Identifying the sources of human campylobacteriosis in Nigeria.

Campylobacter spp. have been shown to be the most common cause of bacterial gastroenteritis worldwide. Cases of human campylobacteriosis are usually reported as sporadic and not part of an outbreak which makes the identification of the source of infection difficult. A study of the relationships within isolate populations in Nigeria and source attribution analysis of Nigerian human Campylobacter spp. to other animal isolates was carried out to determine the possible sources for human Campylobacter infection in Nigeria. The results showed nine sequence types (STs) common to both humans and livestock isolated from abattoirs, farms and live bird markets with similar STs clustering together on a phylogenetic tree, confirming a degree of genetic similarity. Source attribution analysis suggests wild birds as the most important reservoir (38%) for human Campylobacter spp. infection in Nigeria followed by chicken (23%), pig (19%), cattle (11%) and sheep (8%). This might be an indication of the importance of this infection source to humans in Nigeria and probably other low-income countries due to farming practices and human habits.

Nationwide Stepwise Emergence and Evolution of Multidrug-Resistant Campylobacter jejuni Sequence Type 5136, United Kingdom.

We examined whole-genome-sequenced Campylobacter jejuni and C. coli from 2012-2015 isolated from birds and human stool samples in North East Scotland for the presence of antimicrobial resistance genes. We found that sequence type (ST) 5136 (clonal complex 464) was the most prevalent multidrug-resistant strain of C. jejuni exclusively associated with poultry host reservoirs and recovered from human cases of campylobacteriosis. Tetracycline resistance in ST5136 isolates was due to a tet(O/32/O) mosaic gene, ampicillin resistance was conferred by G → T transversion in the -10 promoter region of blaOXA-193, fluoroquinolone resistance was due to C257T change in gyrA, and aminoglycoside resistance was conferred by aac. Whole-genome analysis showed that the strain ST5136 evolved from ST464. The nationwide emergence of ST5136 was probably due to stepwise acquisition of antimicrobial resistance genes selected by high use of ß-lactam, tetracycline, fluoroquinolone, and aminoglycoside classes of drugs in the poultry industry.

Phylogeographic Analysis Reveals Multiple International transmission Events Have Driven the Global Emergence of Escherichia coli O157:H7.

BACKGROUND: Shiga toxin-producing Escherchia coli (STEC) O157:H7 is a zoonotic pathogen that causes numerous food and waterborne disease outbreaks. It is globally distributed, but its origin and the temporal sequence of its geographical spread are unknown. METHODS: We analyzed whole-genome sequencing data of 757 isolates from 4 continents, and performed a pan-genome analysis to identify the core genome and, from this, extracted single-nucleotide polymorphisms. A timed phylogeographic analysis was performed on a subset of the isolates to investigate its worldwide spread. RESULTS: The common ancestor of this set of isolates occurred around 1890 (1845-1925) and originated from the Netherlands. Phylogeographic analysis identified 34 major transmission events. The earliest were predominantly intercontinental, moving from Europe to Australia around 1937 (1909-1958), to the United States in 1941 (1921-1962), to Canada in 1960 (1943-1979), and from Australia to New Zealand in 1966 (1943-1982). This pre-dates the first reported human case of E. coli O157:H7, which was in 1975 from the United States. CONCLUSIONS: Inter- and intra-continental transmission events have resulted in the current international distribution of E. coli O157:H7, and it is likely that these events were facilitated by animal movements (eg, Holstein Friesian cattle). These findings will inform policy on action that is crucial to reduce the further spread of E. coli O157:H7 and other (emerging) STEC strains globally.

Whole Genome Sequencing demonstrates that Geographic Variation of Escherichia coli O157 Genotypes Dominates Host Association.

Genetic variation in an infectious disease pathogen can be driven by ecological niche dissimilarities arising from different host species and different geographical locations. Whole genome sequencing was used to compare E. coli O157 isolates from host reservoirs (cattle and sheep) from Scotland and to compare genetic variation of isolates (human, animal, environmental/food) obtained from Scotland, New Zealand, Netherlands, Canada and the USA. Nei's genetic distance calculated from core genome single nucleotide polymorphisms (SNPs) demonstrated that the animal isolates were from the same population. Investigation of the Shiga toxin bacteriophage and their insertion sites (SBI typing) revealed that cattle and sheep isolates had statistically indistinguishable rarefaction profiles, diversity and genotypes. In contrast, isolates from different countries exhibited significant differences in Nei's genetic distance and SBI typing. Hence, after successful international transmission, which has occurred on multiple occasions, local genetic variation occurs, resulting in a global patchwork of continental and trans-continental phylogeographic clades. These findings are important for three reasons: first, understanding transmission and evolution of infectious diseases associated with multiple host reservoirs and multi-geographic locations; second, highlighting the relevance of the sheep reservoir when considering farm based interventions; and third, improving our understanding of why human disease incidence varies across the world.

Operationalising factors that explain the emergence of infectious diseases: a case study of the human campylobacteriosis epidemic.

A framework of general factors for infectious disease emergence was made operational for Campylobacter utilising explanatory variables including time series and risk factor data. These variables were generated using a combination of empirical epidemiology, case-case and case-control studies, time series analysis, and microbial sub-typing (source attribution, diversity, genetic distance) to unravel the changing/emerging aetiology of human campylobacteriosis. The study focused on Scotland between 1990-2012 where there was a 75% increase in reported cases that included >300% increase in the elderly and 50% decrease in young children. During this period there were three phases 1990-2000 a 75% rise and a 20% fall to 2006, followed by a 19% resurgence. The rise coincided with expansions in the poultry industry, consumption of chicken, and a shift from rural to urban cases. The post-2000 fall occurred across all groups apart from the elderly and coincided with a drop of the prevalence of Campylobacter in chicken and a higher proportion of rural cases. The increase in the elderly was associated with uptake of proton pump inhibitors. During the resurgence the increase was predominantly in adults and the elderly, again there was increasing use of PPIs and high prevalences in chicken and ruminants. Cases associated with foreign travel during the study also increased from 9% to a peak of 16% in 2006 before falling to an estimated 10% in 2011, predominantly in adults and older children. During all three periods source attribution, genetic distance, and diversity measurements placed human isolates most similar to those in chickens. A combination of emergence factors generic for infectious diseases were responsible for the Campylobacter epidemic. It was possible to use these to obtain a putative explanation for the changes in human disease and the potential to make an informed view of how incidence rates may change in the future.

Elucidating the aetiology of human Campylobacter coli infections.

There has been little research on the determinants of Campylobacter coli infection, despite its contributing up to 10% of human Campylobacter infections. A case-control and two case-case study methods explored the aetiology of C. coli over a one year period across Scotland. The case-control multivariate model found an increased risk of C. coli infection in people older than 19 years (O.R. = 3.352), and during the summer months (O.R. = 2.596), while residing in an urban area decreased the risk (O.R. = 0.546). The first case-case study compared C. coli and C. jejuni cases and also showed a higher risk of C. coli during the summer (O.R. = 1.313) and in people older than 19 years (O.R. = 0.791). Living in an urban area was associated with a reduced risk of infection (O.R. = 0.769). Multi-locus sequence typing (MLST) indicated that sheep and chicken C. coli sequence types (STs) were most frequently found in humans whilst those from cattle and pigs were rarer. MLST diversity was high in isolates from pigs and chicken, intermediate in human isolates, and low in ruminant isolates. The second case-case study used MLST data to ascribe putative sources of infection to the cases. The putative source for 40% of cases was chicken, with 60% acquired from other sources (ruminants 54% and pigs 6%). The case-case analysis also showed that female gender was a risk factor (O.R. = 1.940), which may be explained by females being more likely to prepare poultry in the home. These findings indicate differences between the aetiology of C. coli and C. jejuni infections: this should be taken into account by public health professionals when developing strategies to reduce the burden of human campylobacteriosis.

Effects of seasonality and a diet of brassicas on the shedding of Escherichia coli O157 in sheep.

Sheep flocks were tested for Escherichia coli O157 from pooled fecal samples while they grazed on pasture in winter, brassicas in spring, and on pasture during the summer. The winter pasture study reported an average individual prevalence of 3.1% (95% confidence interval [CI]: 0.6-5.6%) and an average farm-level prevalence of 10.4% (95% CI: 2.1-18.8%) over the 3-year study period. The spring brassica study reported a prevalence of 0% and the summer pasture study had an individual prevalence of 6.3% (95% CI: 2.1-12.1%) and a farm prevalence of 36.8% (95% CI: 15.8-57.8%). Analysis showed significant differences between the shedding of E. coli O157 in sheep grazing on brassicas in spring when compared to sheep grazing on pasture in the summer (p<0.01) and in winter (p=0.044; odds ratio [OR]=0.106). Furthermore, sheep excreted a lower prevalence of E. coli O157 in winter while grazing on pasture (p=0.017; OR=0.199). E. coli O157 isolates were characterized using polymerase chain reaction for the presence of known virulence factors; all carried the eae and stx2 gene and 10/11 positive flocks possessed the stx2c gene, suggesting that sheep are a potential source of human infection.

Multiplex T-RFLP allows for increased target number and specificity: detection of Salmonella enterica and six species of Listeria in a single test.

A multiplex T-RFLP test was developed to detect and identify Salmonella enterica and all six species of Listeria inoculated into milk at minimal levels. Extensive in silico analysis was used to design a fifteen-primer, six-amplimer methodology and in vitro application showed target organism DNA, when amplified individually, yielded the predicted terminal restriction fragments (TRFs) following digestion. Non-target organisms were either not-amplified or yielded TRFs which did not interfere with target identification. Multiple target DNA analysis gave over 86% detection of total TRFs predicted, and this was improved to over 90% detection of total TRFs predicted when only two target DNA extracts were combined analysed. Co-inoculation of milk with five strains each of the target species of S. enterica and L. monocytogenes, along with five strains of the non-target species E. coli was followed by enrichment in SEL medium for M-TRFLP analysis. This allowed for detection of both target species in all samples, with detection of one S. enterica and two Listeria TRFs in all cases, and detection of a second S. enterica TRF in 91% of cases. This was from an initial inoculum of <5 cfu per 25 ml milk with a background of competing E. coli present, and gave a result from sampling of under 20 hours. The ability to increase target species number without loss of sensitivity means that extensive screening can be performed at reduced cost due to a reduction in the number of tests required.

Temporal variation and host association in the Campylobacter population in a longitudinal ruminant farm study.

Campylobacter jejuni and C. coli were quantified and typed, using multilocus sequence typing (MLST), from fecal samples collected from a mixed cattle and sheep farm during summer. Cattle had a significantly higher prevalence than sheep (21.9% [74/338] and 14.0% [30/214], respectively), but both decreased over time. There were no differences in the average Campylobacter concentrations shed by cattle (600 CFU g(-1)) and sheep (820 CFU g(-1)), although sheep did show a significant temporal reduction in the number of Campylobacter organisms shed in their feces. A total of 21 different sequence types (STs) (97.7% C. jejuni, 2.3% C. coli) were isolated from cattle, and 9 different STs were isolated from sheep (40.6% C. jejuni, 59.4% C. coli). The Campylobacter population in cattle was relatively stable, and the frequencies of genotypes isolated showed little temporal variation. However, the composition of subtypes isolated from sheep did show significant temporal differences. The cattle and sheep consistently showed significant differences in their carriage of Campylobacter species, STs, and CCs despite the fact that both were exposed to the same farming environment. This work has highlighted the patterns of a Campylobacter population on a ruminant farm by identifying the existence of both temporal and between-host variations.

Campylobacter excreted into the environment by animal sources: prevalence, concentration shed, and host association.

An intensive study of 443 isolates of Campylobacter jejuni and Campylobacter coli from 2031 fecal samples excreted by animal sources including cattle, sheep, and pigs, a range of wild and domesticated avian species and pets is described. The prevalence found in the majority of animal sources ranged from 22% to 28% with poultry being highest at 41% and cats and dogs lowest (<5%). The average count excreted for each animal source was found not to be significantly different ranging from approximately 10(2) to 10(5) cfu/g. Multilocus sequence typing (MLST) identified phylogenies that exhibited host specificity. A number of clonal complexes (CCs) and sequence types (STs) were characteristic of particular hosts (e.g., CC-179, ST-637, and ST-1341 found only in pigeons and gulls). Analysis of genetic distance demonstrated numerous significant differences in the distribution of MLST types (CC, ST, and allele) between animal sources. Host association was quantified using structure that correctly assigned the nine animal sources with accuracies of 28%, 24%, and 55% at the CC, ST, and allele levels, respectively. This is substantially higher than would be expected by random allocation (11%) but farmyard poultry had the lowest assignment accuracy (13%, 13%, and 21%) suggesting that isolates were shared with a wide range of other animals. This study demonstrates the link between MLST type and host and provides data that can be used in risk assessment and food attribution models. Further, it demonstrates the applicability of MLST to characterize Campylobacter strains from a broad range of environmental sources.

Spatiotemporal homogeneity of Campylobacter subtypes from cattle and sheep across northeastern and southwestern Scotland.

Source attribution using molecular subtypes has implicated cattle and sheep as sources of human Campylobacter infection. Whether the Campylobacter subtypes associated with cattle and sheep vary spatiotemporally remains poorly known, especially at national levels. Here we describe spatiotemporal patterns of prevalence, bacterial enumeration, and subtype composition in Campylobacter isolates from cattle and sheep feces from northeastern (63 farms, 414 samples) and southwestern (71 farms, 449 samples) Scotland during 2005 to 2006. Isolates (201) were categorized as sequence type (ST), as clonal complex (CC), and as Campylobacter jejuni or Campylobacter coli using multilocus sequence typing (MLST). No significant difference in average prevalence (cattle, 22%; sheep, 25%) or average enumeration (cattle, 2.7 x 10(4) CFU/g; sheep, 2.0 x 10(5) CFU/g) was found between hosts or regions. The four most common STs (C. jejuni ST-19, ST-42, and ST-61 and C. coli ST-827) occurred in both hosts, whereas STs of the C. coli ST-828 clonal complex were more common in sheep. Neither host yielded evidence for regional differences in ST, CC, or MLST allele composition. Isolates from the two hosts combined, categorized as ST or CC, were more similar within than between farms but showed no further spatiotemporal trends up to 330 km and 50 weeks between farm samples. In contrast, both regions yielded evidence for significant differences in ST, CC, and allele composition between hosts, such that 65% of isolates could be attributed to a known host. These results suggest that cattle and sheep within the spatiotemporal scales analyzed are each capable of contributing homogeneous Campylobacter strains to human infections.

E. coli O157 from sheep in northeast Scotland: prevalence, concentration shed, and molecular characterization by multilocus variable tandem repeat analysis.

We report the prevalence, concentrations, and strain diversity of Escherichia coli O157 shed by sheep fed on root crops during a winter period in northeast Scotland. E. coli O157 was isolated on 6 farms from 14 studied during January to March 2005. The individual sheep prevalence was 5.8% and concentration excreted was <10(2) colony-forming units/g for all but one fecal sample. Verocytotoxigenic E. coli O157, determined by polymerase chain reaction and verocell assay, was recovered from 27% of samples. Four farms had sheep shedding the same strain as determined by multiple-locus variable analysis and no within-farm diversity was observed. The low numbers shed and the high levels of atoxigenic strains indicate a lower risk to human health from these animals compared to many ruminants grazing pasture during summer months. These data will be valuable for quantitative risk assessments and provide preliminary information that feeding sheep on root crops may be a practical intervention to reduce E. coli O157 infection in animals and ultimately humans.

Campylobacter genotypes from food animals, environmental sources and clinical disease in Scotland 2005/6.

A nationwide multi-locus sequence typing (MLST) survey was implemented to analyze patterns of host association among Campylobacter jejuni and Campylobacter coli isolates from clinical disease in Scotland (July 2005-September 2006), food animals (chickens, cattle, sheep, pigs and turkey), non-food animals (wild birds) and the environment. Sequence types (STs) were determined for 5247 clinical isolates and 999 from potential disease sources (augmented with 2420 published STs). Certain STs were over represented among particular sample sets/host groups. These host-associated STs were identified for all sample groups in both Campylobacter species and host associated clonal complexes (groups of related STs) were characterized for C. jejuni. Some genealogical lineages were present in both human disease and food animal samples. This provided evidence for the relative importance of different infection routes/food animal sources in human disease. These results show robust associations of particular genotypes with potential infection sources supporting the contention that contaminated poultry is a major source of human disease.

Has retail chicken played a role in the decline of human campylobacteriosis?

Between 2001 and 2006, the incidence of human Campylobacter infections decreased by 10 and 27% in Scotland and the Grampian region of Scotland, respectively. Contemporaneous collection and analyses of human and retail-chicken isolates from Grampian were carried out over a 10-week period in 2001 and again in 2006 in order to determine whether the fall in the incidence of human infections was related to the retail-chicken exposure route. Rates of carriage of Campylobacter on chicken carcasses from retail outlets in Grampian in 2001 and 2006 were estimated. Chicken-derived Campylobacter isolates from 2001 (n = 84) and 2006 (n = 105) and human-derived isolates from patients with clinical cases of infection in 2001 (n = 172) and 2006 (n = 119) were typed by multilocus sequence typing. We found no evidence for statistically significant changes in prevalence and counts per carcass. We found by rarefaction that although the degree of diversity in humans tended to be higher than that in chickens, these differences were not significant. The genetic distance between chicken and human isolates from 2001 according to sequence type, clonal complex (CC), or allele composition was not significant, whereas the distances between 2006 isolates at the CC and allele levels were significant. This difference was attributable to a lower proportion of CC-21's being found in retail-chicken isolates from 2006 than in chicken isolates from 2001. We conclude that human exposure to Campylobacter via retail chicken is important and that changes in the population structure of campylobacters in this reservoir need to be taken into account in investigating human infection.

Multiple sample flow through immunomagnetic separator for concentrating pathogenic bacteria.

The standard method of immunomagnetic separation for isolating pathogenic bacteria from food and environmental matrices processes 1 ml volumes. Pathogens present at low levels (<0.5 pathogenic bacteria/g) will not be consistently detected by this method. Here a multiple sample flow through immunomagnetic separator has been designed and tested to process large volume samples (50 to 250 ml). Preliminary results show >97% recovery of polydisperse magnetic particles (diameter range 1 to 8 microm) containing 29-33% w/w Fe3O4 content. Between 70 and 130 times more of the pathogenic bacteria Escherichia coli O157 is recovered from PBS compared with the standard 1 ml method. Also, the recovery of E. coli O157 from beef mince homogenates, after a 4 h incubation at 42 degrees C, is between 80 and 180 times higher than the standard 1 ml method.

The detection of Cryptosporidium parvum and Escherichia coli O157 in UK bivalve shellfish.

Optimised immunomagnetic separation methods to detect Cryptosporidium parvum and Escherichia coli O157 in UK shellfish are described. Whole tissue homogenates gave the best recoveries for C. parvum oocysts compared with gill or haemolymph extracts. The sensitivity of recovery from spiked samples was comparable to that achieved when processing water and varied from 12-34% in mussels, 48-69.5% in oysters and 30-65% in scallops. Maximum recovery of E. coli O157 was achieved by enriching in buffered peptone water supplemented with vancomycin at 42 degrees C. Increasing enrichment temperatures from 37 to 42 degrees C gave a significant increase in target number recovery. Implementation of these methods into monitoring programmes and end-product testing will enable shellfish producers to better assess product safety.

Is the prevalence and shedding concentrations of E. coli O157 in beef cattle in Scotland seasonal?

The prevalence of Escherichia coli O157 in Scottish beef cattle at abattoir was found to be greater during the cooler months [11.2% (95% CI, 8.4-13.9%)] compared to the warmer months [7.5% (95% CI, 5.4-9.6%)]; the reverse of seasonality of human infections. However, high shedding beef cattle (excreting 10(-4) g(-1)) appear to shed greater concentrations of E. coli O157 in the warmer months which may partly explain increased human infection seasonality at this time.