Exploring the Bacterial Community in Aged Fecal Sources from Dairy Cows: Impacts on Fecal Source Tracking.

(1) Background: This paper discusses the impact of agricultural activities on stream health, particularly in relation to dairy cow fecal pollution. The study explores the fecal microbiome of cattle and the potential ecological implications of aging fecal pollution on waterways. (2) Methods: The study examines changes in the bacterial community available for mobilization from in-situ decomposing cowpats and the effects of simulated rainfall. The microbiome of individual cowpats was monitored over 5.5 months. We used 16S rRNA metagenomics and machine learning software, FEAST (Fast Expectation-mAximization for microbial Source Tracking), for bacterial and fecal source assignments. (3) Results: The phyla Bacillota and Bacteroidota are dominant in the fecal microbiota of fresh cow feces but shift to Pseudomonodota, Actinomycetota, and environmental Bacteroidota in aged cowpats. Potential impacts of these bacterial community shifts on inputs to local agricultural streams are discussed in relation to water quality monitoring and aging sources of fecal contamination. We identified taxon orders that are potential indicators of fresh cattle sources (Oscillospirales and Bacteroidales) and aged sources (Peptostreptococcales-Tissierellales) in water bodies. (4) The paper highlights that bacterial metagenomic profiling can inform our understanding of the ecology of microbial communities in aquatic environments and the potential impacts of agricultural activities on ecosystem health.

Diagnosis of protozoa diarrhoea in Campylobacter patients increases markedly with molecular techniques.

Cryptosporidium and Giardia are major causes of diarrhoea globally, and two of the most notified infectious diseases in New Zealand. Diagnosis requires laboratory confirmation carried out mostly via antigen or microscopy-based techniques. However, these methods are increasingly being superseded by molecular techniques. Here we investigate the level of protozoa detection by molecular methods in campylobacteriosis cases missed through antigen-based assays and investigate different molecular testing protocols. We report findings from two observational studies; the first among 111 people during a Campylobacter outbreak and the second during normal surveillance activities among 158 people presenting with diarrhoea and a positive Campylobacter test, but negative Cryptosporidium and Giardia antigen-based test results. The molecular methods used for comparison were in-house end-point PCR tests targeting the gp60 gene for Cryptosporidium and gdh gene for Giardia. DNA extraction was performed with and without bead-beating and comparisons with commercial real-time quantitative (qPCR) were made using clinical Cryptosporidium positive sample dilutions down to 10-5. The Cryptosporidium prevalence was 9% (95% CI: 3-15; 10/111) and Giardia prevalence 21% (95% CI: 12-29; 23/111) in the 111 Campylobacter outbreak patients. The Cryptosporidium prevalence was 40% (95% CI: 32-48; 62/158) and Giardia prevalence 1.3% (95% CI: 0.2-4.5; 2/158) in the 158 routine surveillance samples. Sequencing identified Cryptosporidium hominis, C. parvum, and Giardia intestinalis assemblages A and B. We found no statistical difference in positive test results between samples using end-point PCR with or without bead-beating prior to DNA extraction, or between the in-house end-point PCR and qPCR. The qPCR Ct value was 36 (95% CI: 35-37) for 1 oocyst, suggesting a high limit of detection. In conclusion in surveillance and outbreak situations we found diagnostic serology testing underdiagnoses Cryptosporidium and Giardia coinfections in Campylobacter patients, suggesting the impact of protozoa infections may be underestimated through underdiagnosis using antigen-based assays.

A Framework for Public Health Authorities to Evaluate Health Determinants for Wastewater-Based Epidemiology.

BACKGROUND: Wastewater-based epidemiology (WBE) is rapidly developing as a powerful public health tool. It can provide information about a wide range of health determinants (HDs), including community exposure to environmental hazards, trends in consumption of licit and illicit substances, spread of infectious diseases, and general community health. As such, the list of possible candidate HDs for WBE is almost limitless. Consequently, a means to evaluate and prioritize suitable candidates for WBE is useful, particularly for public health authorities, who often face resource constraints. OBJECTIVES: We have developed a framework to assist public health authorities to decide what HDs may be appropriate for WBE and what biomarkers could be used. This commentary reflects the experience of the authors, who work at the interface of research and public health implementation. DISCUSSION: To be suitable for WBE, a candidate HD should address a public health or scientific issue that would benefit from better understanding at the population level. For HDs where information on individual exposures or stratification by population subgroups is required, WBE is less suitable. Where other methodologies are already used to monitor the candidate HD, consideration must be given to whether WBE could provide better or complementary information to the current approach. An essential requirement of WBE is a biomarker specific for the candidate HD. A biomarker in this context refers to any human-excreted chemical or biological that could act as an indicator of consumption or exposure to an environmental hazard or of the human health state. Suitable biomarkers should meet several criteria outlined in this commentary, which requires background knowledge for both the biomarker and the HD. An evaluation tree summarizing key considerations for public health authorities when assessing the suitability of candidate HDs for WBE and an example evaluation are presented. https://doi.org/10.1289/EHP11115.

Sensitivity of wastewater-based epidemiology for detection of SARS-CoV-2 RNA in a low prevalence setting.

To assist public health responses to COVID-19, wastewater-based epidemiology (WBE) is being utilised internationally to monitor SARS-CoV-2 infections at the community level. However, questions remain regarding the sensitivity of WBE and its use in low prevalence settings. In this study, we estimated the total number of COVID-19 cases required for detection of SARS-CoV-2 RNA in wastewater. To do this, we leveraged a unique situation where, over a 4-month period, all symptomatic and asymptomatic cases, in a population of approximately 120,000, were precisely known and mainly located in a single managed isolation and quarantine facility (MIQF) building. From 9 July to 6 November 2020, 24-hr composite wastewater samples (n = 113) were collected daily from the sewer outside the MIQF, and from the municipal wastewater treatment plant (WWTP) located 5 km downstream. New daily COVID-19 cases at the MIQF ranged from 0 to 17, and for most of the study period there were no cases outside the MIQF identified. SARS-CoV-2 RNA was detected in 54.0% (61/113) at the WWTP, compared to 95.6% (108/113) at the MIQF. We used logistic regression to estimate the shedding of SARS-CoV-2 RNA into wastewater based on four infectious shedding models. With a total of 5 and 10 COVID-19 infectious cases per 100,000 population (0.005% and 0.01% prevalence) the predicated probability of SARS-CoV-2 RNA detection at the WWTP was estimated to be 28 and 41%, respectively. When a proportional shedding model was used, this increased to 58% and 87% for 5 and 10 cases, respectively. In other words, when 10 individuals were actively shedding SARS-CoV-2 RNA in a catchment of 100,000 individuals, there was a high likelihood of detecting viral RNA in wastewater. SARS-CoV-2 RNA detections at the WWTP were associated with increasing COVID-19 cases. Our results show that WBE provides a reliable and sensitive platform for detecting infections at the community scale, even when case prevalence is low, and can be of use as an early warning system for community outbreaks.

Genome Typing and Epidemiology of Human Listeriosis in New Zealand, 1999 to 2018.

This study describes the epidemiology of listeriosis in New Zealand between 1999 and 2018 as well as the retrospective whole-genome sequencing (WGS) of 453 Listeria monocytogenes isolates corresponding to 95% of the human cases within this period. The average notified rate of listeriosis was 0.5 cases per 100,000 population, and non-pregnancy-associated cases were more prevalent than pregnancy-associated cases (averages of 19 and 5 cases per annum, respectively). WGS data was assessed using multilocus sequencing typing (MLST), including core-genome and whole-genome MLST (cgMLST and wgMLST, respectively) and single-nucleotide polymorphism (SNP) analysis. Thirty-nine sequence types (STs) were identified, with the most common being ST1 (21.9%), ST4 (13.2%), ST2 (11.3%), ST120 (6.1%), and ST155 (6.4%). A total of 291 different cgMLST types were identified, with the majority (n = 243) of types observed as a single isolate, consistent with the observation that listeriosis is predominately sporadic. Among the 49 cgMLST types containing two or more isolates, 18 cgMLST types were found with 2 to 4 isolates each (50 isolates in total, including three outbreak-associated isolates) that shared low genetic diversity (0 to 2 whole-genome alleles), some of which were dispersed in time or geographical regions. SNP analysis also produced results comparable to those from wgMLST. The low genetic diversity within these clusters suggests a potential common source, but incomplete epidemiological data impaired retrospective epidemiological investigations. Prospective use of WGS analysis together with thorough exposure information from cases could potentially identify future outbreaks more rapidly, including those that may have been undetected for some time over different geographical regions.

A large scale waterborne Campylobacteriosis outbreak, Havelock North, New Zealand.

BACKGROUND: We describe the investigation of a Campylobacter outbreak linked to contamination of an untreated, groundwater derived drinking water supply. METHODS: We analysed epidemiological data collected from clinician-confirmed diarrheal cases and estimated the total burden of Havelock North cases using an age-adjusted cross-sectional telephone survey. Campylobacter isolates from case fecal specimens, groundwater samples, and sheep fecal specimens from paddocks adjacent to the drinking water source were whole genome sequenced. FINDINGS: We estimate between 6260 and 8320 cases of illness including up to 2230 who lived outside the reticulation area, were linked to the contaminated water supply. Of these, 953 cases were physician reported, 42 were hospitalized, three developed Guillain-Barré syndrome, and Campylobacter infection contributed to at least four deaths. Of the 12 genotypes observed in cases, four were also observed in water, three were also observed in sheep and one was also observed in both water and sheep. INTERPRETATION: The contamination of the untreated reticulated water supply occurred following a very heavy rainfall event which caused drainage of sheep feces into a shallow aquifer. The existence of a routine clinical surveillance system for campylobacteriosis facilitated identification of the outbreak, recovery of clinical isolates, and early testing of the water for pathogens. Genotyping of the Campylobacter jejuni helped define the source of the outbreak and confirm outbreak periods and cases. Expected increases in heavy rainfall events and intensification of agriculture mean that additional safeguards are needed to protect populations from such drinking water outbreaks. FUNDING: NZ Ministry of Health, Health Research Council, ESR SSIF, Royal Society.

Genomic Analysis of Fluoroquinolone- and Tetracycline-Resistant Campylobacter jejuni Sequence Type 6964 in Humans and Poultry, New Zealand, 2014-2016.

In 2014, antimicrobial drug-resistant Campylobacter jejuni sequence type 6964 emerged contemporaneously in poultry from 3 supply companies in the North Island of New Zealand and as a major cause of campylobacteriosis in humans in New Zealand. This lineage, not previously identified in New Zealand, was resistant to tetracycline and fluoroquinolones. Genomic analysis revealed divergence into 2 major clades; both clades were associated with human infection, 1 with poultry companies A and B and the other with company C. Accessory genome evolution was associated with a plasmid, phage insertions, and natural transformation. We hypothesize that the tetO gene and a phage were inserted into the chromosome after conjugation, leaving a remnant plasmid that was lost from isolates from company C. The emergence and rapid spread of a resistant clone of C. jejuni in New Zealand, coupled with evolutionary change in the accessory genome, demonstrate the need for ongoing Campylobacter surveillance among poultry and humans.

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.

Fecal source tracking methods to elucidate critical sources of pathogens and contaminant microbial transport through New Zealand agricultural watersheds - A review.

In New Zealand, there is substantial potential for microbial contaminants from agricultural fecal sources to be transported into waterways. The flow and transport pathways for fecal contaminants vary at a range of scales and is dependent on chemical, physical and biological attributes of pathways, soils, microorganisms and landscape characteristics. Understanding contaminant transport pathways from catchment to stream can aid water management strategies. It is not practical, however to conduct direct field measurement for all catchments on the fate and transport of fecal pathogens due to constraints on time, personnel, and material resources. To overcome this problem, fecal source tracking can be utilised to link catchment characteristics to fecal signatures identifying critical sources. In this article, we have reviewed approaches to identifying critical sources and pathways for fecal microorganisms from agricultural sources, and make recommendations for the appropriate use of these fecal source tracking (FST) tools.

A Designed Experiments Approach to Optimizing MALDI-TOF MS Spectrum Processing Parameters Enhances Detection of Antibiotic Resistance in Campylobacter jejuni.

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.

Identifying avian sources of faecal contamination using sterol analysis.

Discrimination of the source of faecal pollution in water bodies is an important step in the assessment and mitigation of public health risk. One tool for faecal source tracking is the analysis of faecal sterols which are present in faeces of animals in a range of distinctive ratios. Published ratios are able to discriminate between human and herbivore mammal faecal inputs but are of less value for identifying pollution from wildfowl, which can be a common cause of elevated bacterial indicators in rivers and streams. In this study, the sterol profiles of 50 avian-derived faecal specimens (seagulls, ducks and chickens) were examined alongside those of 57 ruminant faeces and previously published sterol profiles of human wastewater, chicken effluent and animal meatwork effluent. Two novel sterol ratios were identified as specific to avian faecal scats, which, when incorporated into a decision tree with human and herbivore mammal indicative ratios, were able to identify sterols from avian-polluted waterways. For samples where the sterol profile was not consistent with herbivore mammal or human pollution, avian pollution is indicated when the ratio of 24-ethylcholestanol/(24-ethylcholestanol + 24-ethylcoprostanol + 24-ethylepicoprostanol) is ≥0.4 (avian ratio 1) and the ratio of cholestanol/(cholestanol + coprostanol + epicoprostanol) is ≥0.5 (avian ratio 2). When avian pollution is indicated, further confirmation by targeted PCR specific markers can be employed if greater confidence in the pollution source is required. A 66% concordance between sterol ratios and current avian PCR markers was achieved when 56 water samples from polluted waterways were analysed.

Same-day subtyping of Campylobacter jejuni and C. coli isolates by use of multiplex ligation-dependent probe amplification-binary typing.

Campylobacteriosis is the most commonly reported form of human bacterial gastroenteritis in the world. Sound identification of infectious sources requires subtyping, but the most widely used methods have turnaround times measured in days and require specialist equipment and skills. A multiplex ligation-dependent probe amplification-binary typing (MBiT) assay was developed for subtyping Campylobacter jejuni and Campylobacter coli. It was tested on 245 isolates, including recent isolates from Belgium and New Zealand, and compared to multilocus sequence typing (MLST). When used in an outbreak setting, MBiT identified the predominant genotype and possible additional cases days before pulsed-field gel electrophoresis (PFGE) results were available. MBiT was more discriminatory than MLST and, being a single assay with results produced within 6 h, was more rapid and cost-effective than both MLST and PFGE. In addition, MBiT requires only basic molecular biology equipment and skills.

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.

Sunlight inactivation of human polymerase chain reaction markers and cultured fecal indicators in river and saline waters.

Decay rates for sunlight inactivation of polymerase chain reaction (PCR) markers for total Bacteroidales, human-specific Bacteroidales, Escherichia coli, and Bifidobacterium adolescentis relative to cultured E. coli were investigated. The experiment used 100-L chambers of fresh water and seawater (paired with dark controls) seeded with human sewage and exposed to natural sunlight over three summer days. Culturable E. coli levels in sunlight-exposed chambers decreased by at least 3 logs on day 1, and by day 3 a total reduction of 4.5 to 5.5 logs was achieved in fresh water and seawater, respectively. In contrast, PCR detection of the four gene targets in sunlight-exposed chambers reduced by no more than 2 logs over the duration of the study (k(t) < 0.071 log(e) units h(-1)). Under these experimental conditions, PCR markers are considerably more conservative than culturable E. coli and can persist for extended periods of time following inactivation of E. coli.

A PCR marker for detection in surface waters of faecal pollution derived from ducks.

Detection of the faecal pollution contribution from wildfowl is an important adjunct in determining the sources of faecal pollution in waterways. This is particularly true, where human waste and other animal faecal sources have been eliminated as the pollution source. A polymerase chain reaction (PCR) marker was developed as a duck-specific marker of faecal pollution. The semi-nested primer system targeted an unknown bacterium (E2) isolated from mallard ducks. E2 had the closest 16S rRNA sequence similarity to members of the Desulfovibrio genus, which was further confirmed by phenotypic characterisation of the bacterium. Testing of the prevalence of E2 identified the marker in 76% of duck faecal samples (n=42), 20% of swan faecal samples (n=10) and 15% of Canada geese faecal samples (n=20). It was also identified in the faeces of two out of 15 domestic goats (13%). The marker was not detected in any samples derived from human faeces or effluent, dairy cows or sheep. It is proposed that this PCR marker would be useful in conjunction with faecal taxation indicators in the determination of pollution derived from duck faecal inputs in waterways.