Campylobacter immunity and coinfection following a large outbreak in a farming community.

An outbreak of campylobacteriosis affected approximately one-half of 165 people attending an annual farmers' dance in Montrose, Scotland, in November 2005. Epidemiological investigations, including a cohort study (n = 164), identified chicken liver paté as the most likely vehicle of infection. Paté preparation involved deliberate undercooking of chicken livers by flash-frying, followed by mechanical homogenization. Typing of 32 Campylobacter strains (isolated from submitted stools) by multilocus sequence typing identified four distinct clades of Campylobacter jejuni. There was good agreement when isolates were typed by Penner serotyping, pulsed-field gel electrophoresis, and flaA short variable region sequencing but poorer agreement with phage and antibiotic susceptibility testing. At least three attendees were coinfected with two Campylobacter strains each. The outbreak was probably due to several livers contributing Campylobacter strains that survived undercooking and were dispersed throughout the paté. The study highlights improper culinary procedures as a potential human health risk and provides a striking counterexample to the "dominant outbreak strain" view of point source outbreaks of food-borne infections. It also demonstrates that previous exposure to biologically plausible sources of Campylobacter may confer protection against subsequent infection.

Persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline.

OBJECTIVES: The aim of this study was to investigate the persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline. METHODS: Three commercially reared broiler flocks, naturally colonized with Campylobacter, were treated with chlortetracycline under experimental conditions. The numbers of Campylobacter isolated, and the species, flaA short variable region allele, and antimicrobial resistance of isolates were determined. RESULTS: For two of three flocks, tetracycline-resistant strains predominated prior to chlortetracycline exposure. Presence of the antibiotic had no discernible effect on the numbers or types of Campylobacter and the tetracycline-resistant strains persisted in numbers similar to those observed before treatment. With all flocks, some faecal samples were obtained that contained no Campylobacter, irrespective of exposure to chlortetracycline; this was more common as the birds grew older. For the third flock, tetracycline-resistant Campylobacter were in the minority of samples before and during exposure to chlortetracycline, but at sampling times after this, no resistant strains were found in the treated (or untreated) birds, irrespective of exposure to the antibiotic. All tetracycline-resistant isolates (MICs 16 to >128 mg/L) contained tet(O) and, for some isolates, this was transferable to Campylobacter jejuni 81116. The efflux pump inhibitor PAbetaN reduced the MICs of tetracycline for these isolates by 4-fold, suggesting that an intact efflux pump, presumably CmeABC, is required for high-level tetracycline resistance. CONCLUSIONS: Our data indicate that chlortetracycline treatment does not eradicate tetracycline-resistant Campylobacter spp. from poultry. However, if a low number of resistant isolates are present, then the antibiotic pressure appears insufficient to select such strains as the dominant population.

Campylobacter and Salmonella in raw red meats in the United Kingdom: prevalence, characterization and antimicrobial resistance pattern, 2003-2005.

The prevalence of Campylobacter and Salmonella was assessed in 3959 raw red meats in the UK during 2003-2005. Meats were more frequently contaminated with Campylobacter (7.2%) than with Salmonella (2.4%). Lamb and other meats (e.g. mutton, rabbit) exhibited the highest contamination from Campylobacter (12.6% and 19.8%, respectively), compared with pork (6.3%) and beef (4.9%). Pork however had the highest contamination from Salmonella (3.9%), followed by lamb (2.0%), other meats (2.0%) and beef (1.3%). Offal samples (36.6%) were more frequently contaminated with Campylobacter or Salmonella than muscle tissue (7.0%). C. jejuni predominated in all meat types. C. coli isolates were more likely to exhibit antimicrobial drug resistance, including quinolones, than C. jejuni. Salmonella typhimurium was the most frequent Salmonella serotype isolated from meats; S. typhimurium DT104/104b isolates exhibited higher rates of multiple drug resistance than other serotypes. The findings reinforce the importance of adequate cooking of meat and good hygiene to avoid cross-contamination.

Surveillance of primary antibiotic resistance of Helicobacter pylori at centres in England and Wales over a six-year period (2000-2005).

Antibiotic resistance is a key factor in the failure of Helicobacter pylori eradication therapy, yet few sentinel schemes exist to monitor trends in resistance at local, national or international levels. This study aimed, over a six-year period, to monitor resistance levels of H. pylori in England and Wales to the four antibiotics used in its treatment. A total of 1,310 isolates from Gwynedd in north Wales and from mid-Essex in south-east England were collected from 2000 to 2005 and tested for susceptibilities to metronidazole, clarithromycin, amoxicillin and tetracycline. Overall, metronidazole and clarithromycin resistance rates were 28.6% and 8.3% in Gwynedd and significantly higher (36.3%, p=0.0031, and 12.7%, p=0.0112) in mid-Essex. Rates of resistance to metronidazole and clarithromycin increased in both areas over this six-year period. Resistance rates were higher in female compared with male patients (38.1% vs 26.6% for metronidazole, p<0.0001, and 12.9% vs 7.5% for clarithromycin, p=0.0024), and were higher in patients <45 years compared with those ?45 years (44.0% vs 29.0% for metronidazole, p=0.0002, and 15.0% vs 9.4% for clarithromycin, p=0.0233). This study highlights the importance of antibiotic resistance surveillance in H. pylori for providing information on local resistance rates for test and treat strategies.

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Specific detection of Campylobacter jejuni from faeces using single nucleotide polymorphisms.

Specimens of human faeces were tested by a rapid strategy for detection of Campylobacter jejuni lineages by the presence of specific single nucleotide polymorphisms (SNPs) based on the C. jejuni multi locus sequence typing (MLST) scheme. This strategy was derived from analysis of the MLST databases to identify clonal complex specific SNPs followed by the design of real-time PCR assays to enable identification of six major C. jejuni clonal complexes associated with cases of human infection. The objective was to use the MLST SNP-based assays for the direct detection of C. jejuni by clonal complex from specimens of human faeces, and then confirm the accuracy of the clonal complex designation from the SNP-based assays by performing MLST on the cultured faecal material, this targeted at determining the validity of direct molecular specimen identification. Results showed it was possible to identify 38% of the isolates to one of the six major MLST clonal complexes using a rapid DNA extraction method directly from faeces in under 3 h. This method provides a novel strategy for the use of real-time PCR for detection and characterization beyond species level, supplying real-time epidemiological data, which is comparable with MLST results.

Culture of Helicobacter pylori from domestic water samples--the impact of strain variation on growth on solid and in liquid media.

Helicobacter pylori is an important global human pathogen and there is growing evidence from PCR assays that contaminated drinking water might be a possible source of infection in some circumstances. There are no validated protocols for direct isolation but various culture media have been developed for possible environmental sampling. Our aim here was to investigate how inter-strain variation might affect the interpretation of results with such media. Two laboratory adapted reference strains and four recent clinical isolates were tested on four solid media and in ten liquid media. Considerable variation was found between strains in their ability to recover on the different media after stress exposure (suspension in sterile tap water). Generally, clinical isolates were less robust than the laboratory-adapted strains and, overall, the former required longer recovery times. Our findings highlighted the importance of using a range of isolates for evaluations, as examination of laboratory-adapted strains alone did not provide an accurate representation of the utility of media that may be used to recover H. pylori from water.

An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy.

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution.

Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England.

AIMS: To investigate treated water distribution systems in England as a source of Helicobacter pylori. METHODS AND RESULTS: Water and biofilms were obtained from 11 domestic and seven educational properties and from hydrants, reservoirs and water meters supplied by three water utilities. Samples were cultured on nonselective and antibiotic containing media combined with immunomagnetic separation concentration. Viable helicobacters were not detected in any of the 151 samples but Helicobacter-specific PCR assays detected DNA in 26% of samples from domestic properties, schools and hydrants with the highest frequency in biofilms (42%). Direct sequencing of six selected amplicons confirmed >95% sequence homology to H. pylori. CONCLUSIONS: While viable helicobacters were not isolated, evidence was obtained for the presence of Helicobacter DNA, including that of H. pylori. Biofilms on surfaces within water distribution systems may act either as sites for the passive accumulation of helicobacters or as potentially important reservoirs of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings strengthen evidence that H. pylori may be transmitted through drinking water. However, there is currently no evidence that viable cells can survive the disinfection levels used in UK mains supplies and the health risk from this source remains unclear.

Evaluation of Helicobacter species in inflammatory bowel disease.

BACKGROUND: Bacteria have been implicated in the pathogenesis of inflammatory bowel disease. Helicobacter species have been shown to cause colitis in animal models and have been identified in human diarrhoeal illness and Crohn's disease. AIM: To determine whether Helicobacter species are present in human inflammatory bowel disease tissue. METHODS: Thirty patients undergoing colonoscopy for clinical reasons were studied. Nine had Crohn's disease, 11 had ulcerative colitis and 10 had histologically normal colons. Tissue was snap-frozen at -70 degrees C. DNA was extracted and examined by five different polymerase chain reaction (PCR) assays that were either genus or species specific for Helicobacter. RESULTS: Analyses of colonic biopsies by two Helicobacter genus-specific PCR assays, two H. pylori-specific assays and a PCR assay designed to amplify fragments of 'H. heilmannii'-like organisms demonstrated that product was not generated by any test. Internal control PCR demonstrated that PCR results for the five assays were not negative due to the presence of residual substances inhibitory to PCR. CONCLUSIONS: Helicobacter species were not identified in this study, using multiple PCRs to eliminate the problems of non-specific cross-reaction. This suggests that Helicobacter species do not play a role in the pathogenesis of inflammatory bowel disease.

Surveillance and clinical relevance of vacA genotypes of Helicobacter pylori infecting dyspeptic patients in mid-Essex.

The Helicobacter pylori vacuolating cytotoxin is a putative pathogenicity factor encoded by vacA, a mosaic gene with a global distribution. The vacA type prevalence and diversity of H. pylori isolated from antral gastric biopsies of 360 dyspeptic patients in mid-Essex, and of 79 patients from other locations, were investigated in order to test for links with disease severity. Mid (m)-region genotyping and subtyping by vacA HaeIII RFLP (restriction fragment length polymorphism) analysis showed that the m1 and m2 alleles were diverse, with 191 different subtypes. Variation in 44% of strains was accounted for by ten subtypes of which subtype v-1 represented a conserved core (33%) of the m1 form. Prevalence rates for combined mid and signal (s)-region genotypes were 40% for s1/m1, 46% for s1/m2, and 11% for s2/m2. Overall, vacA genotyping provided high typability and discrimination, but no specific RFLP markers could reliably predict a clinically significant presentation due to an H. pylori infection.

Determination of Helicobacter pylori vacA allelic types by single-step multiplex PCR.

AIMS: To develop and evaluate a novel multiplex PCR assay that enables definition of Helicobacter pylori vacA allelic type in a single reaction. METHODS AND RESULTS: Application of the one-step system to DNA extracts from 22 cultures of known vacA genotype demonstrated that it was highly accurate. Analysis of 15 matched gastric biopsy/culture pairs generated exactly correlating genotype profiles. vacA genotypes were determined from an additional 62/70 gastric biopsies from dyspeptic patients of known H. pylori positive status by the one-step assay, compared with 63/70 by the original two-reaction test. Types s1/m1, s1/m2 and s2/m2 were identified in 51.9%, 31.2% and 16.9% of biopsies, respectively. CONCLUSIONS: The multiplex PCR system developed enables rapid one-step vacA genotyping that is accurate, easy to interpret and more economical than the alternative multiple-reaction tests. Application of this system to gastric biopsies from patients in South-east England demonstrated that s1/m1 was the most common genotype, while s1/m2 and s2/m2 were less prevalent. SIGNIFICANCE AND IMPACT OF THE STUDY: This simple one-step system can be applied direct to antral gastric biopsies without the need for culture, thereby facilitating rapid surveillance of vacA genotype in relation to geographical location and disease status.

Molecular testing for antibiotic resistance in Helicobacter pylori.

An estimated 7.5 million individuals in England and Wales are actively infected with Helicobacter pylori and hence knowledge of local resistance rates is of growing importance. Also, information on strain resistance following treatment failure is crucial in selecting an appropriate regimen as the development of bacterial resistance to antibiotics makes retreatment difficult. Molecular test methods may have an impact on improving the availability and accuracy of information on H pylori antimicrobial resistance to guide in the selection of primary as well as secondary backup treatment regimens.

Impact of transport crate reuse and of catching and processing on Campylobacter and Salmonella contamination of broiler chickens.

The influence of transport, catching, and processing on contamination of broiler chickens with Salmonella and Campylobacter was investigated. Transport crates were reused with high frequency and were often still contaminated with Salmonella and Campylobacter when they arrived at the farm despite the fact that they were washed at the factory, and thus they were a potential route of infection. These organisms contaminated the feathers of previously Campylobacter- and Salmonella-negative birds going to the processing plant and were isolated from processed carcasses, albeit at a low frequency. The Campylobacter types which were the predominant organisms on the live birds when they arrived at the processing plant were not necessarily the types that were most frequently isolated from processed carcasses. This finding may reflect cross-contamination that occurred during processing or differences in the tolerance of the strains to the hostile environments that the bacteria experienced. The process of catching and putting the birds in crates significantly increased the chance of contamination with Campylobacter (P < 0.001).

Screening the human protocadherin 8 (PCDH8) gene in schizophrenia.

Abnormalities in synaptic connectivity and plasticity have been implicated in the pathophysiology of schizophrenia. Molecules involved in the development and maintenance of neural circuitry include the recently cloned protocadherins. Human protocadherin 8 (PCDH8) is homologous to 'arcadlin', a molecule shown to play a role in hippocampal synaptic function in the rat. The gene encoding PCDH8 maps to a region on chromosome 13 where linkage to schizophrenia has been reported. In this study, the entire expressed sequence of the PCDH8 gene and over 800 bp of the 5' flanking region were screened for polymorphisms in 30 DSM-IV schizophrenia individuals using Denaturing High Performance Liquid Chromatography (DHPLC). A total of nine single nucleotide polymorphisms were identified, including three in the first exon that are predicted to change the amino acid sequence. One polymorphism, causing the Trp7Arg change in the putative signal peptide, showed a trend towards excess of the arginine encoding allele in a case-control sample consisting of 520 DSM-IV schizophrenia patients and 535 matched controls from the UK (chi2=3.72, P [1 df]= 0.054). However, this polymorphism did not show preferential transmission to schizophrenic individuals in a separate sample of 203 proband-parent trios from Bulgaria. A second, rare single nucleotide variation, predicting the non-conservative amino acid change Glu39Ala, was found in one schizophrenic individual and their affected sibling but not in a further 352 affected individuals, nor 357 controls. These results suggest that any contribution of PCDH8 polymorphisms to schizophrenia susceptibility is likely to be weak, although the existence of rare variations of stronger effect cannot be excluded.

Conservation and microdiversity of the phospholipase A (pldA) gene of Helicobacter pylori infecting dyspeptics from different countries.

Phospholipase activity is important in bacterial pathogenicity and could contribute to the pathogenic role of Helicobacter pylori by degradation of the gastric mucus, and in maintaining long-term colonisation. Our aim was to determine the degree of variation in the phospholipase A gene (pldA) of H. pylori from different geographic locations, and to investigate links between pldA genotype and clinical disease severity, as well as with variation in cagA status and vacA genotypes. PCR-restriction fragment length polymorphism (RFLP) analysis with MboI and HaeIII was used to study 124 isolates from 10 countries that included the two genome-sequenced strains (26695 and J99), as well as Tx30a and NCTC 11637 (type strain). The 925-bp pldA fragment was amplified with a frequency of 90%. The presence of pldA was confirmed in the other strains using an alternative forward primer. Isolates were distinguished by PCR-RFLP analysis with 10 MboI and four HaeIII restriction patterns that combined to give 25 distinct pldA RFLP types. The pldA M2H2 strain genotype was most common (20%) in the UK but similar strains came from several other countries. Microdiversity was evident in pldA sequences of strains representing different RFLP types, and five M2H2 strains each had a distinct pldA sequence type. Intragenic variation was independent of gastric disease severity as well as strain cagA status and vacA genotype, with the exception of eight geographically diverse strains all with the pldA M4H3/cagA+/vacA s1m1 genotype predominantly from peptic ulcer patients. The study indicated a spectrum of genotypic variants and was supportive of a pldA function in H. pylori colonisation and persistence rather than in chronicity of infection.

Searching for susceptibility genes in schizophrenia.

The existence of an important genetic contribution to the aetiology of schizophrenia is well established from genetic epidemiological studies. However, the mode of transmission is complex and non-Mendelian. The main approaches used to identify susceptibility genes are linkage and association studies and the study of cytogenetic abnormalities associated with or linked to schizophrenia. Many linkage studies have been reported but have failed as yet to produce unequivocal, replicated demonstrations of linkage. However, modest evidence for several regions has been reported in more than one data set. Areas implicated include chromosome 22q11-12, 6p24-22, 6q, 8p22-21, 13q14.1-q32 and 1q21-q22, but in every case there are positive as well as negative findings. Most candidate gene studies have been based upon neuropharmacological studies suggesting that abnormalities in monoamine neurotransmission play a role in the aetiology of schizophrenia. Overall, the results have been disappointing, but it should be noted that the sample sizes in many of the older studies would now generally be regarded as inadequate. Finally, recent work has suggested that velo-cardio-facial syndrome (VCFS) is associated with rates of psychosis possibly as high as 30%. VCFS is caused by small interstitial deletions of chromosome 22q11 in 80-85% of individuals. Work is now under way to try and identify whether a gene or genes within the deleted region are of more general relevance to schizophrenia. Future directions in schizophrenia research include collecting larger samples to increase power of findings and applying novel methods for large-scale genotyping of single-nucleotide polymorphisms.