Comparative Analysis of Campylobacter jejuni and C. coli Isolated from Livestock Animals to C. jejuni and C. coli Isolated from Surface Water Using DNA Sequencing and MALDI-TOF.

This study evaluated the contribution of cattle, sheep, poultry and pigs to the contamination of surface water from rivers by Campylobacter jejuni and C. coli using MLST, cgMLST and considered MALDI-TOF MS as an alternative technique. The 263 strains isolated from cattle (n = 61), sheep (n = 42), poultry (n = 65), pigs (n = 60) and surface water (n = 35) were distributed across 115 sequence types (STs), 49 for C. jejuni and 66 for C. coli. Considering MLST data, 14.2%, 11.4% and 2.8% of the surface water strains could be attributed to cattle, poultry and sheep, respectively, none to pigs, and 85.7% were non-attributed. Analysis of cg-MLST data with STRUCTURE indicated that C. jejuni strains from water were predominantly attributed to poultry (93.5%), weakly to sheep (<1%) and 6.3% non-attributed, and that conversely, C. coli strains from water were predominantly non-attributed (94.3%) and 5.7% attributed to poultry. Considering the protein profiles with a threshold of 94% and 97% of similarity, respectively, strains from surface water could be attributed to poultry (31.4% and 17.1%), and to cattle (17.1% and 5.7%); 54.1% and 77.1% were non-attributed. This study confirmed these livestock animals might contribute to the contamination of surface water, with a level of contribution depending on the typing technique and the method of analysis. MALDI-TOF could potentially be an alternative approach for source attribution.

Genomic Diversity of Campylobacter lari Group Isolates from Europe and Australia in a One Health Context.

Members of the Campylobacter lari group are causative agents of human gastroenteritis and are frequently found in shellfish, marine waters, shorebirds, and marine mammals. Within a One Health context, we used comparative genomics to characterize isolates from a diverse range of sources and geographical locations within Europe and Australia and assess possible transmission of food, animal, and environmental isolates to the human host. A total of 158 C. lari isolates from Australia, Denmark, France, and Germany, which included 82 isolates from human stool and blood, 12 from food, 14 from domestic animal, 19 from waterbirds, and 31 from the environment were analyzed. Genome-wide analysis of the genetic diversity, virulence, and antimicrobial resistance (AMR) traits was carried-out. Most of the isolates belonged to C. lari subsp. lari (Cll; 98, 62.0%), while C. lari subsp. concheus and C. lari urease-positive thermotolerant Campylobacter (UPTC) were represented by 12 (7.6%) and 15 (9.5%) isolates, respectively. Furthermore, 33 (20.9%) isolates were not assigned a subspecies and were thus attributed to distant Campylobacter spp. clades. Whole-genome sequence-derived multilocus sequence typing (MLST) and core-genome MLST (cgMLST) analyses revealed a high genetic diversity with 97 sequence types (STs), including 60 novel STs and 14 cgMLST clusters (≤10 allele differences), respectively. The most prevalent STs were ST-21, ST-70, ST-24, and ST-58 (accounting for 13.3%, 4.4%, 3.8%, and 3.2% of isolates, respectively). A high prevalence of the 125 examined virulence-related loci (from 76.8 to 98.4% per isolate) was observed, especially in Cll isolates, suggesting a probable human pathogenicity of these strains. IMPORTANCE Currently, relatedness between bacterial isolates impacting human health is easily monitored by molecular typing methods. These approaches rely on discrete loci or whole-genome sequence (WGS) analyses. Campylobacter lari is an emergent human pathogen isolated from diverse ecological niches, including fecal material from humans and animals, aquatic environments, and seafood. The presence of C. lari in such diverse sources underlines the importance of adopting an integrated One Health approach in studying C. lari population structure for conducting epidemiological risk assessment. This retrospective study presents a comparative genomics analysis of C. lari isolates retrieved from two different continents (Europe and Australia) and from different sources (human, domestic animals, waterbirds, food, and environment). It was designed to improve knowledge regarding C. lari ecology and pathogenicity, important for developing effective surveillance and disease prevention strategies.

Free-living and particle-attached bacterial community composition, assembly processes and determinants across spatiotemporal scales in a macrotidal temperate estuary.

Bacteria play an important role in biogeochemical cycles as they transform and remineralize organic matter. Particles are notable hotspots of activity, hosting particle-attached (PA) communities that can differ largely from their free-living (FL) counterparts. However, long-standing questions remain concerning bacterial community assembly processes and driving factors. This study investigated the FL and PA community compositions and determinants within the Aulne estuary and the Bay of Brest coastal waters (France). Our results revealed that the FL and PA community compositions greatly varied with salinity and season, explaining a larger part of the variance than the sampling fraction. Both the FL and PA communities were driven by deterministic assembly processes and impacted by similar factors. The FL-PA dissimilarity varied across space and time. It decreased in the estuarine stations compared to the freshwater and marine ends, and in summer. Interestingly, a significant proportion of the FL and PA communities' ß-diversity and dissimilarity was explained by cohesion, measuring the degree of taxa co-occurrence. This suggested the importance of co-occurrence patterns in shaping the FL and PA community compositionss. Our results shed light on the factors influencing estuarine bacterial communities and provide a first step toward understanding their biogeochemical impacts.

Quorum sensing disruption regulates hydrolytic enzyme and biofilm production in estuarine bacteria.

Biofilms of heterotrophic bacteria cover organic matter aggregates and constitute hotspots of mineralization, primarily acting through extracellular hydrolytic enzyme production. Nevertheless, regulation of both biofilm and hydrolytic enzyme synthesis remains poorly investigated, especially in estuarine ecosystems. In this study, various bioassays, mass spectrometry and genomics approaches were combined to test the possible involvement of quorum sensing (QS) in these mechanisms. QS is a bacterial cell-cell communication system that relies notably on the emission of N-acylhomoserine lactones (AHLs). In our estuarine bacterial collection, we found that 28 strains (9%), mainly Vibrio, Pseudomonas and Acinetobacter isolates, produced at least 14 different types of AHLs encoded by various luxI genes. We then inhibited the AHL QS circuits of those 28 strains using a broad-spectrum lactonase preparation and tested whether biofilm production as well as ß-glucosidase and leucine-aminopeptidase activities were impacted. Interestingly, we recorded contrasted responses, as biofilm production, dissolved and cell-bound ß-glucosidase and leucine-aminopeptidase activities significantly increased in 4%-68% of strains but decreased in 0%-21% of strains. These findings highlight the key role of AHL-based QS in estuarine bacterial physiology and ultimately on biogeochemical cycles. They also point out the complexity of QS regulations within natural microbial assemblages.

Comparative Analysis of Fecal Microbiomes From Wild Waterbirds to Poultry, Cattle, Pigs, and Wastewater Treatment Plants for a Microbial Source Tracking Approach.

Fecal pollution in coastal areas is of a high concern since it affects bathing and shellfish harvesting activities. Wild waterbirds are non-negligible in the overall signal of the detectable pollution. Yet, studies on wild waterbirds' gut microbiota focus on migratory trajectories and feeding impact on their shape, rare studies address their comparison to other sources and develop quantitative PCR (qPCR)-based Microbial Source Tracking (MST) markers to detect such pollution. Thus, by using 16S rRNA amplicon high-throughput sequencing, the aims of this study were (i) to explore and compare fecal bacterial communities from wild waterbirds (i.e., six families and 15 species, n = 275 samples) to that of poultry, cattle, pigs, and influent/effluent of wastewater treatment plants (n = 150 samples) and (ii) to develop new MST markers for waterbirds. Significant differences were observed between wild waterbirds and the four other groups. We identified 7,349 Amplicon Sequence Variants (ASVs) from the hypervariable V3-V4 region. Firmicutes and Proteobacteria and, in a lesser extent, Actinobacteria and Bacteroidetes were ubiquitous while Fusobacteria and Epsilonbacteraeota were mainly present in wild waterbirds. The clustering of samples in non-metric multidimensional scaling (NMDS) ordination indicated a by-group clustering shape, with a high diversity within wild waterbirds. In addition, the structure of the bacterial communities was distinct according to bird and/or animal species and families (Adonis R 2 = 0.13, p = 10-4, Adonis R 2 = 0.11, p = 10-4, respectively). The Analysis of Composition of Microbiomes (ANCOM) showed that the wild waterbird group differed from the others by the significant presence of sequences from Fusobacteriaceae (W = 566) and Enterococcaceae (W = 565) families, corresponding to the Cetobacterium (W = 1427) and Catellicoccus (W = 1427) genera, respectively. Altogether, our results suggest that some waterbird members present distinct fecal microbiomes allowing the design of qPCR MST markers. For instance, a swan- and an oystercatcher-associated markers (named Swan_2 and Oyscab, respectively) have been developed. Moreover, bacterial genera harboring potential human pathogens associated to bird droppings were detected in our dataset, including enteric pathogens, i.e., Arcobacter, Clostridium, Helicobacter, and Campylobacter, and environmental pathogens, i.e., Burkholderia and Pseudomonas. Future studies involving other wildlife hosts may improve gut microbiome studies and MST marker development, helping mitigation of yet unknown fecal pollution sources.

The Spatiotemporal Dynamics and Microevolution Events That Favored the Success of the Highly Clonal Multidrug-Resistant Monophasic Salmonella Typhimurium Circulating in Europe.

The European epidemic monophasic variant of Salmonella enterica serovar Typhimurium (S. 1,4,[5],12:i:-) characterized by the multi locus sequence type ST34 and the antimicrobial resistance ASSuT profile has become one of the most common serovars in Europe (EU) and the United States (US). In this study, we reconstructed the time-scaled phylogeny and evolution of this Salmonella in Europe. The epidemic S. 1,4,[5],12:i:- ST34 emerged in the 1980s by an acquisition of the Salmonella Genomic Island (SGI)-4 at the 3' end of the phenylalanine phe tRNA locus conferring resistance to copper and arsenic toxicity. Subsequent integration of the Tn21 transposon into the fljAB locus gave resistance to mercury toxicity and several classes of antibiotics used in food-producing animals (ASSuT profile). The second step of the evolution occurred in the 1990s, with the integration of mTmV and mTmV-like prophages carrying the perC and/or sopE genes involved in the ability to reduce nitrates in intestinal contents and facilitate the disruption of the junctions of the host intestinal epithelial cells. Heavy metals are largely used as food supplements or pesticide for cultivation of seeds intended for animal feed so the expansion of the epidemic S. 1,4,[5],12:i:- ST34 was strongly related to the multiple-heavy metal resistance acquired by transposons, integrative and conjugative elements and facilitated by the escape until 2011 from the regulatory actions applied in the control of S. Typhimurium in Europe. The genomic plasticity of the epidemic S. 1,4,[5],12:i:- was demonstrated in our study by the analysis of the plasmidome. We were able to identify plasmids harboring genes mediating resistance to phenicols, colistin, and fluoroquinolone and also describe for the first time in six of the analyzed genomes the presence of two plasmids (pERR1744967-1 and pERR2174855-2) previously described only in strains of enterotoxigenic Escherichia coli and E. fergusonii.

Passive Samplers, a Powerful Tool to Detect Viruses and Bacteria in Marine Coastal Areas.

The detection of viruses and bacteria which can pose a threat either to shellfish health or shellfish consumers remains difficult. The current detection methods rely on point sampling of water, a method that gives a snapshot of the microorganisms present at the time of sampling. In order to obtain better representativeness of the presence of these microorganisms over time, we have developed passive sampling using the adsorption capacities of polymer membranes. Our objectives here were to assess the feasibility of this methodology for field detection. Different types of membrane were deployed in coastal waters over 2 years and the microorganisms tested using qPCR were: human norovirus (NoV) genogroups (G)I and II, sapovirus, Vibrio spp. and the species Vibrio alginolyticus, V. cholerae, V. vulnificus, and V. parahaemolyticus, OsHV-1 virus, and bacterial markers of fecal contamination. NoV GII, Vibrio spp., and the AllBac general Bacteroidales marker were quantified on the three types of membrane. NoV GII and OsHV-1 viruses followed a seasonal distribution. All membranes were favorable for NoV GII detection, while Zetapor was more adapted for OsHV-1 detection. Nylon was more adapted for detection of Vibrio spp. and the AllBac marker. The quantities of NoV GII, AllBac, and Vibrio spp. recovered on membranes increased with the duration of exposure. This first application of passive sampling in seawater is particularly promising in terms of an early warning system for the prevention of contamination in oyster farming areas and to improve our knowledge on the timing and frequency of disease occurence.

AB_SA: Accessory genes-Based Source Attribution - tracing the source of Salmonella enterica Typhimurium environmental strains.

The partitioning of pathogenic strains isolated in environmental or human cases to their sources is challenging. The pathogens usually colonize multiple animal hosts, including livestock, which contaminate the food-production chain and the environment (e.g. soil and water), posing an additional public-health burden and major challenges in the identification of the source. Genomic data opens up new opportunities for the development of statistical models aiming to indicate the likely source of pathogen contamination. Here, we propose a computationally fast and efficient multinomial logistic regression source-attribution classifier to predict the animal source of bacterial isolates based on 'source-enriched' loci extracted from the accessory-genome profiles of a pangenomic dataset. Depending on the accuracy of the model's self-attribution step, the modeller selects the number of candidate accessory genes that best fit the model for calculating the likelihood of (source) category membership. The Accessory genes-Based Source Attribution (AB_SA) method was applied to a dataset of strains of Salmonella enterica Typhimurium and its monophasic variant (S. enterica 1,4,[5],12:i:-). The model was trained on 69 strains with known animal-source categories (i.e. poultry, ruminant and pig). The AB_SA method helped to identify 8 genes as predictors among the 2802 accessory genes. The self-attribution accuracy was 80 %. The AB_SA model was then able to classify 25 of the 29 S. enterica Typhimurium and S. enterica 1,4,[5],12:i:- isolates collected from the environment (considered to be of unknown source) into a specific category (i.e. animal source), with more than 85 % of probability. The AB_SA method herein described provides a user-friendly and valuable tool for performing source-attribution studies in only a few steps. AB_SA is written in R and freely available at https://github.com/lguillier/AB_SA.

Four European Salmonella Typhimurium datasets collected to develop WGS-based source attribution methods.

Zoonotic Salmonella causes millions of human salmonellosis infections worldwide each year. Information about the source of the bacteria guides risk managers on control and preventive strategies. Source attribution is the effort to quantify the number of sporadic human cases of a specific illness to specific sources and animal reservoirs. Source attribution methods for Salmonella have so far been based on traditional wet-lab typing methods. With the change to whole genome sequencing there is a need to develop new methods for source attribution based on sequencing data. Four European datasets collected in Denmark (DK), Germany (DE), the United Kingdom (UK) and France (FR) are presented in this descriptor. The datasets contain sequenced samples of Salmonella Typhimurium and its monophasic variants isolated from human, food, animal and the environment. The objective of the datasets was either to attribute the human salmonellosis cases to animal reservoirs or to investigate contamination of the environment by attributing the environmental isolates to different animal reservoirs.

Complete Genome Sequence of Campylobacter armoricus CA639, Which Carries Two Plasmids, Compiled Using Oxford Nanopore and Illumina Sequencing Technologies.

As determined by a hybrid approach combining Oxford Nanopore MinION and Illumina MiniSeq sequence data, Campylobacter armoricus strain CA639 harbored a circular chromosome of 1,688,169 bp with a G+C content of 28.47% and two plasmids named pCA639-1 and pCA639-2, with lengths of 51,123 and 28,139 bp, and G+C contents of 26.5% and 28.45%, respectively.

Campylobacter armoricus sp. nov., a novel member of the Campylobacter lari group isolated from surface water and stools from humans with enteric infection.

During a study on the prevalence and diversity of members of the genus Campylobacter in a shellfish-harvesting area and its catchment in Brittany, France, six urease-positive isolates of members of the genus Campylobacter were recovered from surface water samples, as well as three isolates from stools of humans displaying enteric infection in the same period. These strains were initially identified as members of the Campylobacter lari group by MALDI-TOF mass spectrometry and placed into a distinct group in the genus Campylobacter, following atpA gene sequence analysis based on whole-genome sequencing data. This taxonomic position was confirmed by phylogenetic analysis of the 16S rRNA, rpoB and hsp60 (groEL) loci, and an analysis of the core genome that provided an improved phylogenetic resolution. The average nucleotide identity between the representative strain CA656T (CCUG 73571T=CIP 111675T) and the type strain of the most closely related species Campylobacter ornithocola WBE38T was 88.5 %. The strains were found to be microaerobic and anaerobic, motile, non-spore-forming, Gram-stain-negative, spiral-shaped bacteria that exhibit catalase, oxidase and urease activities but not nitrate reduction. This study demonstrates clearly that the nine isolates represent a novel species within the C. lari group, for which the name Campylobacter armoricus is proposed. Here, we present phenotypic and morphological features of the nine strains and the description of their genome sequences. The proposed type strain CA656T has a 1.589 Mbp chromosome with a DNA G+C content of 28.5 mol% and encodes 1588 predicted coding sequences, 38 tRNAs, and 3 rRNA operons.

Occurrence of Bacterial Pathogens and Human Noroviruses in Shellfish-Harvesting Areas and Their Catchments in France.

During a 2-year study, the presence of human pathogenic bacteria and noroviruses was investigated in shellfish, seawater and/or surface sediments collected from three French coastal shellfish-harvesting areas as well as in freshwaters from the corresponding upstream catchments. Bacteria isolated from these samples were further analyzed. Escherichia coli isolates classified into the phylogenetic groups B2, or D and enterococci from Enterococcus faecalis and E. faecium species were tested for the presence of virulence genes and for antimicrobial susceptibility. Salmonella members were serotyped and the most abundant serovars (Typhimurium and its monophasic variants and Mbandaka) were genetically characterized by high discriminative subtyping methods. Campylobacter and Vibrio were identified at the species level, and haemolysin-producing Vibrio parahaemolyticus were searched by tdh- and trh- gene detection. Main results showed a low prevalence of Salmonella in shellfish samples where only members of S. Mbandaka were found. Campylobacter were more frequently isolated than Salmonella and a different distribution of Campylobacter species was observed in shellfish compared to rivers, strongly suggesting possible additional inputs of bacteria. Statistical associations between enteric bacteria, human noroviruses (HuNoVs) and concentration of fecal indicator bacteria revealed that the presence of Salmonella was correlated with that of Campylobacter jejuni and/or C. coli as well as to E. coli concentration. A positive correlation was also found between the presence of C. lari and the detection of HuNoVs. This study highlights the importance of simultaneous detection and characterization of enteric and marine pathogenic bacteria and human noroviruses not only in shellfish but also in catchment waters for a hazard assessment associated with microbial contamination of shellfish.

Application of a microbial source tracking based on bacterial and chemical markers in headwater and coastal catchments.

This study identified sources of fecal contamination in three different French headwater and coastal catchments (the Justiçou, Pen an Traon, and La Fresnaye) using a combination of microbial source tracking tools. The tools included bacterial markers (three host-associated Bacteroidales) and chemical markers (six fecal stanols), which were monitored monthly over one or two years in addition to fecal indicator bacteria. 168 of the 240 freshwater and marine water samples had Escherichia coli (E. coli) or enterococci concentrations higher than "excellent" European water quality threshold. In the three catchments, the results suggested that the fecal contamination appeared to be primarily from an animal origin and particularly from a bovine origin in 52% (Rum2Bac) and 46% (Bstanol) of the samples and to a lesser extent from a porcine origin in 19% (Pig2Bac) and 21% (Pstanol) of the samples. Our results suggested a human fecal contamination in 56% (HF183) and 32% (Hstanol) of the samples. Rainfall also impacted the source identification of microbial contamination. In general, these findings could inform effective implementation of microbial source tracking strategies, specifically that the location of sampling points must include variability at the landscape scale.

Change in the Structure of Escherichia coli Population and the Pattern of Virulence Genes along a Rural Aquatic Continuum.

The aim of this study was to investigate the diversity of the Escherichia coli population, focusing on the occurrence of pathogenic E. coli, in surface water draining a rural catchment. Two sampling campaigns were carried out in similar hydrological conditions (wet period, low flow) along a river continuum, characterized by two opposite density gradients of animals (cattle and wild animals) and human populations. While the abundance of E. coli slightly increased along the river continuum, the abundance of both human and ruminant-associated Bacteroidales markers, as well as the number of E. coli multi-resistant to antibiotics, evidenced a fecal contamination originating from animals at upstream rural sites, and from humans at downstream urban sites. A strong spatial modification of the structure of the E. coli population was observed. At the upstream site close to a forest, a higher abundance of the B2 phylogroup and Escherichia clade strains were observed. At the pasture upstream site, a greater proportion of both E and B1 phylogroups was detected, therefore suggesting a fecal contamination of mainly bovine origin. Conversely, in downstream urban sites, A, D, and F phylogroups were more abundant. To assess the occurrence of intestinal pathogenic strains, virulence factors [afaD, stx1, stx2, eltB (LT), estA (ST), ipaH, bfpA, eae, aaiC and aatA] were screened among 651 E. coli isolates. Intestinal pathogenic strains STEC O174:H21 (stx2) and EHEC O26:H11 (eae, stx1) were isolated in water and sediments close to the pasture site. In contrast, in the downstream urban site aEPEC/EAEC and DAEC of human origin, as well as extra-intestinal pathogenic E. coli belonging to clonal group A of D phylogroup, were sampled. Even if the estimated input of STEC (Shiga toxin-producing E. coli) - released in water at the upstream pasture site - at the downstream site was low, we show that STEC could persist in sediment. These results show that, the run-off of small cattle farms contributed, as much as the wastewater effluent, in the dissemination of pathogenic E. coli in both water and sediments, even if the microbiological quality of the water was good or to average quality according to the French water index.

Human-Driven Microbiological Contamination of Benthic and Hyporheic Sediments of an Intermittent Peri-Urban River Assessed from MST and 16S rRNA Genetic Structure Analyses.

Rivers are often challenged by fecal contaminations. The barrier effect of sediments against fecal bacteria was investigated through the use of a microbial source tracking (MST) toolbox, and by Next Generation Sequencing (NGS) of V5-V6 16S rRNA gene (rrs) sequences. Non-metric multi-dimensional scaling analysis of V5-V6 16S rRNA gene sequences differentiated bacteriomes according to their compartment of origin i.e., surface water against benthic and hyporheic sediments. Classification of these reads showed the most prevalent operating taxonomic units (OTU) to be allocated to Flavobacterium and Aquabacterium. Relative numbers of Gaiella, Haliangium, and Thermoleophilum OTU matched the observed differentiation of bacteriomes according to river compartments. OTU patterns were found impacted by combined sewer overflows (CSO) through an observed increase in diversity from the sewer to the hyporheic sediments. These changes appeared driven by direct transfers of bacterial contaminants from wastewaters but also by organic inputs favoring previously undetectable bacterial groups among sediments. These NGS datasets appeared more sensitive at tracking community changes than MST markers. The human-specific MST marker HF183 was strictly detected among CSO-impacted surface waters and not river bed sediments. The ruminant-specific DNA marker was more broadly distributed but intense bovine pollution was required to detect transfers from surface water to benthic and hyporheic sediments. Some OTU showed distribution patterns in line with these MST datasets such as those allocated to the Aeromonas, Acinetobacter, and Pseudomonas. Fecal indicators (Escherichia coli and total thermotolerant coliforms) were detected all over the river course but their concentrations were not correlated with MST ones. Overall, MST and NGS datasets suggested a poor colonization of river sediments by bovine and sewer bacterial contaminants. No environmental outbreak of these bacterial contaminants was detected.

Prevalence and Characterization of Shiga Toxin-Producing and Enteropathogenic Escherichia coli in Shellfish-Harvesting Areas and Their Watersheds.

more strains formed a strong biofilm at 18 than at 30°C. Finally, more than 85% of analyzed strains were found to be sensitive to the 16 tested antibiotics. These data suggest the low risk of human infection by STEC if shellfish from these shellfish-harvesting areas were consumed.

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman(®), HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman(®) was found to be the most effective marker of human fecal contamination in this California-based study.

Evaluation of the repeatability and reproducibility of a suite of qPCR-based microbial source tracking methods.

Many PCR-based methods for microbial source tracking (MST) have been developed and validated within individual research laboratories. Inter-laboratory validation of these methods, however, has been minimal, and the effects of protocol standardization regimes have not been thoroughly evaluated. Knowledge of factors influencing PCR in different laboratories is vital to future technology transfer for use of MST methods as a tool for water quality management. In this study, a blinded set of 64 filters (containing 32 duplicate samples generated from 12 composite fecal sources) were analyzed by three to five core laboratories with a suite of PCR-based methods utilizing standardized reagents and protocols. Repeatability (intra-laboratory variability) and reproducibility (inter-laboratory variability) of observed results were assessed. When standardized methodologies were used, intra- and inter-laboratory %CVs were generally low (median %CV 0.1-3.3% and 1.9-7.1%, respectively) and comparable to those observed in similar inter-laboratory validation studies performed on other methods of quantifying fecal indicator bacteria (FIB) in environmental samples. ANOVA of %CV values found three human-associated methods (BsteriF1, BacHum, and HF183Taqman) to be similarly reproducible (p > 0.05) and significantly more reproducible (p < 0.05) than HumM2. This was attributed to the increased variability associated with low target concentrations detected by HumM2 (approximately 1-2 log10copies/filter lower) compared to other human-associated methods. Cow-associated methods (BacCow and CowM2) were similarly reproducible (p > 0.05). When using standardized protocols, variance component analysis indicated sample type (fecal source and concentration) to be the major contributor to total variability with that from replicate filters and inter-laboratory analysis to be within the same order of magnitude but larger than inherent intra-laboratory variability. However, when reagents and protocols were not standardized, inter-laboratory %CV generally increased with a corresponding decline in reproducibility. Overall, these findings verify the repeatability and reproducibility of these MST methods and highlight the need for standardization of protocols and consumables prior to implementation of larger scale MST studies involving multiple laboratories.