and 26.1%, respectively. Yersinia enterocolitica biotype (BT) 1a was the most frequently isolated biotype from livestock (58%) and was the predominant biotype (53%) isolated from human cases over the same period. The main recognized pathogenic Y. enterocolitica biotype isolated from livestock was BT3 (O:5,27) (35% of sheep, 22% of pigs and 4% of cattle) but this biotype was not detected in any of the human isolates investigated. The major pathogenic biotypes of strains isolated from humans were BT3 (O:9) (24%) and BT4 (O:3) (19%) whereas of the veterinary isolates investigated, only pigs (11%) carried BT3 (O:9) strains. CONCLUSIONS: Because of significant overlaps in phenotypes of the veterinary and human strains it is not possible to comment on the correlation between host and pathogenicity, especially of biotype 1a. SIGNIFICANCE AND IMPACT OF THE STUDY: The data suggest that further investigations using methods with greater discriminatory power are required. However the data also suggests that pigs may be the primary reservoir for human pathogenic Y. enterocolitica infection.

The Campylobacter jejuni general glycosylation system is important for attachment to human epithelial cells and in the colonization of chicks.

It has recently been shown that the enteropathogen Campylobacter jejuni has an N-linked general protein glycosylation pathway (Pgl) that modifies many of the organism's proteins. To determine the role of the N-linked general glycosylation in C jejuni, the authors studied the pglH gene, which shows high similarity to a family of sugar transferases. pglH mutants were constructed in strains 81116 and 11168H. Both mutants were shown to be deficient in their ability to glycosylate a number of C. jejuni proteins, but their lipooligosaccharide and capsule were unaffected. The pglH mutants had significantly reduced ability to adhere to and invade human epithelial Caco-2 cells. Additionally, the 81116 pglH mutant was severely affected in its ability to colonize chicks. These results suggest that glycosylation is important for the attachment of C. jejuni to human and chicken host cells and imply a role for glycoproteins in the pathogenesis of C. jejuni.

Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experience.

AIMS: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP (fla typing) methods previously described for Campylobacter. METHODS AND RESULTS: The sample set (n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two of the three flaA typing methods amplified flaA alone, whereas one, a multiplex assay, amplified flaB in addition to flaA. DdeI restriction enzyme was employed for all methods, but HinfI was also investigated. 98-100% typeability was obtained for flaA-based methods, but only 93% for the multiplex assay, due to inconsistent amplification of a non-specific product. In addition, there appeared to be selective amplification of flaA over flaB. More DdeI types were generated using a longer flaA PCR amplicon, whilst additional use of HinfI increased the number of types by ca 25%. Inter-laboratory reproducibility for both flaA-based methods was defined at 100%. CONCLUSIONS: Fla typing requires standardization with respect to PCR primers and restriction enzymes. This study identified an assay, employing the full flaA gene and DdeI digestion, as an appropriate method on which to standardize. 100% inter-laboratory reproducibility was demonstrated using that method. SIGNIFICANCE AND IMPACT OF THE STUDY: This work should facilitate progress towards inter-laboratory standardization of fla typing.

Prevalence of multiple antibiotic resistance in 443 Campylobacter spp. isolated from humans and animals.

AIMS: In view of recent findings that a multidrug efflux pump CmeABC exists in Campylobacter jejuni, 391 C. jejuni and 52 Campylobacter coli of human and animal origin were examined for a multidrug resistance phenotype. MATERIALS AND METHODS: The MICs of ampicillin, chloramphenicol, ciprofloxacin, erythromycin, kanamycin, tetracycline, cetrimide, triclosan, acridine orange, paraquat and ethidium bromide were determined. Resistance to organic solvents and the effect of salicylate (known inducer of the marRAB operon in Escherichia coli and Salmonella) were also examined. RESULTS: Two C. coli and 13 C. jejuni isolates, mainly from pigs or poultry, were resistant to three or more antibiotics and 12 of these strains had reduced susceptibility to acridine orange and/or ethidium bromide. Strains (n = 20) that were less susceptible to acridine orange, ethidium bromide and triclosan were significantly more resistant (P < 0.05) to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, nalidixic acid and tetracycline, with two- to four-fold increases in MIC values compared with strains (n = 20) most susceptible to acridine orange, ethidium bromide and triclosan. Growth of strains with 1 mM salicylate caused a small (up to two-fold) but statistically significant (P < or = 0.005) increase in the MICs of chloramphenicol, ciprofloxacin, erythromycin and tetracycline. CONCLUSIONS: These data indicate that multiple antibiotic resistant (MAR)-like Campylobacter strains occur and it may be postulated that these may overexpress cmeABC or another efflux system.

Role of S-layer protein antigenic diversity in the immune responses of sheep experimentally challenged with Campylobacter fetus subsp. fetus.

Surface layer proteins (SLPs) are essential for induction of abortion by Campylobacter fetus subsp. fetus in experimentally challenged ewes. These proteins are encoded by multiple sap genes and vary in size and antigenicity. The role of SLP antigenic variation during experimental ovine infection was investigated. Following subcutaneous challenge, the SLPs were highly antigenic, and antibodies were detected in serum, milk, bile, and urine. Fecal anti-SLP antibodies were detected only in animals challenged orally. Ewes challenged with wild-type strain 23D with variable SLPs developed detectable circulating anti-SLP immunoglobulin G (IgG) antibodies by 2 weeks postchallenge. In contrast, ewes challenged with mutants of 23D that had fixed expression of a single SLP developed antibodies within 1 week postchallenge, suggesting that antigenic variation in SLPs may delay the host antibody response. Although not statistically significant, the data from challenge experiments in which vaccinated ewes were used suggested that SLP-expressing vaccines could protect animals from abortion and that this effect was independent of the SLP expressed, indicating involvement of conserved epitopes in the SLP. The conserved 184-amino-acid N-terminal region of the SLP, identified from previously published sequences, was epitope mapped with rabbit anti-SLP antisera by using overlapping synthetic 20-mer peptides. Two putative epitopes were identified at amino acids 81 to 110 and 141 to 160. Amino acids 81 to 100 also bound serum IgG antibodies from experimentally challenged sheep. Conserved antigenic regions of the SLP that induce protective immune responses may enable development of synthetic vaccine candidates for C. fetus subsp. fetus-associated ovine abortion.

Long-term antibody responses following human infection with Campylobacter jejuni.

Epidemiological evidence suggests prior infection of humans by Campylobacter jejuni leads to protection against disease following further exposure. It is known that infections elicit strong antibody responses following the onset of disease and that antibody levels are elevated in putatively immune populations. To determine if systemic and mucosal antibodies induced by a confirmed infection remain at elevated levels for prolonged periods, repeat serum, saliva and urine samples were taken from campylobacter patients from 1 week and up to a year postinfection. Antibodies were monitored by ELISAs using three different antigen preparations: acid-glycine extracts (AE) of C. jejuni strain 81116 and an aflagellate mutant (R2), and a whole-cell R2 sonicate, and by Western blotting. Levels of serum IgG antibodies against 81116AE and R2 sonicate, but not R2AE, remained significantly raised over time when compared to a comparison population. Serum anti-sonicate IgA antibody levels were initially significantly raised but decreased over time to levels similar to the comparison group. There were no significant differences in levels of salivary IgA against the AEs. Anti-sonicate salivary IgA and IgG levels were initially significantly higher than in the comparison group. Both declined over time but the IgG levels remained significantly higher. Significant correlations were seen between serum IgG levels and age and duration of illness. Serum antibodies against flagellin, 40 kDa and 29 kDa antigens were still detectable in most patients up to a year postinfection, as were salivary antibodies to flagellin, the major outer-membrane protein and a 40 kDa antigen.

The carry-over of Campylobacter isolates between sequential poultry flocks.

The carry-over of Campylobacter strains from one flock to a subsequent flock in the same broiler house has been studied using molecular epidemiological techniques. In all, 524 Campylobacter strains, isolated from two sequential broiler flocks from 60 broiler houses, were typed by restriction fragment polymorphism of the polymerase chain reaction (PCR) product of the flaA and flaB genes (fla typing). Selected strains were also typed using pulsed field gel electrophoresis (PFGE). By fla typing, 15 (21%) of the 60 houses with Campylobacter-positive sequential flocks had identical genotypes. In 10 (16% overall) of these houses the strains were also identical by PFGE. The difference in PFGE patterns in the strains from the three remaining houses may be indicative of genetic instability. Overall, these results suggest that carry-over from one flock to a subsequent flock in the same house is a relatively infrequent event and, therefore, that routine broiler house cleansing and/or disinfection is largely adequate to eliminate Campylobacter contamination. An alternative explanation of the low level carry-over is a persistent source or reservoir, external to the environment of the broiler houses.