Markers Of Human Fecal Contamination Research Articles

Environmental Surveillance for Salmonella Typhi and its Association With Typhoid Fever Incidence in India and Malawi.

Environmental surveillance (ES) for Salmonella Typhi potentially offers a low-cost tool to identify communities with a high burden of typhoid fever. We developed standardized protocols for typhoid ES, including sampling site selection, validation, characterization; grab or trap sample collection, concentration; and quantitative PCR targeting Salmonella genes (ttr, staG, and tviB) and a marker of human fecal contamination (HF183). ES was implemented over 12 months in a historically high typhoid fever incidence setting (Vellore, India) and a lower incidence setting (Blantyre, Malawi) during 2021-2022. S. Typhi prevalence in ES samples was higher in Vellore compared with Blantyre; 39/520 (7.5%; 95% confidence interval [CI], 4.4%-12.4%) vs 11/533 (2.1%; 95% CI, 1.1%-4.0%) in grab and 79/517 (15.3%; 95% CI, 9.8%-23.0%) vs 23/594 (3.9%; 95% CI, 1.9%-7.9%) in trap samples. Detection was clustered by ES site and correlated with site catchment population in Vellore but not Blantyre. Incidence of culture-confirmed typhoid in local hospitals was low during the study and zero some months in Vellore despite S. Typhi detection in ES. ES describes the prevalence and distribution of S. Typhi even in the absence of typhoid cases and could inform vaccine introduction. Expanded implementation and comparison with clinical and serological surveillance will further establish its public health utility.

Can shellfish be used to monitor SARS-CoV-2 in the coastal environment?

The emergence and worldwide spread of SARS-CoV-2 raises new concerns and challenges regarding possible environmental contamination by this virus through spillover of human sewage, where it has been detected. The coastal environment, under increasing anthropogenic pressure, is subjected to contamination by a large number of human viruses from sewage, most of them being non-enveloped viruses like norovirus. When reaching coastal waters, they can be bio-accumulated by filter-feeding shellfish species such as oysters. Methods to detect this viral contamination were set up for the detection of non-enveloped enteric viruses, and may need optimization to accommodate enveloped viruses like coronaviruses (CoV).Here, we aimed at assessing methods for the detection of CoV, including SARS-CoV-2, in the coastal environment and testing the possibility that SARS-CoV-2 can contaminate oysters, to monitor the contamination of French shores by SARS-CoV-2 using both seawater and shellfish.Using the porcine epidemic diarrhea virus (PEDV), a CoV, as surrogate for SARS-CoV-2, and Tulane virus, as surrogate for non-enveloped viruses such as norovirus, we assessed and selected methods to detect CoV in seawater and shellfish. Seawater-based methods showed variable and low yields for PEDV. In shellfish, the current norm for norovirus detection was applicable to CoV detection. Both PEDV and heat-inactivated SARS-CoV-2 could contaminate oysters in laboratory settings, with a lower efficiency than a calicivirus used as control. Finally, we applied our methods to seawater and shellfish samples collected from April to August 2020 in France, where we could detect the presence of human norovirus, a marker of human fecal contamination, but not SARS-CoV-2.Together, our results validate methods for the detection of CoV in the coastal environment, including the use of shellfish as sentinels of the microbial quality of their environment, and suggest that SARS-CoV-2 did not contaminate the French shores during the summer season.

CrAssphage and its bacterial host in cat feces

CrAssphages are a diverse group of related phages detected in human feces where they are the most prevalent and abundant prokaryotic virus. CrAssphages’ cellular host has been identified as the anaerobic Bacteroides intestinalis. CrAssphage has also been reported in non-human primates and environmental samples and has been proposed as a marker of human fecal contamination. Here we describe crAssphage DNA in a feline fecal sample. 95% of the ~ 100 Kb genome could be assembled and classified in genus 1 of the recently proposed Alphacrassvirinae subfamily. The cat origin of the fecal sample was confirmed by partial mitochondrial DNA sequencing. High levels of Bacteroides intestinalis DNA could also be detected in this cat’s feces. Fecal samples longitudinally collected over a 4-week period showed the continuous shedding of crAssphage DNA. We therefore report the first genome sequence-confirmed detection of crAssphage in fecal samples of a non-primate mammal.

Bifidobacterias como indicadoras de contaminación fecal en aguas tropicales

Human fecal pollution in water constitute a serious risk to the public health, nevertheless the indicator microorganisms commonly used to detect the levels of fecal pollution does not identify the specific source. The detection of certain Bifidobacterium species as B. adolescentis and B. dentium have been proposed as an effective marker of human fecal contamination, but this has not yet been demonstrated in tropical environmental conditions. The aim of the present work was to determine the Bifidobacteria profile in one sample of water from the Mesolandia Swamp in the Colombian Caribbean and feces samples of 260 human and 94 domestic animals from a human settlement around the swamp. DNA from all the samples was amplified by PCR using specific specie primers based on the 16S rRNA gene sequence and separated by DGGE (Denaturing Gradient Gel Electrophoresis). DGGE bands were excised, reamplified, sequenced, and compared to GenBank database. Bifidobacterial DGGE profiles showed that eight species of Bifidobacterium that were found in the water sample, were also present in the human and animal feces. Bifidobacterium adolescentis and B. dentium described as potential human fecal pollution indicators were found in domestic animals. In this study under the environmental and experimental conditions evaluated was not possible to find a Bifidobacterium species as specific marker of human fecal contamination in tropical areas. However, the applied method in this study could be useful to detect fecal pollution in tropical waters allowing a nearest approximation to the origin of the fecal pollution compared with cultural traditional methods, since it was possible to find identical DNA sequences in the water and in the fecal samples.

Fecal pollution can explain antibiotic resistance gene abundances in anthropogenically impacted environments

Discharge of treated sewage leads to release of antibiotic resistant bacteria, resistance genes and antibiotic residues to the environment. However, it is unclear whether increased abundance of antibiotic resistance genes in sewage and sewage-impacted environments is due to on-site selection pressure by residual antibiotics, or is simply a result of fecal contamination with resistant bacteria. Here we analyze relative resistance gene abundance and accompanying extent of fecal pollution in publicly available metagenomic data, using crAssphage sequences as a marker of human fecal contamination (crAssphage is a bacteriophage that is exceptionally abundant in, and specific to, human feces). We find that the presence of resistance genes can largely be explained by fecal pollution, with no clear signs of selection in the environment, with the exception of environments polluted by very high levels of antibiotics from manufacturing, where selection is evident. Our results demonstrate the necessity to take into account fecal pollution levels to avoid making erroneous assumptions regarding environmental selection of antibiotic resistance.

Exposure to Human-Associated Chemical Markers of Fecal Contamination and Self-Reported Illness among Swimmers at Recreational Beaches

Anthropogenic chemicals have been proposed as potential markers of human fecal contamination in recreational water. However, to date, there are no published studies describing their relationships with illness risks. Using a cohort of swimmers at seven U.S. beaches, we examined potential associations between the presence of chemical markers of human fecal pollution and self-reported gastrointestinal (GI) illness, diarrhea, and respiratory illness. Swimmers were surveyed about their beach activities, water exposure, and baseline symptoms on the day of their beach visit, and about any illness experienced 10-12 days later. Risk differences were estimated using model-based standardization and adjusted for the swimmer's age, beach site, sand contact, rainfall, and water temperature. Sixty-two chemical markers were analyzed from daily water samples at freshwater and marine beaches. Of those, 20 were found consistently. With the possible exception of bisphenol A and cholesterol, no chemicals were consistently associated with increased risks of illness. These two chemicals were suggestively associated with 2% and 1% increased risks of GI illness and diarrhea in both freshwater and marine beaches. Additional research using the more sensitive analytic methods currently available for a wider suite of analytes is needed to support the use of chemical biomarkers to quantify illness risk and identify fecal pollution sources.

Distribution of human fecal marker GB-124 bacteriophages in urban sewage and reclaimed water of São Paulo city, Brazil.

Bacteriophages infecting Bacteroides fragilis GB-124 have been described as potential markers of human fecal contamination in water sources. The aim of this study was to evaluate the occurrence of GB-124 phages in raw sewage, secondary effluents and reclaimed water of the São Paulo city using a low-cost microbial source tracking method. Samples were collected monthly from April 2015 to March 2016 in four municipal wastewater treatment plants that operate with activated sludge processes followed by different tertiary treatments (sand-anthracite filtration, membrane bioreactor/reverse osmosis) and final chlorination. GB-124 phages were detected in 100% of the raw sewage samples, with viral loads varying from 7.5 × 103 to 1.32 × 106 PFU/L. Virus removal efficiency in activated sludge processes ranged from 1.89 to 2.31 log10. Frequencies of phage detection were lower in reclaimed water samples (0-22.2%). The results indicated that GB-124 phage could be a complementary low-cost viral marker for the detection of human fecal pollution in waters impacted with urban sewage in this region. However, the datasets of tertiary effluents resulted in several samples with concentrations below the detection limit (DL ≤1 PFU/mL) suggesting the need to obtain analytical methods with lower DL for greater accuracy of negative results.

Assessment of water quality in a border region between the Atlantic forest and an urbanised area in Rio de Janeiro, Brazil.

The preservation of water resources is one of the goals of the designation of parks that act as natural reservoirs. In order to assess the impact of the presence of humans in an environmental preservation area bordering urban areas, the presence of four pathogenic enteric viruses [group A rotavirus (RV-A), norovirus (NoV), human adenoviruses (HAdV), and hepatitis A virus (HAV)], as well as the physico-chemical parameters, and Escherichia coli levels were assessed in riverine water samples. From June 2008 to May 2009, monthly monitoring was performed along the Engenho Novo River. RV-A, NoV, and HAdV were observed in 29% (31/108) of the water samples, with concentrations of up to 10(3) genome copies/liter. The natural occurrence of infectious HAdV was demonstrated by Integrated Cell Culture-PCR (ICC-PCR). This study confirms the suitability of using the detection of fecal-oral transmitted viruses as a marker of human fecal contamination in water matrices and indicates the spread of pathogenic viruses occurring in an alleged area of environmental protection.

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.

Assessment of burden of virus agents in an urban sewage treatment plant in Rio de Janeiro, Brazil

Sewage discharge is considered to be the main source of virus contamination in aquatic environments. There is no correlation between the presence of viruses and the presence of fecal coliforms in water; therefore virological markers are needed when monitoring contamination. This study investigates DNA and RNA virus concentrations in wastewater and evaluates a potential virus marker of human contamination. Influent and effluent samples were collected twice a month throughout a 1-year period. Viruses were detected using quantitative polymerase chain reaction protocols; nucleotide sequencing was carried out for virus genotyping. Human adenovirus (HAdV) and polyomavirus JC (JCPyV) were the most prevalent viruses found in influent samples (100%) with a virus load that ranged from 10(6) to 10(5) genome copies per liter (gc l(-1)). Norovirus genogroup II (NoV GII) and human astrovirus (HAstV) were less prevalent, and ranged from 10(4) to 10(3)gc l(-1). Quantitative data on virus profiles in wastewaters stress the high level of rotavirus species A environmental dissemination and address the potential of HAdV as a useful virological marker of virus contamination in aquatic environments. This study corroborates other studies performed in developed countries on DNA viruses as good markers of human fecal contamination.

Assessment of sources of human pathogens and fecal contamination in a Florida freshwater lake

We investigated the potential for a variety of environmental reservoirs to harbor or contribute fecal indicator bacteria (FIB), DNA markers of human fecal contamination, and human pathogens to a freshwater lake. We hypothesized that submerged aquatic vegetation (SAV), sediments, and stormwater act as reservoirs and/or provide inputs of FIB and human pathogens to this inland water. Analysis included microbial source tracking (MST) markers of sewage contamination (Enterococcus faecium esp gene, human-associated Bacteroides HF183, and human polyomaviruses), pathogens (Salmonella, Cryptosporidium, Giardia, and enteric viruses), and FIB (fecal coliforms, Escherichia coli, and enterococci). Bayesian analysis was used to assess relationships among microbial and physicochemical variables. FIB in the water were correlated with concentrations in SAV and sediment. Furthermore, the correlation of antecedent rainfall and major rain events with FIB concentrations and detection of human markers and pathogens points toward multiple reservoirs for microbial contaminants in this system. Although pathogens and human-source markers were detected in 55% and 21% of samples, respectively, markers rarely coincided with pathogen detection. Bayesian analysis revealed that low concentrations (<45 CFU × 100 ml−1) of fecal coliforms were associated with 93% probability that pathogens would not be detected; furthermore the Bayes net model showed associations between elevated temperature and rainfall with fecal coliform and enterococci concentrations, but not E. coli. These data indicate that many under-studied matrices (e.g. SAV, sediment, stormwater) are important reservoirs for FIB and potentially human pathogens and demonstrate the usefulness of Bayes net analysis for water quality assessment.

Characterization of Enterococcus faecalis-infecting phages (enterophages) as markers of human fecal pollution in recreational waters

Enterophages are a novel group of phages that specifically infect Enterococcus faecalis and have been recently isolated from environmental water samples. Although enterophages have not been conclusively linked to human fecal pollution, we are currently characterizing enterophages to propose them as viral indicators and possible surrogates of enteric viruses in recreational waters. Little is known about the morphological or genetic diversity which will have an impact on their potential as markers of human fecal contamination. In the present study we are determining if enterophages can be grouped by their ability to replicate at different temperatures, and if different groups are present in the feces of different animals. As one of the main objectives is to determine if these phages can be used as indicators of the presence of enteric viruses, the survival rate under different conditions was also determined as was their prevalence in sewage and a large watershed. Coliphages were used as a means of comparison in the prevalence and survival studies. Results indicated that the isolates are mainly DNA viruses. Their morphology as well as their ability to form viral plaques at different temperatures indicates that several groups of enterophages are present in the environment. Coliphage and enterophage concentrations throughout the watershed were lower than those of thermotolerant coliforms and enterococci. Enterophage concentrations were lower than coliphages at all sampling points. Enterophages showed diverse inactivation rates and T 90 values across different incubation temperatures in both fresh and marine waters and sand. Further molecular characterization of enterophages may allow us to develop probes for the real-time detection of these alternative indicators of human fecal pollution.

Rapid detection of human fecal contamination in estuarine environments by PCR targeting of Bifidobacterium adolescentis

Detection of Bifidobacterium adolescentis was used as an effective genetic marker of human fecal contamination in Georgia estuaries. Enterococci enumerations on mEI media indicated that a tributary to the Little Satilla River with 516 CFU/100 ml was the most polluted of all the rivers tested. Extracted DNA from eight river water samples was subjected to a two-step nested PCR protocol using genus and species specific primers for Bifidobacterium spp. and B. adolescentis. B. adolescentis was detected from extracted DNA in Dunbar River, Black Bank Creek, and in a Little Satilla River tributary which demonstrates the presence of human fecal contamination in these three rivers. In the five other estuaries tested including West Point–Federica River and the Altamaha River, which both had less than 16 CFU/100 ml of enterococci, B. adolescentis was not detected.

Multitiered Approach Using Quantitative PCR To Track Sources of Fecal Pollution Affecting Santa Monica Bay, California

The ubiquity of fecal indicator bacteria such as Escherichia coli and Enterococcus spp. in urban environments makes tracking of fecal contamination extremely challenging. A multitiered approach was used to assess sources of fecal pollution in Ballona Creek, an urban watershed that drains to the Santa Monica Bay (SMB) near Los Angeles, Calif. A mass-based design at six main-stem sites and four major tributaries over a 6-h period was used (i) to assess the flux of Enterococcus spp. and E. coli by using culture-based methods (tier 1); (ii) to assess levels of Enterococcus spp. by using quantitative PCR and to detect and/or quantify additional markers of human fecal contamination, including a human-specific Bacteroides sp. marker and enterovirus, using quantitative reverse transcriptase PCR (tier 2); and (iii) to assess the specific types of enterovirus genomes found via sequence analysis (tier 3). Sources of fecal indicator bacteria were ubiquitous, and concentrations were high, throughout Ballona Creek, with no single tributary dominating fecal inputs. The flux of Enterococcus spp. and E. coli averaged 10(9) to 10(10) cells h(-1) and was as high at the head of the watershed as at the mouth prior to discharge into the SMB. In addition, a signal for the human-specific Bacteroides marker was consistently detected: 86% of the samples taken over the extent during the study period tested positive. Enteroviruses were quantifiable in 14 of 36 samples (39%), with the highest concentrations at the site furthest upstream (Cochran). These results indicated the power of using multiple approaches to assess and quantify fecal contamination in freshwater conduits to high-use, high-priority recreational swimming areas.