Christopher Staley

Microbial source tracking markers for detection of fecal contamination in environmental waters: relationships between pathogens and human health outcomes

Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of fecal pollution sources in environmental waters that rely on the association of certain fecal microorganisms with a particular host. MST is used to assess recreational water quality and associated human health risk, and total maximum daily load allocations. Many methods rely on signature molecules (markers) such as DNA sequences of host-associated microorganisms. Human sewage pollution is among the greatest concerns for human health due to (1) the known risk of exposure to human waste and (2) the public and regulatory will to reduce sewage pollution; however, methods to identify animal sources are receiving increasing attention as our understanding of zoonotic disease potential improves. Here, we review the performance of MST methods in initial reports and field studies, with particular emphasis on quantitative PCR (qPCR). Relationships among human-associated MST markers, fecal indicator bacteria, pathogens, and human health outcomes are presented along with recommendations for future research. An integrated understanding of the advantages and drawbacks of the many MST methods targeting human sources advanced over the past several decades will benefit managers, regulators, researchers, and other users of this rapidly growing area of environmental microbiology.

Effect of Fecal Microbiota Transplantation on Recurrence in Multiply Recurrent Clostridium difficile Infection: A Randomized Trial

Clostridium difficile infection (CDI) is the most common health careassociated infection in U.S. hospitals, with approximately 453000 infections and 29000 deaths in 2011 (1). Antibiotics are frequently ineffective (2, 3), with recurrence rates of 15% to 35% after a first episode and up to 65% after treatment of a second recurrence (4, 5). Recurrences are clinically challenging and are typically treated with prolonged courses of antibiotics, which maintain and exacerbate intestinal dysbiosis (6). Fecal microbiota transplantation (FMT) restores the normal composition of gut microbiota and is recommended when antibiotics fail to clear the infection (3). However, the evidence for FMT rests largely on case series and several open-label clinical trials that have suggested cure rates of 81% to 100% in recurrent CDI (712). To date, there has not been an adequately controlled and blinded trial of FMT for CDI treatment. Furthermore, the optimal method for administering FMT has not been determined. Evidence suggests that colonoscopic delivery has advantages in terms of efficacy (9), safety (13), and patient acceptance and tolerability (14) compared with administration via the nasoenteric route. We therefore performed a double-blind, randomized, controlled study of colonoscopic FMT for treatment of recurrent CDI. Cure rates and adverse events (AEs) were compared between donor FMT and autologous FMT (given as a placebo) in patients who had at least 3 CDI recurrences. Methods Design Overview This prospective, dual-center, double-blind, randomized, controlled trial compared FMT using donor stool or the patients own stool administered by colonoscopy. Patients treated with autologous FMT whose CDI relapsed during the 8-week follow-up were offered FMT using donor stool. Those who underwent donor FMT and had relapse were offered repeated FMT using stool from a different donor. Patients were enrolled between 15 November 2012 and 10 March 2015 at 2 academic hospitals: Montefiore Medical Center in the Bronx, New York (NY), and The Miriam Hospital in Providence, Rhode Island (RI). Microbiome analyses on donor and patient fecal specimens were performed at the University of Minnesota in Minneapolis. The institutional review board at each center approved the study protocol (Supplement). A data and safety monitoring board monitored the trial using halting rules for serious, unexpected, and related adverse safety outcomes, and all authors performed data analysis. Supplement. Supplemental Information Study Population The study population comprised adult outpatients who had 3 or more documented CDI recurrences (defined as 3 unformed stools over 24 hours for 2 consecutive days and either a positive stool test result for C difficile or pseudomembranes on colonoscopy) and who did not maintain cure after a course of tapered or pulsed vancomycin or were unable to taper or discontinue vancomycin (or an alternative antibiotic with activity against CDI) without recurrent diarrhea requiring anti-CDI treatment. All patients had completed at least 10 days of vancomycin therapy for the most recent CDI and continued therapy until 2 to 3 days before the procedure. Major exclusion criteria included age 75 years or older; inflammatory bowel disease, irritable bowel syndrome (IBS), or chronic diarrheal disorder; any immunocompromised state or immunodeficiency; anaphylactic food allergy; previous FMT; untreated, in situ colorectal cancer; and inability to undergo colonoscopy. Donor Identification and Screening Patients were permitted to identify a donor or choose to be treated with stool from healthy volunteers who were recruited at each site. All prospective donors underwent a medical interview and physical examination and were excluded if they had a known communicable disease, features of the metabolic syndrome, a diarrheal disorder, an autoimmune or atopic disease, a tumor, a neurologic disorder, or chronic pain syndrome or if they had used antibiotics for any indication within 3 months. Potential donors also completed a modified AABB full-length donor history questionnaire, and those with risk factors for infectious agents were excluded (Supplement). Testing for HIV-1 and HIV-2 was performed within 2 weeks before donation for FMT. Other serologic and stool testing was performed within 1 month before FMT and included testing for hepatitis A, B, and C viruses; testing for Treponema pallidum; polymerase chain reaction (PCR) testing for detection of C difficile toxin; culture for enteric pathogens (Escherichia coli, Salmonella, Shigella, Yersinia, Campylobacter, Listeria monocytogenes, Vibrio parahaemolyticus, and V cholerae); testing for fecal Giardia and Cryptosporidium antigens; acid-fast stain for detection of Cyclospora and Isospora; ova and parasite testing; and enzyme immunoassay for detection of Rotavirus. Patients also had baseline testing for HIV-1 and HIV-2; hepatitis A, B, and C viruses; and T pallidum. Randomization and Interventions Donors took an osmotic laxative (magnesium hydroxide) the evening before and provided fresh stool the day of FMT. All donor specimens were transported on ice and processed within 6 hours of collection. Patients were given a standard bowel purge (sodium sulfate, potassium sulfate, and magnesium sulfate oral solution) the evening before the procedure. For patient convenience, sodium sulfate, potassium sulfate, and magnesium sulfate oral solution was substituted for the polyethylene glycol (PEG) bowel purge described in the study protocol. After initiating the preparation, patients were required to collect the first stool passed (for possible use in autologous FMT), transfer it to a clean container, and keep it either refrigerated or on ice. Within 1 hour before the scheduled FMT procedure, the nonblinded research assistant took possession of the stool specimens from both the donor and the patient. Patients were equally allocated to the donor and autologous FMT groups via block randomization by C difficile positivity at baseline, with stratification by study site. The protocol specified a dose of 100 g of stool diluted in 500 mL of nonbacteriostatic 0.9% normal saline immediately before the procedure, but the study relied on fresh stool, which has unpredictable weight and volume, and most provided specimens were less than 100 g. Because patients had discontinued use of vancomycin, had completed bowel lavage, and had been randomly assigned, we elected to use the lesser amount of fecal material. In those circumstances, available stool was weighed and suspended in a proportionately reduced volume of saline. A mean stool dose of 64 g (SD, 25 g; range, 20 to 100 g) was infused for donor FMT. Colonoscopies were performed within the endoscopy units of the study centers. Endoscopic findings and the depth of insertion were recorded. The study physician administered 300 mL of the fecal suspension through the instruments working channel into the furthest point reached (terminal ileum or cecum). After FMT, patients were transferred to the recovery area and encouraged to retain the stool for at least 1 hour. If they were unable to do so, the time of the first postprocedure bowel movement was recorded. Follow-up Patients were instructed to contact the clinical team if they had recurrence of diarrhea and were provided with a thermometer for daily oral temperature readings and a diary card to record solicited AEs for 7 days and unsolicited AEs for 30 days after transplantation. They were contacted by telephone 2 and 7 days after FMT and then biweekly for 8 weeks and questioned about stool form and frequency. All patients were seen in the clinic for follow-up 2 and 8 weeks after treatment, where they were assessed for infectious and gastrointestinal symptoms and underwent physical examination. They submitted stool specimens at baseline and at each post-FMT visit for C difficile PCR testing and microbiome analysis. All patients were contacted by telephone by a study representative 6 months after the last treatment to record any serious AEs (SAEs), new medical conditions or diagnoses, or changes in medical conditions or medications since the last study contact. End Points The primary efficacy end point was the rate of clinical cure in the intention-to-treat (ITT) population 8 weeks after FMT or at the time of early withdrawal. Clinical cure was defined as lack of CDI recurrence with maintenance of resolution (that is, <3 unformed stools per day) for 8 weeks without requirement for further antibiotics (metronidazole, vancomycin, or fidaxomicin). Because nucleic acid testing does not distinguish colonized patients from those with symptomatic disease (15), it cannot test for cure (2, 16); therefore, patients meeting the definition of clinical cure were considered to be cured regardless of the results of follow-up PCR testing of stool for C difficile. Safety end points were evaluated by review of SAEs, AEs, new medical conditions or diagnoses, or changes in medical conditions at the 6-month follow-up. Analysis of Fecal Microbiota Fecal microbiota was analyzed from patients at least 5 days before and 2 and 8 weeks after FMT, as well as from donor samples. DNA extraction, 16S ribosomal RNA gene amplification, and sequencing were performed by the University of Minnesota Genomics Center. Processing details are described in the Supplement, and data are archived in the Sequence Read Archive at the National Center for Biotechnology Information under BioProject accession number SRP066964. Taxonomic assignments were made against the Ribosomal Database Project using mothur software, version 1.34.0 (17). Alpha and beta diversity of bacterial communities were evaluated using the same software. Shannon indices and abundance-based coverage estimate parameters were calculated to assess alpha diversity. Differences in beta diversity and abundances of genera were performed using analysis of similarity and KruskalWallis analysis, respectively (18). Statis

Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism

Targeted deletion of CD103+CD11b+ LP DCs results in reduced LP Th17 cells at steady state, but has no impact on Citrobacter infection or the composition of the intestinal microbiota.

Application of Illumina next-generation sequencing to characterize the bacterial community of the Upper Mississippi River.

A next‐generation, Illumina‐based sequencing approach was used to characterize the bacterial community at ten sites along the Upper Mississippi River to evaluate shifts in the community potentially resulting from upstream inputs and land use changes. Furthermore, methodological parameters including filter size, sample volume and sample reproducibility were evaluated to determine the best sampling practices for community characterization.

Performance of Two Quantitative PCR Methods for Microbial Source Tracking of Human Sewage and Implications for Microbial Risk Assessment in Recreational Waters

ABSTRACT Before new, rapid quantitative PCR (qPCR) methods for assessment of recreational water quality and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant source has been diluted in environmental waters is needed. This study determined the limits of detection and quantification of the human-associated Bacteroides sp. (HF183) and human polyomavirus (HPyV) qPCR methods for sewage diluted in buffer and in five ambient, Florida water types (estuarine, marine, tannic, lake, and river). HF183 was quantifiable in sewage diluted up to 10−6 in 500-ml ambient-water samples, but HPyVs were not quantifiable in dilutions of >10−4. Specificity, which was assessed using fecal composites from dogs, birds, and cattle, was 100% for HPyVs and 81% for HF183. Quantitative microbial risk assessment (QMRA) estimated the possible norovirus levels in sewage and the human health risk at various sewage dilutions. When juxtaposed with the MST marker detection limits, the QMRA analysis revealed that HF183 was detectable when the modeled risk of gastrointestinal (GI) illness was at or below the benchmark of 10 illnesses per 1,000 exposures, but the HPyV method was generally not sensitive enough to detect potential health risks at the 0.01 threshold for frequency of illness. The tradeoff between sensitivity and specificity in the MST methods indicates that HF183 data should be interpreted judiciously, preferably in conjunction with a more host-specific marker, and that better methods of concentrating HPyVs from environmental waters are needed if this method is to be useful in a watershed management or monitoring context.

Microbes in Beach Sands: Integrating Environment, Ecology and Public Health.

Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.

Interlaboratory Comparison of Real-Time PCR Protocols for Quantification of General Fecal Indicator Bacteria

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol.

Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

ABSTRACT Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 102 to 103V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R2 = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.

Species sorting and seasonal dynamics primarily shape bacterial communities in the Upper Mississippi River.

Bacterial community structure (BCS) in freshwater ecosystems varies seasonally and due to physicochemical gradients, but metacommunity structure of a major river remains understudied. Here we characterize the BCS along the Mississippi River and contributing rivers in Minnesota over three years using Illumina next-generation sequencing, to determine how changes in environmental conditions as well as inputs from surrounding land and confluences impacted community structure. Contributions of sediment to water microbial diversity were also evaluated. Long-term variation in community membership was observed, and significant shifts in relative abundances of major freshwater taxa, including α-Proteobacteria, Burkholderiales, and Actinomycetales, were observed due to temporal and spatial variations. Environmental parameters (e.g. temperature, rainfall, and nutrient concentrations) primarily contributed to differences in phyla abundances (88% of variance), with minimal influence from spatial distance alone (<1% of variance). Furthermore, an annually-recurrent BCS was observed in late summer, further suggesting that seasonal dynamics strongly influence community composition. Sediment communities differed from those in the water, but contributed up to 50% to community composition in the water column. Among water sampling sites, 34% showed significant variability in BCS of replicate samples indicating variability among riverine communities due to heterogeneity in the water column. Results of this study highlight the need for a better understanding of spatial and temporal variations in riverine bacterial diversity associated with physicochemical gradients and reveal how communities in sediments, and potentially other environmental reservoirs, impact waterborne BCS. Techniques used in this study may prove useful to determine sources of microbes from sediments and soils to waterways, which will facilitate best management practices and total maximum daily load determinations.

Bacterial community structure is indicative of chemical inputs in the Upper Mississippi River

Local and regional associations between bacterial communities and nutrient and chemical concentrations were assessed in the Upper Mississippi River in Minnesota to determine if community structure was associated with discrete types of chemical inputs associated with different land cover. Bacterial communities were characterized by Illumina sequencing of the V6 region of 16S rDNA and compared to >40 chemical and nutrient concentrations. Local bacterial community structure was shaped primarily by associations among bacterial orders. However, order abundances were correlated regionally with nutrient and chemical concentrations, and were also related to major land coverage types. Total organic carbon and total dissolved solids were among the primary abiotic factors associated with local community composition and co-varied with land cover. Escherichia coli concentration was poorly related to community composition or nutrient concentrations. Abundances of 14 bacterial orders were related to land coverage type, and seven showed significant differences in abundance (P ≤ 0.046) between forested or anthropogenically-impacted sites. This study identifies specific bacterial orders that were associated with chemicals and nutrients derived from specific land cover types and may be useful in assessing water quality. Results of this study reveal the need to investigate community dynamics at both the local and regional scales and to identify shifts in taxonomic community structure that may be useful in determining sources of pollution in the Upper Mississippi River.