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.

An outbreak of multiple genotypes of Listeria monocytogenes in New Zealand linked to contaminated ready-to-eat meats-a retrospective analysis using whole-genome sequencing.

Four cases of listeriosis in a hospital (A) in New Zealand were identified in 2012. Pulsed-field gel electrophoresis (PFGE) used at the time identified four pulsotypes amongst the clinical isolates. Two of the pulsotypes matched to Listeria monocytogenes isolates obtained from ready-to-eat (RTE) meat samples from a RTE producer tested during a nationwide microbiological survey the month prior. The outbreak investigation confirmed that the RTE producer had supplied product to the hospital and additional testing confirmed the presence of L.  monocytogenes in RTE meats from the hospital kitchen. Two further listeriosis cases presented in another hospital (B) with one clinical isolate identified as the same pulsotype as identified for one case in hospital A, but the epidemiology information concluded that the clinical cases from hospital B were not linked to the outbreak. Retrospective whole-genome sequencing confirmed that epidemiologically linked isolates belonging to three different genotypes for clinical cases from hospital A and RTE meats samples from the hospital kitchen differed by 0-1 core-genome locus or single nucleotide polymorphisms (SNP). The use of core-genome multilocus sequence typing and SNP analysis provided a greater degree of discrimination between isolates compared to PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a listeriosis outbreak associated with a hospital in New Zealand and attributed to contaminated ready-to-eat (RTE) meat supplied to the hospital by a single producer. Retrospective whole-genome sequence analysis of outbreak isolates was found to provide a greater degree of discrimination between isolates compared to pulsed-field gel electrophoresis and supported the conclusions made at the time of the outbreak. The multiple genotypes identified from clinical cases and the RTE meats obtained during the outbreak highlight the importance of epidemiological concordance alongside genotyping.

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.

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading.

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.

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.

Preliminary development and validation of a real-time reverse transcription PCR assay for the semi-quantification of viable Campylobacter jejuni in water samples.

A TaqMan-based real-time reverse transcription PCR (RT-PCR) assay was developed for semi-quantification of viable Campylobacter jejuni in water samples. This preliminary assay is based on measuring the heat-shock induction of groEL messenger RNA (mRNA); the logic being that only viable cells can synthesise new mRNA. A 128-bp C. jejuni groEL DNA fragment was cloned and used as a positive control standard in TaqMan runs. The assay could detect as few as 13 genome equivalent copies of groEL plasmid DNA and 1-2 colony forming unit (CFU) of viable C. jejuni. A multi-step concentration technique was developed for collecting C. jejuni from large volumes of water samples. An average recovery of 20% (range: 8-47%) was obtained at spiking levels of 750-6,500 CFU per 10-litre of river water. Starting from concentration, the enumeration of viable C. jejuni in river water samples was achieved in approximately 12 hours. Quantification of viable C. jejuni in natural river water samples by this method showed similar trends to a culture-based double enrichment most probable number (MPN) -PCR method. There is potential to apply this fast, specific and sensitive semi-quantitative method to monitor a range of water samples for viable C. jejuni.

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.

Chronic abruption-oligohydramnios sequence.

OBJECTIVE: To determine outcome in patients with chronic abruption. STUDY DESIGN: A retrospective review was performed of all patients delivering at a tertiary medical center during a 54-month period. All patients with a diagnosis of placental abruption with oligohydramnios or ruptured membranes were included. Chronic abruption-oligohydramnios sequence (CAOS) was defined by the following criteria: (1) clinically significant vaginal bleeding in the absence of placenta previa or other identifiable source of bleeding, (2) amniotic fluid volume initially documented as normal, and (3) oligohydramnios (amniotic fluid index < or = 5) eventually developing without concurrent evidence of ruptured membranes. RESULTS: Twenty-four patients with CAOS were identified. Fourteen had first evidence of abruption at < 20 weeks' gestational age. A clot was identified between the chorion and uterus in 18/24. The mean gestational age at the first bleeding episode was 19.4 +/- 5.5 (SD) weeks, with the mean gestational age at delivery 28.1 +/- 4.5 weeks. Preterm premature membrane rupture occurred in 15/24. In these 15 there was a mean of 11.5 +/- days between the diagnosis of oligohydramnios and of ruptured membranes. Patients whose first blood occurred at < 20 weeks' gestation delivered at a gestational age of 26.1 +/- 3.9 weeks versus 33.0 +/- 5.3 weeks for the control group. CONCLUSION: CAOS can occur in pregnancies complicated by abruptio placentae. If it develops, the mean gestational age at delivery is 28 weeks.

Elucidation of potential transmission routes of Campylobacter in New Zealand.

Campylobacter is the most commonly reported notifiable disease in New Zealand. The cost of Campylobacter infections in the country during 1994 was estimated as dollar 61.7M although the true cost was probably higher. Investigation of the main environmental reservoirs and routes of transmission to humans is necessary to formulate the most appropriate intervention strategies. This project investigated the reservoirs of Campylobacter in a defined geographical area within New Zealand and compared strains isolated from humans and environmental sources within this area as a prelude to investigating the likely transmission routes to humans. Campylobacter jejuni was commonly found in faeces from dairy cows, beef cattle, sheep and ducks, chicken carcasses, sheep offal and surface waters and C. coli was commonly found in sheep faeces. Preliminary analysis of Penner types was suggestive of transmission to humans from dairy and beef cattle and possibly from sheep.

The use of chemical and molecular microbial indicators for faecal source identification.

Identifying the source of faecal pollution is important to enable appropriate management of faecal pollution of water. We are developing and evaluating a combination of these microbial and chemical indicators better able to identify the source of faecal pollution. These assays make use of a combination of direct PCR, culturing, and colony hybridisation to identify source specific species of Bifidobacterium, Rhodococcus and Bacteroides. In conjunction with assays for (a) fluorescent whitening agents and (b) faecal sterols and stanols, these indicators were able to identify human derived faecal pollution in river water containing inputs from septic tanks, municipal oxidation ponds, farmed animals and feral animals. Differentiating amongst the animal sources was more difficult and will require development of molecular assays for organisms specific to each animal group.

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.

Survival of Escherichia coli, enterococci and Campylobacter jejuni in Canada goose faeces on pasture.

Freshly excreted Canada goose faeces pose a public health risk as they contain pathogenic microorganisms. Accordingly, a study was carried out on the growth and survival of resident indicator bacteria (enterococci and Escherichia coli) and inoculated Campylobacter jejuni in freshly excreted faeces over summer and winter. Canada goose faeces were collected, mixed thoroughly and inoculated with 108 g⁻¹ C. jejuni. The faeces were mixed again before making the Canada goose dropping. The simulated goose droppings (N = 70) were placed on pasture, and the concentrations of E. coli, enterococci and the pathogen, C. jejuni, were monitored. In summer only, the molecular marker of E. coli LacZ and the avian-associated bacteria E2 was also monitored. Results of the survival study indicated that significant growth of enterococci and E. coli occurred in summer, before concentrations decreased to less than 15% of the original concentration (day 77) for enterococci and 0.01% for E. coli. LacZ followed a similar pattern to E. coli, while the E2 marker dropped to below 0.1% of the original concentration within 4 days. In winter, enterococci grew slightly, while no growth of E. coli occurred. In both summer and winter, C. jejuni was rapidly inactivated. This research highlights the ability of bacterial indicators to replicate and survive in the environment when harboured by avian faeces, and the limited risk aged Canada goose faeces pose as an environmental source of Campylobacter spp.

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).