Fecal indicator bacteria from environmental sources; strategies for identification to improve water quality monitoring.

In tropical to temperate environments, fecal indicator bacteria (FIB), such as enterococci and Escherichia coli, can persist and potentially multiply, far removed from their natural reservoir of the animal gut. FIB isolated from environmental reservoirs such as stream sediments, beach sand and vegetation have been termed "naturalized" FIB. In addition, recent research suggests that the intestines of poikilothermic animals such as fish may be colonized by enterococci and E. coli, and therefore, these animals may contribute to FIB concentrations in the aquatic environment. Naturalized FIB that are derived from fecal inputs into the environment, and subsequently adapted to maintain their population within the non-host environment are termed "naturalized enteric FIB". In contrast, an additional theory suggests that some "naturalized" FIB diverged from enteric FIB many millions of years ago and are now normal inhabitants of the environment where they are referred to as "naturalized non-enteric FIB". In the case of the Escherichia genus, the naturalized non-enteric members are identified as E. coli during routine water quality monitoring. An over-estimation of the health risk could result when these naturalized, non-enteric FIB, (that is, not derived from avian or mammalian fecal contamination), contribute to water quality monitoring results. It has been postulated that these environmental FIB belonging to the genera Escherichia and Enterococcus can be differentiated from enteric FIB by genetic methods because they lack some of the genes required for colonization of the host intestine, and have acquired genes that aid survival in the environment. Advances in molecular tools such as next generation sequencing will aid the identification of genes peculiar or "enriched" in particular habitats to discriminate between enteric and environmental FIB. In this appraisal, we have reviewed the research studying "naturalized" FIB, and discussed the techniques for their differentiation from enteric FIB. This differentiation includes the important distinction between enteric FIB derived from fresh and non-recent fecal inputs, and those truly non-enteric environmental microbes, which are currently identified as FIB during routine water quality monitoring. The inclusion of tools for the identification of naturalized FIB (enteric or environmental) would be a valuable resource for future studies assessing water quality.

Spatio-temporal analysis of differences in campylobacteriosis incidence between urban and rural areas in the Southern District Health Board, New Zealand.

The objective of this paper is to investigate differences in campylobacteriosis incidence between urban and rural areas in the Southern District Health Board of New Zealand between 2000 and 2015. The data were analysed using a Bayesian change-point model to evaluate how campylobacteriosis incidence changed over time and to see whether the dynamics differed between rural and urban areas. A conditional auto regressive error term was introduced to account for any spatial effects. The results of our analysis showed that campylobacteriosis incidence increased between 2000 and 2005, decreased between 2006 and 2008 then stabilised from 2009 onward. In addition we found that the changes in incidence were greater in urban areas than in rural ones.

Relationships between chemical and microbial faecal source tracking markers in urban river water and sediments during and post-discharge of human sewage.

This study explores the relationships between faecal source tracking (FST) markers (quantitative Polymerase Chain Reaction (qPCR) markers and steroids), microbial indicators, the faecal ageing ratio of atypical colonies/total coliforms (AC/TC) and potential human pathogens (Giardia, Cryptosporidium and Campylobacter). Faecal source PCR markers tested were GenBac3, HumM3, HumBac (HF183-Bac708R); Bifidobacterium adolescentis, wildfowl and canine-associated markers. Sediment and water samples from the Avon River were collected during and post-discharge of untreated human sewage inputs, following a series of earthquakes, which severely damaged the Christchurch sewerage system. Significant, positive Spearman Ranks (rs) correlations were observed between human-associated qPCR markers and steroid FST markers and Escherichia coli and F-specific RNA bacteriophage (rs 0.57 to 0.84, p < 0.001) in water samples. These human source indicative FST markers demonstrated that they were also effective predictors of potentially pathogenic protozoa in water (rs 0.43-0.74, p ≤ 0.002), but correlated less well with Campylobacter. Human-associated qPCR and steroid markers showed significant, substantial agreement between the two FST methods (Cohen's kappa, 0.78, p = 0.023), suggesting that water managers could be confident in the results using either method under these contamination conditions. Low levels of fluorescent whitening agents (FWA) (mean 0.06 µg/L, range 0.01-0.40 µg/L) were observed in water throughout the study, but steroids and FWA appeared to be retained in river sediments, months after continuous sewage discharges had ceased. No relationship was observed between chemical FST markers in sediments and the overlying water, and few correlations in sediment between chemical FST markers and target microorganisms. The low values observed for the faecal ageing ratio, AC/TC in water, were significantly, negatively correlated with increasing pathogen detection. This study provides support for the use of the AC/TC ratio, and qPCR and steroid FST markers as indicators of health risks associated with the discharge of raw human sewage into a freshwater system.

Evaluating statistical model performance in water quality prediction.

Exposure to contaminated water while swimming or boating or participating in other recreational activities can cause gastrointestinal and respiratory disease. It is not uncommon for water bodies to experience rapid fluctuations in water quality, and it is therefore vital to be able to predict them accurately and in time so as to minimise population's exposure to pathogenic organisms. E. coli is commonly used as an indicator to measure water quality in freshwater, and higher counts of E. coli are associated with increased risk to illness. In this case study, we compare the performance of a wide range of statistical models in prediction of water quality via E. coli levels for the weekly data collected over the summer months from 2006 to 2014 at the recreational site on the Oreti river in Wallacetown, New Zealand. The models include naive model, multiple linear regression, dynamic regression, regression tree, Markov chain, classification tree, random forests, multinomial logistic regression, discriminant analysis and Bayesian network. The results show that Bayesian network was superior to all the other models. Overall, it had a leave-one-out and k-fold cross validation error rate of 21%, while predicting the majority of instances of E. coli levels classified as unsafe by the Microbiological Water Quality Guidelines for Marine and Freshwater Recreational Areas 2003, New Zealand. Because Bayesian networks are also flexible in handling missing data and outliers and allow for continuous updating in real time, we have found them to be a promising tool, and in the future, plan to extend the analysis beyond the current case study site.

Concentrations of Campylobacter spp., Escherichia coli, Enterococci, and Yersinia spp. in the Feces of Farmed Red Deer in New Zealand.

Intensive deer farming can cause environmental issues, mainly by its impact on soils and water quality. In particular, there is a risk to the microbial quality of water, as high quantities of suspended sediment and fecal bacteria can enter into water systems. The feces of farmed red deer (, = 206) from Canterbury and Southland, New Zealand, were analyzed with regard to the presence of spp., , enterococci, and spp.. Enterococci and were isolated from all samples, with mean concentrations of 4.5 × 10 (95% CI 3.5 × 10, 5.6 10) and 1.3 × 10 (95% CI 1.1 × 10, 1.5 × 10) per gram of dry feces, respectively. spp. were isolated from 27 fecal samples, giving an overall prevalence of 13.1%. isolation rates were variable within and between regions (Canterbury 7.95% [95% CI 2-14%], Southland 16.95% [95% CI 10-24%]). Five out of 42 composite samples were positive for , and one sample for The overall prevalence ranges on a per-animal basis were therefore 2.43 to 11.17% and 0.49 to 2.91%, respectively. This study is the first to quantify the concentration of spp. present in healthy deer farmed in New Zealand. Deer feces are a potential source of human campylobacteriosis, with all genotypes isolated also previously observed among human cases. The fecal outputs from deer should be regarded as potentially pathogenic to humans and therefore be appropriately managed.

Human-, Ovine-, and Bovine-Specific Viral Source Tracking Tools to Discriminate Between the Major Fecal Sources in Agricultural Waters.

This study evaluated the sources of fecal contamination in different river catchments, using a combination of microbial source tracking tools, for human, ruminant, ovine and bovine livestock, in order to define appropriate water management strategies. Every source of waterway pollution was evaluated in river water samples from one urban river catchment and two important farming regions in New Zealand. Fecal pollution was initially measured by testing Escherichia coli and evaluating the presence of human- and ruminant-associated DNA markers of Bacteroidales (BiAdo, BacHum-UCD, BacH, and BacR) and human and ruminant fecal sterols/stanols ratios. Then specific fecal pollution sources were assessed with previously reported quantitative PCR assays targeting human-, bovine-, and ovine-specific viruses: human adenoviruses (HAdV), human JC polyomaviruses, bovine polyomaviruses (BPyV), and ovine polyomaviruses (OPyV). High level of ruminant fecal contamination was detected all over the farming areas, whereas no ruminant sources were identified in the urban river sampling sites. BacR was the most frequently observed ruminant marker and OPyV and BPyV allowed the identification of ovine and bovine fecal sources. The human fecal viral marker (HAdV) was the most frequently observed human marker, highly abundant in the urban sites, and also present in farming areas. This is the first study using simultaneously the ovine and the bovine viral markers to identify and quantify both bovine and ovine fecal pollution.

The impact of major earthquakes and subsequent sewage discharges on the microbial quality of water and sediments in an urban river.

A series of large earthquakes struck the city of Christchurch, New Zealand in 2010-2011. Major damage sustained by the sewerage infrastructure required direct discharge of up to 38,000 m(3)/day of raw sewage into the Avon River of Christchurch for approximately six months. This allowed evaluation of the relationship between concentrations of indicator microorganisms (Escherichia coli, Clostridium perfringens and F-RNA phage) and pathogens (Campylobacter, Giardia and Cryptosporidium) in recreational water and sediment both during and post-cessation of sewage discharges. Giardia was the pathogen found most frequently in river water and sediment, although Campylobacter was found at higher levels in water samples. E. coli levels in water above 550 CFU/100 mL were associated with increased likelihood of detection of Campylobacter, Giardia and Cryptosporidium, supporting the use of E. coli as a reliable indicator for public health risk. The strength of the correlation of microbial indicators with pathogen detection in water decreased in the following order: E. coli>F-RNA phage>C. perfringens. All the microorganisms assayed in this study could be recovered from sediments. C. perfringens was observed to accumulate in sediments, which may have confounded its usefulness as an indicator of fresh sewage discharge. F-RNA phage, however, did not appear to accumulate in sediment and in conjunction with E. coli, may have potential as an indicator of recent human sewage discharge in freshwater. There is evidence to support the low-level persistence of Cryptosporidium and Giardia, but not Campylobacter, in river sediments after cessation of sewage discharges. In the event of disturbances of the sediment, it is highly probable that there could be re-mobilisation of microorganisms beyond the sediment-water exchange processes occurring under base flow conditions. Re-suspension events do, therefore, increase the potential risk to human health for those who participate in recreational and work-related activities in the river environment.

Coevolution of Cryptosporidium tyzzeri and the house mouse (Mus musculus).

Two house mouse subspecies occur in Europe, eastern and northern Mus musculus musculus (Mmm) and western and southern Mus musculus domesticus (Mmd). A secondary hybrid zone occurs where their ranges meet, running from Scandinavia to the Black Sea. In this paper, we tested a hypothesis that the apicomplexan protozoan species Cryptosporidium tyzzeri has coevolved with the house mouse. More specifically, we assessed to what extent the evolution of this parasite mirrors divergence of the two subspecies. In order to test this hypothesis, we analysed sequence variation at five genes (ssrRNA, Cryptosporidium oocyst wall protein (COWP), thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1), actin and gp60) in C. tyzzeri isolates from Mmd and Mmm sampled along a transect across the hybrid zone from the Czech Republic to Germany. Mmd samples were supplemented with mice from New Zealand. We found two distinct isolates of C. tyzzeri, each occurring exclusively in one of the mouse subspecies (C. tyzzeri-Mmm and C. tyzzeri-Mmd). In addition to genetic differentiation, oocysts of the C. tyzzeri-Mmd subtype (mean: 4.24×3.69µm) were significantly smaller than oocysts of C. tyzzeri-Mmm (mean: 4.49×3.90 µm). Mmm and Mmd were susceptible to experimental infection with both C. tyzzeri subtypes; however, the subtypes were not infective for the rodent species Meriones unguiculatus, Mastomys coucha, Apodemus flavicollis or Cavia porcellus. Overall, our results support the hypothesis that C. tyzzeri is coevolving with Mmm and Mmd.

Survival of Escherichia coli, Enterococci, and Campylobacter spp. in sheep feces on pastures.

The survival of enteric bacteria in 10 freshly collected sheep fecal samples on pastures was measured in each of four seasons. Ten freshly collected feces were placed on pasture, and concentrations of Escherichia coli, enterococci, and Campylobacter spp. were monitored until exhaustion of the fecal samples. In all four seasons, there was an increase in enterococcal concentrations by up to 3 orders of magnitude, with peak concentrations recorded between 11 and 28 days after deposition. E. coli concentrations increased in three out of four seasons by up to 1.5 orders of magnitude, with peak concentrations recorded between 8 and 14 days after deposition. The apparent growth of E. coli and enterococci was strongly influenced by the initial water content of the feces and the moisture gained during periods of rehydration following rainfalls. Conversely, the results suggested that dehydration promoted inactivation. Campylobacter spp. did not grow and were rapidly inactivated at a rate that tended to be faster at higher temperatures. Pulsed-field gel electrophoresis (PFGE) of a selection of Campylobacter spp. suggested that these survival data are applicable to a range of Campylobacter spp., including the most frequently isolated PFGE genotype from sheep in New Zealand, and to genotypes previously observed to cause disease in humans. The results of this study are currently being incorporated into a fecal microbe reservoir model that is designed to assist water managers' abilities to estimate microbial loads on pastures grazed by sheep, including the influence of factors such as rainfall and temperature.

Evidence for growth of enterococci in municipal oxidation ponds, obtained using antibiotic resistance analysis.

The Christchurch wastewater treatment plant uses a series of six oxidation ponds to reduce the bacterial load of treated effluent before it is discharged into the local estuary. To ensure that this discharge does not adversely affect water quality in the receiving environment, local regulations specify maximum levels in the discharge for a number of parameters, including enterococci. Between 2001 and 2006, regulations required fewer than 300 enterococci per 100 ml in summer. During this period, the discharge intermittently exceeded this limit, with unexplained levels of enterococci of up to 180,000/100 ml. Characterization of these enterococci by antibiotic resistance analysis showed that enterococci sampled over 4 months had almost identical resistance profiles. In contrast, enterococci from raw sewage and wildfowl from around the oxidation ponds had a diverse range of antibiotic resistance profiles that could be distinguished from each other and also from those of enterococci from the discharge. The hypothesis of a clonal nature of the enterococci in the discharge was supported by molecular genotype analysis, suggesting that these bacteria may have replicated in the pond environment rather than being reflective of breakthrough in the sewage treatment process or the result of recent wildfowl inputs to the ponds. This study highlights the usefulness of antibiotic resistance analysis in identifying this phenomenon and is the first report of apparent replication of a specific type of enterococci in an oxidation pond environment.

Cryptosporidium and its potential as a food-borne pathogen.

Cryptosporidium species are intestinal protozoan parasites and are excreted in animal feces as stable oocysts. Cryptosporidium has now been detected in the feces of a wide range of ruminant and non-ruminant farmed animals, wild animals, domestic pets and birds and the parasite appears to be well adapted to survive and persist in feces for extended periods, ranging from several weeks to many months. Because of this persistence, these materials are important as potential vehicles of transmission within herds, farms, the water chain, the fresh food chain, and the wider environment. Appropriate handling of animal waste is necessary to control spread of this pathogen and to limit the significant risks of human infection. While water is a well-recognized vector of Cryptosporidium, it has only recently emerged that food may play a more significant role than previously realized in the transmission of the Cryptosporidium to humans. In the last 3-5 years, research efforts have been directed both at the development of suitable methods for isolation and detection of the parasite in foods and at the application of these methods to assess the prevalence and persistence of the parasite in a range of foods. Additionally, molecular subtyping methods have been used to establish the transmission routes of the parasite. This paper summarizes the general biology of Cryptosporidium and overviews the current research on C. parvum in the food chain. The risks posed by certain foods, such as salad/vegetable crops and beef, are discussed and control measures which may be useful in the farm-to-fork chain for these products are described.