Isolation and characterization of Clostridium difficile from a small survey of wastewater, food and animals in New Zealand.

The objective of this study was to undertake a microbiological survey of foods, animal faeces and wastewater samples for Clostridium difficile, and determine the genotypes and antimicrobial susceptibilities of isolates. A total of 211 samples were tested for C. difficile using culture methods. Thirteen toxigenic C. difficile isolates were obtained; ten from wastewater samples, one each from pig and duck faeces and another from a raw meat product. Eight PCR-ribotypes (RTs) were identified, including two novel RTs (878 and 879). Single-nucleotide polymorphism analysis using WGS data for all isolates provided greater discrimination between C. difficile isolates within the same RT and multilocus sequence typing (MLST) profiles. All C. difficile isolates were found to be susceptible to the first-line human antimicrobials used to treat C. difficile infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report the isolation of Clostridium difficile from animals, food and wastewater in New Zealand (NZ) and provides important data with respect to ribotypes and multilocus sequence typing profiles, whole genome sequence and antimicrobial susceptibilities. The results highlight the need for further investigations into the epidemiology of C. difficile in NZ and to elucidate the role of the environmental and food sources as transmission routes of human infection.

Evaluation of the representativeness of a sentinel surveillance site for campylobacteriosis.

It is important to assess the suitability of sentinel sites for human disease; however, there have been few publications documenting the process of formal evaluation. We describe an approach to examining the representativeness of a single sentinel site employed for campylobacteriosis surveillance and source attribution, utilizing a selection of data sources and statistical comparisons of demographic, epidemiological and pathogen genotyping data across selected regions of New Zealand. Our findings showed that while this region captured the national variability in many variables, for example by containing sizable urban and rural populations, the relative frequency of these features did vary from other regions of New Zealand. We discuss the value of choosing a sentinel site that represents the national distribution of key variables, compared to a site that captures the broad features of the wider population, but provides greater power for the monitoring of sub-populations.

Leaching of Escherichia coli from sheep faeces during simulated rainfall events.

UNLABELLED: Sheep faeces are known to harbour to a high concentration of microbial indicators and pathogens. These can be released under rainfall and may result in contamination of waterways, potentially leading to illnesses in humans. A study was designed to determine the concentration of Escherichia coli released from fresh and aged (0-21 days old) ovine faeces. In summer and autumn, ovine faeces were subjected to simulated rainfall and the resultant run-off collected. Escherichia coli were enumerated in both the run-off and the faeces. In autumn total suspended solids (TSS) and turbidity were also monitored in the run-off. This study provides quantitative evidence that E. coli in aged sheep faeces is mobilized by rainfall events. Simulated rainfall events released between 10(3) and 10(4) CFU E. coli ml(-1) throughout the 21 days. TSS or turbidity with fresh faeces may be indicative of microbial contamination, but from aged faeces, this may not be the case. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that faecal bacteria can be released from fresh and aged ovine faeces under stimulated rainfall. It demonstrates that aged faeces remain a source of faecal bacteria, which under rainfall can release the bacteria and result in pollution of waterways. This study aids in our understanding of the potential impact of grazing sheep on the microbial quality of surface waters in NZ.

Application of molecular epidemiology to understanding campylobacteriosis in the Canterbury region of New Zealand.

Pulsed-field gel electrophoresis genotypes of Campylobacter isolates from 603 human patients were compared with 485 isolates from retail offal (primarily chicken and lamb) to identify temporal clusters and possible sources of campylobacteriosis. Detailed epidemiological information was collected from 364 of the patients, and when combined with genotyping data allowed a putative transmission pathway of campylobacteriosis to be assigned for 88% of patients. The sources of infection were 47% food, 28% direct animal contact, 7% overseas travel, 4% person-to-person transmission and 3% water-related. A significant summer increase in campylobacteriosis cases was primarily attributed to an increase in food-related cases. Genotyping of isolates was essential for identifying the likely cause of infection for individuals. However, a more rapid and cheaper typing tool for Campylobacter is needed, which if applied to human and animal isolates on a routine basis could advance greatly our understanding of the ongoing problem of Campylobacter infection in New Zealand.

Distinguishing human and possum faeces using PCR markers.

Specificity testing of two published polymerase chain reaction (PCR) markers for the detection of human faecal pollution, revealed 100% false-positive rates to brush-tailed possum faeces (n = 10), but low false-positive rates against other potential pollution sources. Cross-reaction with possums could be a problem with other human-specific markers; therefore, a possum PCR marker was developed for use in conjunction with human PCR markers. The possum PCR marker was based on Bacteroidales 16S ribosomal ribonucleic acid sequences, and was tested on 233 individual faecal samples from 11 other animal species. Sensitivity of the possum marker in possum faeces (n = 36) was high at 83.3%. Cross-reactivity of the possum marker was limited to black swan (7/20 samples), human (2/48 samples) and rabbit (1/10) faecal samples, all at marker concentrations at least four orders of magnitude lower than possum faeces. The possum marker was not detected in human sewage or the faeces of other animal species. Specificity of the possum PCR marker, therefore, was high at 95.7%. To exclude the possibility that only possum pollution is being detected, additional testing by other faecal source tracking methods is required where the water sample is positive for both human and possum markers.

Survival of Campylobacter spp. in bovine faeces on pasture.

AIMS: To determine the survival on pasture of Campylobacter spp. naturally present in bovine faeces and compare this with a previously published study using laboratory-cultured Campylobacter spp. METHODS AND RESULTS: Ten freshly collected cow pats were deposited on pasture during summer, and Campylobacter spp. were enumerated by enrichment broth culture. The counts in three pats were below detection limits. Counts of Campylobacter spp. in the other seven pats fell below detection limits within 14 days. The geometric means of the counts up to 7 days produced a T(90) of 2.2 days. Characterization of Campylobacter spp. by PCR and pulsed field gel electrophoresis indicated the presence of at least six genotypes of Campylobacter jejuni, Campylobacter coli and Campylobacter lari. CONCLUSIONS: Campylobacter spp. naturally present in cow faeces exhibited a similar survival rate to that previously determined using laboratory-cultured strains. The highly variable counts of naturally occurring Campylobacter spp., and the predominance of lower counts, also support the earlier decision to use laboratory-cultured strains in survival experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reaffirms the short survival of Campylobacter spp. in cow faeces deposited on pasture. This information will be incorporated into a 'reservoir model' for Campylobacter spp. in cow pats on New Zealand pastures.

Comparison of Campylobacter jejuni genotypes from dairy cattle and human sources from the Matamata-Piako District of New Zealand.

AIMS: To identify the prevalence and types of Campylobacter jejuni carried by dairy cattle and the extent of overlap of these types with those causing disease in humans. METHODS AND RESULTS: Faecal samples from 410 dairy cattle were collected from 36 farms in the Matamata-Piako district in New Zealand. Campylobacter jejuni was isolated on all 36 farms, with a prevalence of 51% (95% CI 45-57) in dairy cattle and 65% (95% CI 58-72) in calves. Eighty-nine of these isolates were typed using Penner serotyping and pulsed field gel electrophoresis and were compared with 58 human C. jejuni isolates from people resident within this study area. CONCLUSIONS: Campylobacter jejuni were found in the faeces of over half of the dairy cows and calves examined. Twenty-one per cent of the bovine isolates and 43% of the human isolates formed indistinguishable clusters of at least one bovine and one human isolate. SIGNIFICANCE AND IMPACT OF THE STUDY: While a direct link between bovine isolates and human cases was not demonstrated, the finding of indistinguishable genotypes among C. jejuni isolates from bovine and human sources confirms that dairy cows and calves are a potential source of human campylobacteriosis. Barriers to separate bovine faecal material from the general public are therefore important public health measures.

Concentrations of faecal coliforms, Escherichia coli, enterococci and Campylobacter spp. in equine faeces.

AIMS: To determine the concentration of Campylobacter spp. as well as faecal indicator bacteria; faecal coliforms, Escherichia coli and enterococci in the faeces of healthy adult horses in a sample of properties in the Canterbury region of New Zealand. METHODS: The faeces of healthy adult horses (n=59), including ponies, pleasure horses and Thoroughbreds, were collected from eight properties around Christchurch, New Zealand. The faeces were analysed for concentrations of Campylobacter spp and faecal indicator bacteria; faecal coliforms, Escherichia coli and enterococci. The presence of other animals on the properties sampled as well as the age, feed and health of the horses at the time of sampling was recorded. RESULTS: Enterococci and faecal coliforms were isolated from all samples, and E. coli was isolated from 58/59 samples. Mean concentrations of faecal coliforms and E. coli did not differ between properties, but there was a significant difference in mean concentration of enterococci between properties. Campylobacter spp. were detected in two faecal samples with one isolate being determined by PCR analysis to be a thermotolerant Campylobacter species, the other C. jejuni. CONCLUSIONS: This is the first known report quantifying the concentration of Campylobacter spp. present in healthy adult horses in New Zealand. The presence of equine faecal material in water could elevate concentrations of faecal bacteria and therefore needs to be considered as a source of water contamination. The access of horses to waterways and coastal environments may also need to be restricted to prevent transmission of faecal indicator bacteria and potentially zoonotic agents.

Tissue-specific expression of the human laminin alpha5-chain, and mapping of the gene to human chromosome 20q13.2-13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation.

To investigate the function of the laminin alpha5-chain, previously identified in mice, cDNA clones encoding the 953-amino-acid carboxy terminal G-domain of the human laminin alpha5-chain were characterized. Northern blot analysis showed that the laminin alpha5-chain is expressed in human placenta, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin alpha5-chain gene (LAMA5) was assigned to chromosome 20q13.2-q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra) mutation.

Application of polymerase chain reaction (PCR) and TaqMan PCR techniques to the detection and identification of Rhodococcus coprophilus in faecal samples.

Rhodococcus coprophilus, a natural inhabitant of herbivore faeces, has been suggested as a good indicator of animal (as opposed to human) faecal contamination of aquatic environments. However, conventional detection methods limit its use for this as they require up to 21 days to obtain a result. In this paper an optimised method for extracting R. coprophilus DNA from faecal samples is described. PCR and 5'-nuclease (TaqMan) PCR methods were developed to allow the detection and enumeration of R. coprophilus in faecal samples within 2-3 days. Both PCR methods targeted the 16S rRNA gene, producing an amplicon of 443 bp which was specific for R. coprophilus. Sixty cells were required to produce an amplification product by conventional PCR, while as little as one cell was required for the TaqMan PCR method. The latter approach gave a linear quantitative response over at least four log units with both bacterial cells and DNA. Successful amplification by PCR was achieved using DNA extracted from cow, sheep, horse and deer faeces but was negative for samples from humans, pig, possum, duck and rabbit. These PCR methods enhance the feasibility of using R. coprophilus to distinguish faecal pollution of farmed herbivores from human pollution.

The limitations of pulsed-field gel electrophoresis for analysis of Yersinia enterocolitica isolates.

This study describes the analysis of 432 isolates of Yersinia enterocolitica by pulsed-field gel electrophoresis (PFGE). PFGE had a high level of discrimination with biotype 1A isolates (Simpson's Diversity Index 0.997), but with the clinically important biotypes 2, 3 and 4, the discriminatory ability of PFGE was so low as to severely limit its usefulness (DI <0.6). For biotypes 2, 3 and 4, 79% or more of isolates of each biotype were of just three different PFGE profiles. Because of this, four known outbreaks of yersiniosis would not have been identified by PFGE analysis. However, a previously unrecognized potential outbreak of yersiniosis caused by biotype 4 isolates was identified on the basis of a rare PFGE genotype with spatial and temporal clustering. We conclude that PFGE has a very limited application to the genotyping of Y. enterocolitica biotypes 2, 3 and 4, and inferences based on finding indistinguishable PFGE profiles among cases or between cases and sources need to be substantiated using alternative typing tools, or strong epidemiological evidence.

The prevalence and genetic diversity of Campylobacter spp. in domestic 'backyard' poultry in Canterbury, New Zealand.

Campylobacteriosis is the most commonly notified illness in New Zealand. Whilst the importance of commercial poultry in campylobacteriosis is well established, little is known about the possible role of chickens kept at home as a direct animal/faecal contact or consumption exposure pathway. The aim of this study was to determine the prevalence and genetic diversity of Campylobacter spp. in domestic backyard chicken flocks in the Canterbury region of New Zealand. Poultry faecal samples were collected from 35 domestic 'backyard' poultry flocks from urban and rural properties around the Canterbury Region of New Zealand. A total of 291 samples were collected and tested for the presence of thermotolerant Campylobacter spp. and positive isolates were analysed using pulsed-field gel electrophoresis (PFGE) using both SmaI and KpnI enzymes. There was a high prevalence of Campylobacter spp. with 86% of flocks testing positive. Campylobacter jejuni alone, Campylobacter coli alone and both C. jejuni and C. coli were detected in 20 (57%), 2 (6%) and 8 (23%) of the flocks respectively. SmaI/KpnI PFGE analysis identified 50 different genotypes across the 35 flocks. Genotype diversity richness was highest on the lifestyle block and farm properties with 43 different genotypes isolated, whilst urban properties displayed the least richness with 12 genotypes isolated. Rural flocks tended to have more different genotypes in a given flock than urban flocks. Comparison of the genotypes with the PulseNet Aotearoa Campylobacter database showed that 28 of the genotypes had previously been isolated from human cases of campylobacteriosis. Many of these were also indistinguishable from Campylobacter spp. previously isolated from retail chicken. Therefore, contact with backyard poultry or their faecal material is a potential additional infection pathway outside of exposure to the established pathways associated with the consumption of Campylobacter-contaminated commercial meat or foods cross-contaminated from contaminated poultry.

The transmission of thermotolerant Campylobacter spp. to people living or working on dairy farms in New Zealand.

New Zealand has one of the highest rates of campylobacteriosis in the developed world with an incidence rate of 383.5 cases per 100,000 in 2006. Dairy farming has been suggested as a potential source of campylobacteriosis. To explore this connection, seven farm investigations were undertaken at dairy farms on which a campylobacteriosis case had been notified. Campylobacter spp. were isolated from a range of sources on the farm (including 66% of bovine faecal samples) and genotypes compared with that of the clinical isolate of the index case. In depth, epidemiological questionnaires were also administered to determine exposure risks from a wide range of possible sources. Contact with dairy cow faeces was the most likely source of infection in four of the seven cases investigated, and occurred exclusively in new farm workers and children. In one of the cases investigated, infection was likely to have been acquired from non-dairy related sources, and in two cases the source could not be determined. The relative risk of dairy farm worker being notified with campylobacteriosis was estimated to be 1.88 (95% confidence interval=1.6-2.2).

Statistical comparison of Campylobacter jejuni subtypes from human cases and environmental sources.

AIM: To analyse Campylobacter jejuni typing data to define statistically which potential reservoirs and transmission sources contain isolates that are most similar to one another and to isolates from human infections. METHODS AND RESULTS: Serotyping and SmaI macrorestriction profiling data for C. jejuni isolates from human campylobacteriosis cases, chicken carcass rinses, duck, sheep, dairy and beef cattle faeces, river water, and sheep, beef and pork offal obtained from a defined rural area of New Zealand were compared using the Czekanowski proportional similarity index. Subtypes of isolates from ruminant animals, whether derived from their faeces or offals, were generally similar to one another. The spectrum of isolate subtypes from human cases was more similar to that from ruminant faeces than the other matrices considered. Isolate subtypes from chicken rinses, pork offal, water and duck faeces were not highly similar to those from other matrices. CONCLUSIONS: Results from a combination of phenotypic and genotypic approaches suggest that, for this rural population, exposures associated with a rural lifestyle may be significant sources of human campylobacteriosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The Czekanowski index was applied to subtyping data and supported the greater importance of contact with livestock in campylobacteriosis cases associated with a rural setting, in comparison with urban studies that have identified poultry-related factors.

Genotypic characterization of bacteria cultured from duck faeces.

AIMS: To characterize the bacterial composition of mallard duck faeces and determine if novel bacterial species are present that could be utilized as potential indicators of avian faecal contamination. METHODS AND RESULTS: Combined samples of fresh faeces from four ducks were serially diluted and plated onto six different media selected to allow the growth of a range of organisms at 42 degrees C under three atmospheric conditions: aerobic, microaerophilic and anaerobic. Forty-seven morphologically dissimilar isolates were purified and partial sequencing of the16S rRNA indicated at least 31 bacterial species. Twenty of these could be identified to the species level including pathogenic species of Bacillus, Campylobacter, Clostridium and Streptococcus. Other species identified included: Enterococcus, Escherichia, Megamonas, Cellulosimicrobium, Neisseria, Staphylococcus and Veillonella. Potentially novel species, which could represent bacteria specific to avian fauna included Bacillus, Corynebacterium, Macrococcus and Peptostreptococcus, while four isolates had <97% similarity to known bacterial species in the available databases. CONCLUSION: A survey of the natural microflora of the mallard duck and its hybrid with the grey duck identified both bacteria that are potentially human pathogenic and putative novel bacteria species as determined by 16S rRNA sequencing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides further evidence that duck faeces is a potential human health hazard, and has identified bacteria potentially useful for distinguishing duck faeces from other faecal sources.

The occurrence of Campylobacter subtypes in environmental reservoirs and potential transmission routes.

AIM: To identify potential reservoirs and transmission routes of human pathogenic Campylobacter spp. METHODS AND RESULTS: An enrichment PCR method for the detection and identification of Campylobacter jejuni and/or Campylobacter coli in faecal, food and river water samples was applied to 1450 samples of 12 matrix types obtained from a defined geographical area. PCR-positive samples were cultured to yield isolates for typing, and the data for 616 C. jejuni isolates obtained. Serotyping and SmaI macrorestriction profiling using pulsed field gel electrophoresis revealed a high level of diversity within the isolates from each matrix. Campylobacter jejuni and C. coli subtypes indistinguishable from those obtained from human cases were detected in most of the matrices examined. No Campylobacter isolates were isolated from possum faeces. CONCLUSIONS: Ten of the 12 matrices examined may be involved in the transmission of human campylobacteriosis as they contained Campylobacter subtypes also isolated from clinical cases. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate that, for this rural population, a range of potential transmission routes that could lead to campylobacteriosis exist. Their relative importance needs to be assessed from an exposure assessment standpoint.

Characterization of erythromycin resistance in Campylobacter coli and Campylobacter jejuni isolated from pig offal in New Zealand.

AIMS: To determine the level and mechanism(s) of antimicrobial resistance in Campylobacter isolates obtained from human and environmental sources from South Canterbury, New Zealand. METHODS AND RESULTS: A total of 251 Campylobacter isolates were tested for susceptibility to ciprofloxacin, erythromycin, nalidixic acid and tetracycline using disc diffusion assays. Five pig offal isolates were observed to be highly erythromycin resistant, with minimal inhibitory concentrations determined to be >/=256 microg ml(-1). Nucleotide sequencing of the 23S ribosomal DNA (rDNA) in these resistant isolates identified an A --> G change at Escherichia coli position 2059 that has been previously implicated in erythromycin resistance in Campylobacter coli. Macrorestriction profiling using pulsed-field gel electrophoresis showed these isolates were nonclonal. CONCLUSIONS: The majority of Campylobacter isolates from South Canterbury remain sensitive to the most clinically relevant antimicrobial agents. Our results support other reports showing that specific variations in the 23S rDNA contribute to erythromycin resistance. SIGNIFICANCE AND IMPACTS OF THE STUDY: This study defines the baseline frequency of antimicrobial resistance associated with Campylobacter isolates from South Canterbury, and discusses the likely molecular mechanisms conferring erythromycin resistance in this organism. Resistance to erythromycin in these isolates is not linked to a dominant Campylobacter clone and has likely arisen independently in different genetic lines exposed to selective antimicrobial pressure.

Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion.

The ADAMs (A disintegrin and metalloprotease) comprise a family of membrane-anchored cell surface proteins with a putative role in cell-cell and/or cell-matrix interactions. By immunostaining, ADAM 12 (meltrin alpha) was up-regulated in several human carcinomas and could be detected along the tumor cell membranes. Because of this intriguing staining pattern, we investigated whether human ADAM 12 supports tumor cell adhesion. Using an in vitro assay using recombinant polypeptides expressed in Escherichia coli, we examined the ability of individual domains of human ADAM 12 and ADAM 15 to support tumor cell adhesion. We found that the disintegrin-like domain of human ADAM 15 supported adhesion of alphavbeta3-expressing A375 melanoma cells. In the case of human ADAM 12, however, recombinant polypeptides of the cysteine-rich domain but not the disintegrin-like domain supported cell adhesion of a panel of carcinoma cell lines. On attachment to recombinant polypeptides from the cysteine-rich domain of human ADAM 12, most tumor cell lines, such as MDA-MB-231 breast carcinoma cells, were rounded and associated with numerous actin-containing filopodia and used a cell surface heparan sulfate proteoglycan to attach. Finally, we demonstrated that authentic full-length human ADAM 12 could bind to heparin Sepharose. Together these results suggest a novel role of the cysteine-rich domain of ADAM 12 -- that of supporting tumor cell adhesion.

Human ADAM 12 (meltrin alpha) is an active metalloprotease.

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM 12 is catalytically active. We used the trapping mechanism of alpha2-macroglobulin to assay for protease activity of wild-type and mutant ADAM 12 proteins produced in a COS cell transfection system. We found that ADAM 12 is synthesized as a zymogen, with the prodomain maintaining the metalloprotease in a latent form, probably by means of a cysteine switch. The zymogen could be activated chemically by alkylation with N-ethylmaleimide. Cleavage of the prodomain at a site for a furin-like endopeptidase resulted in an ADAM 12 protein with proteolytic activity. The protease activity was sensitive to inhibition by 1,10-phenanthroline and could be eliminated by mutation of the critical glutamate residue at the active site. The demonstration that the ADAM 12 metalloprotease domain is functional may have important implications for future studies that explore the role of ADAM 12 protein in development and disease.

The detection of Bifidobacterium adolescentis by colony hybridization as an indicator of human faecal pollution.

AIMS: To develop an improved method for the detection of Bifidobacterium adolescentis as an indicator of human faecal pollution. METHODS AND RESULTS: Bifidobacterium medium (BFM) was identified as the optimal medium for the recovery of bifidobacteria from human effluent. Dilutions of faeces and effluent from both humans and animals were filtered, grown on BFM and human specific B. adolescentis identified via colony hybridization with a digoxigenin (DIG)-labelled oligonucleotide probe. CONCLUSIONS: The combination of BFM with colony probing allows the detection of B. adolescentis, a specific indicator of human faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: It is now technically feasible to use B. adolescentis as indicators of human faecal pollution, and studies to examine the survival and appropriateness of bifidobacteria in this role can be initiated.