Bacteroidales as a fecal contamination indicator in fresh produce industry: A baseline measurement.

Foodborne outbreaks caused by fecal contamination of fresh produce represent a serious concern to public health and the economy. As the consumption of fresh produce increases, public health officials and organizations have pushed for improvements in food safety procedures and environmental assessments to reduce the risk of contamination. Visual inspections and the establishment of "buffer zones" between animal feeding operations and producing fields are the current best practices for environmental assessments. However, a generalized distance guideline and visual inspections may not be enough to account for all environmental risk variables. Here, we report a baseline measurement surveying the background Bacteroidales concentration, as a quantitative fecal contamination indicator, in California's Salinas Valley. We collected a total of 1632 samples from two romaine lettuce commercial fields at the time of harvesting through two seasons in a year. The quantification of Bacteroidales concentration was performed using qPCR, revealing a notably low concentration (0-2.00 copies/cm2) in the commercial fields. To further enhance the applicability of our findings, we developed a user-friendly method for real-world fecal contamination risk assessment that seamlessly integrates with industry practices. Through the generation of heatmaps that visually illustrate varying risk levels across fields, this approach can identify site-specific risks and offer fresh produce stakeholders a more comprehensive understanding of their land. We anticipate this work can encourage the use of Bacteroidales in the fresh produce industry to monitor fecal contamination and prevent future foodborne outbreaks.

Helicobacter pylori virulence genotypes in Bogotá River and wastewater treatment plants in Colombia.

BACKGROUND: Helicobacter pylori is a bacterium that infects 70%-80% of the population in Colombia, causing chronic gastritis in all those infected and gastric cancer in 1%-2% of those infected. In Colombia, some studies have identified the presence of vacA and cagA genes in environmental samples such as treated, surface, and wastewater, but they have not been evaluated in the Bogotá River. For this, the aim of this study was to identify the virulence genotypes of H. pylori present in samples from the Bogotá River and domestic wastewater treatment plants (WWTPs). MATERIALS AND METHODS: A total of 75 water samples (51 from the Bogotá River and 24 from wastewater treatment plants) were collected. The presence of H. pylori DNA and its virulence genotypes was determined by polymerase chain reaction (PCR). RESULTS: The presence of H. pylori DNA was demonstrated in 44% (33/75) of the samples, obtaining 63.6% (21/33) from the Bogotá River and 36.4% (12/33) from the WWTPs. The most prevalent H. pylori genotype was cagA (-) and vacAm1/s1/i1 being the most virulent of the vacA gene. CONCLUSIONS: This is the first study in Colombia that determines the cagA and vacA genotypes in surface water and WWTPs, indicating the circulation of virulent genotypes in the population. The presence of this pathogen in the waters can be represent a risk to the health of the surrounding population since these waters are reused by the communities for different purposes.

Human Norovirus Detection: How Much Are We Prepared?

Norovirus (NoV) is known to be the second nonbacterial enteric pathogen after rotavirus that causes acute gastroenteritis. They can be spread from person to person through fecal-oral routes. Infection can lead to severe diarrhea, causing stomach pain, vomiting, and nausea. Rapid detection of NoV can control huge economic and productive losses. Genotyping various emerging NoV strains is important to compare the severity among different strains. Conventional immunological and molecular methods have evolved and contributed to developing detection techniques. Immunological (enzyme-linked immunosorbent assay) and molecular detection (reverse transcriptase polymerase chain reaction [RT-PCR], RT-quantitative PCR, loop-mediated isothermal amplification, nucleic acid sequence-based alignment, recombinase polymerase amplification) methods have been mainly used. The development of biosensors using aptasensor, affinity peptides, nanoparticles, microfluidics, and so on, are currently the most researched topics. The availability of next-generation sequencing technologies has greatly influenced the diagnosis of NoV. The complementation of advanced technologies is helpful in identification of new variants. In this study, techniques that are useful in detecting NoV are discussed. This review has investigated the availability of recent methods used in the detection, present status, and futuristic plan of action in case of outbreak and pandemic.

Meta-analysis of microbial source tracking for the identification of fecal contamination in aquatic environments based on data-mining.

Microbial source tracking (MST) technology represents an innovative approach employed to trace fecal contamination in environmental water systems. The performance of primers may be affected by amplification techniques, target primer categories, and regional differences. To investigate the influence of these factors on primer recognition performance, a meta-analysis was conducted on the application of MST in water environments using three databases: Web of Science, Scopus, and PubMed (n = 2291). After data screening, 46 studies were included in the final analysis. The investigation encompassed Polymerase Chain Reaction (PCR)/quantitative PCR (qPCR) methodologies, dye-based (SYBR)/probe-based (TaqMan) techniques, and geographical differences of a human host-specific (HF183) primer and other 21 additional primers. The results indicated that the primers analyzed were capable of differentiating host specificity to a certain degree. Nonetheless, by comparing sensitivity and specificity outcomes, it was observed that virus-based primers exhibited superior specificity and recognition capacity, as well as a stronger correlation with human pathogenicity in water environments compared to bacteria-based primers. This finding highlights an important direction for future advancements. Moreover, within the same category, qPCR did not demonstrate significant benefits over conventional PCR amplification methods. In comparing dye-based and probe-based techniques, it was revealed that the probe-based method's advantage lay primarily in specificity, which may be associated with the increased propensity of dye-based methods to produce false positives. Furthermore, the heterogeneity of the HF183 primer was not detected in China, Canada, and Singapore respectively, indicating a low likelihood of regional differences. The variation among the 21 other primers may be attributable to regional differences, sample sources, detection techniques, or alternative factors. Finally, we identified that economic factors, climatic conditions, and geographical distribution significantly influence primer performance.

Role of pepper mild mottle virus as a tracking tool for fecal pollution in aquatic environments.

The plant pathogen pepper mild mottle virus (PMMoV) has recently been proposed as a water quality indicator, it is a RNA virus belonging to the genus Tobamovirus in the family Virgoviridae that causes harm to the pepper crops. After consuming processed food products containing infected peppers, such as hot sauces, PMMoV is excreted in high concentrations in feces; therefore, this is the most common RNA virus, constantly found in the feces of humans. The fecal-oral pathway is emerging as an environmental problem. The presence of high concentrations of pathogens associated with human excreta in environmental waters or water reuse supplies poses a threat to public health. Due to the difficulty in determining the presence of pathogens effectively in water, attempts to monitor microbial water quality often use surrogates or indicator organisms that can be easily detected; therefore, PMMoV is used as a viral surrogate in aquatic environment. This paper describes the incidence and persistence of PMMoV in aquatic environments and in waste treatment plants and its usefulness for quantifying virus reductions by advanced water treatment technologies. In recent research, SARS-CoV-2 was reported to be found in wastewater and utilized for the purpose of monitoring coronavirus illness outbreaks. Since PMMoV is readily identified in the human feces and can also serve as an indicator of human waste, the determined PMMoV concentrations may be utilized to give the normalized report of the SARS-CoV-2 concentration, so that, the amount of human waste found in the wastewater can be taken into consideration.

Modeling Exposure to Fecal Contamination in Drinking Water due to Multiple Water Source Use.

The Joint Monitoring Programme estimated that 71% of people globally had access to "safely managed" drinking water in 2017. However, typical data collection practices focus only on a household's primary water source, yet some households in low- and middle-income countries (LMICs) engage in multiple water source use, including supplementing improved water supplies with unimproved water throughout the year. Monte Carlo simulations and previously published data were used to simulate exposure to fecal contamination (as measured by E. coli) along a range of supplemental unimproved source use rates (e.g., 0-100% improved water use, with the remainder made up with unimproved water). The model results revealed a statistically significant increase in annual exposure to E. coli when individuals supplement their improved water with unimproved water just 2 days annually. Additionally, our analysis identified scenarios-realistic for the data set study setting-where supplementing with unimproved water counterintuitively decreases exposure to E. coli. These results highlight the need for evaluating the temporal dynamics in water quality and availability of drinking water sources in LMICs as well as capturing the use of multiple water sources for monitoring global access to safe drinking water.

Occurrence of bacterial and viral fecal markers in municipal sewage sludge and their removal during sludge conditioning processes.

Fecal contamination in wastewater treatment system may pose severe threats to human health, but the detailed contamination of fecal bacterial and viral pathogens in municipal sewage sludge remains unclear. In addition, it is also unclear how sludge conditioning treatments would impact the distribution of fecal markers in conditioned sewage sludge. Before addressing these two issues, the possible polymerase chain reaction (PCR) inhibition effect when determining the abundances of fecal markers in both sludge solids and sludge supernatants should be solved, and methods of effectively concentrating fecal markers from sludge supernatant should also be developed. In the present study, we found that the serial tenfold dilution effectively reduced the PCR inhibition effect when determining the abundances of fecal markers including cross-assembly phages (CrAssphage), JC polyomavirus (JCPyV), human-specific HF183 bacteroides (HF183), human BK polyomavirus (BKPyV), human adenovirus (HAdV) and Escherichia coli (EC), while the utilization of negatively charged HA membrane was effective to recover fecal markers from sludge supernatant. The results of a six-month monitoring revealed that gene markers of CrAssphage, JCPyV, HF183, BKPyV, HAdV, and EC can be detected in municipal sewage sludge collected from a local wastewater treatment plant. Among the investigated four chemical conditioning methods, i.e., chemical conditioning with polyacrylamide (PAM), Fe[III]/CaO, or Fenton's reagent, and chemical acidification conditioning, chemical conditioning with Fenton's reagent was much more effective than the other three conditioning methods to reduce the abundances of fecal markers in the supernatant and solid of conditioned sewage sludge. Furthermore, the investigated fecal markers in the conditioned sewage sludge can be simultaneously attenuated by employing suitable conditioning methods, consequently reducing the associated environmental risks.

DNA fingerprinting using BOX-A1R and (GTG)5 primers identify spatial variations of fecal contamination along Pasig River, Philippines.

Pasig River is one of the most economically important rivers in Metro Manila, Philippines. It traverses some of the region's major cities, and because of its strategic location, it is utilized as a means of transportation, as a source of water for domestic and industrial uses, and for recreational purposes. However, due to population growth, industrialization, and land use, the river's water quality is deteriorating. Wastes that pollute the river pose health risks to the people that benefit from it. To prevent the river's further degradation, it is essential to identify the origin of contamination. In this study, the sources of fecal contamination in Pasig River were identified using BOX-A1R and (GTG)5 primers in the DNA fingerprinting of Escherichia coli isolated from the river. Results showed the dominance of human contamination (percent composition = 65.55%), followed by agricultural sources (percent composition = 23.48%), and the lowest was from sewage (percent composition = 10.98%). The results of this research can help in evaluating public health risks and can be used as a scientific basis for policymaking and implementation for the rehabilitation and improvement of Pasig River.

Occurrence of Helicobacter spp. and Fecal Bacterial Contamination in High-altitude Aquatic Environments from the Andes.

The occurrence of Helicobacter spp. and fecal bacterial contamination was investigated in high-altitude environments from the Northeastern Andes of Venezuela. Helicobacter DNA was detected by PCR in streams, drinking and irrigation waters, and one culture from drinking water by the HP enrichment medium for selection of Helicobacter pylori, which displayed 98.98% homology to this pathogen based on 16S rRNA gene sequencing. FISH demonstrated predominant coccoid cells of the target bacteria indicative of the viable but nonculturable (VBNC) state in all water samples and HP cultures. Our work reveals for the first time Helicobacter spp. in waters from one of the highest places in the world. These results, together with the presence of fecal coliforms (2-160 MPN/100 mL) from the headwaters of rivers to drinking and irrigation waters, alert fecal contamination and epidemiological implications in this area of ecological and economic importance for the region.

Ingestion of Fecal Bacteria along Multiple Pathways by Young Children in Rural Bangladesh Participating in a Cluster-Randomized Trial of Water, Sanitation, and Hygiene Interventions (WASH Benefits).

Quantifying the contribution of individual exposure pathways to a child's total ingestion of fecal matter could help prioritize interventions to reduce environmental enteropathy and diarrhea. This study used data on fecal contamination of drinking water, food, soil, hands, and objects and second-by-second data on children's contacts with these environmental reservoirs in rural Bangladesh to assess the relative contribution of different pathways to children's ingestion of fecal indicator bacteria and if ingestion decreased with the water, sanitation, and hygiene interventions implemented in the WASH Benefits Trial. Our model estimated that rural Bangladeshi children <36 months old consume 3.6-4.9 log10 most probable number E. coli/day. Among children <6 months, placing objects in the mouth accounted for 60% of E. coli ingested. For children 6-35 months old, mouthing their own hands, direct soil ingestion, and ingestion of contaminated food were the primary pathways of E. coli ingestion. The amount of E. coli ingested by children and the predominant pathways of E. coli ingestion were unchanged by the water, sanitation, and hygiene interventions. These results highlight contaminated soil, children's hands, food, and objects as primary pathways of E. coli ingestion and emphasize the value of intervening along these pathways.

Children Are Exposed to Fecal Contamination via Multiple Interconnected Pathways: A Network Model for Exposure Assessment.

In recent decades, quantitative microbial risk assessment (QMRA) has been widely used to assess exposure to fecal microbes and associated health risks. In this study, a multipathway exposure assessment model was developed to evaluate exposure to fecal microbes for children under 5 in highly contaminated urban environments. Children had contact with various environmental compartments. The contamination levels of these compartments were estimated from fecal indicator counts in the environmental samples. Structured observations of child behavior (including activities, locations, and time) were used to model behavioral sequences as a dynamic network. The exposure model combines behavior sequences with environmental contamination, using additional exposure factors when needed, to estimate the number of fecal microbes transferred from environmental sources to human oral ingestion. As fecal exposure in a highly contaminated urban environment consists of contributions from multiple pathways, it is imperative to study their relative importance. The model helps us better understand the characteristics of the exposure pathways that may be driven by variation in contamination and by variable behavior, like hygiene and high-risk activities. Importantly, the model also allows prediction of the quantitative effects of an intervention-the expected reduction in exposure due to infrastructural or behavioral changes-by means of scenario studies. Based on experience with this exposure model, we make specific recommendations for additional studies of child behavior and exposure factors in order to fill critical information gaps and improve the model structure and assumptions.

The determination of E. coli levels and pathotypes in water sources around Isparta province Turkey.

In this study, a total of 57 water samples were collected over different months around the Isparta region. The levels of total and fecal coliform in these water samples were determined. According to an analysis of the results, although the pollution level of these water sources are not high in terms of their total coliform counts, they cannot be used as drinking water. In the water samples taken from Andik Creek, Dari Creek  Dam, and Egirdir Lake, fecal counts were found to be similar to each other, it is possible to use these waters after refinement. However fecal coliform levels are high in water samples taken from Bezirgan Creek, and these waters cannot be used as drinking water. After the total and fecal coliform counts were determined, the most probable colonies that can be identified to be E. coli were chosen and confirmation tests were performed. In total, 397 colonies were isolated from water samples, and 55 of those were confirmed as E. coli strains. Also, 151 strains were determined to be part of the coliform group. E. coli strains were studied with a real-time PCR to determine some virulence genes. Analysis of the results showed that 29.2% of E. coli strains contained enteropathogenic/enterohemorrhagic E. coli virulence genes, and 14.5% of the isolated E. coli strains contained verotoxigenic E. coli virulence factors. Enteroinvasive E. coli virulence genes were contained in 1.8% of the E. coli strains, and it was found that 54.5% of the isolated E. coli strains contained none of the gene regions investigated.

Prevalent fecal contamination in drinking water resources and potential health risks in Swat, Pakistan.

Fecal bacteria contaminate water resources and result in associated waterborne diseases. This study assessed drinking water quality and evaluated their potential health risks in Swat, Pakistan. Ground and surface drinking water were randomly collected from upstream to downstream in the River Swat watershed and analyzed for fecal contamination using fecal indicator bacteria (Escherichia coli) and physiochemical parameters (potential of hydrogen, turbidity, temperature, electrical conductivity, total dissolved solid, color, odor and taste). The physiochemical parameters were within their safe limits except in a few locations, whereas, the fecal contaminations in drinking water resources exceeded the drinking water quality standards of Pakistan Environmental Protection Agency (Pak-EPA), 2008 and World Health Organization (WHO), 2011. Multivariate and univariate analyses revealed that downstream urbanization trend, minimum distance between water sources and pit latrines/sewerage systems, raw sewage deep well injection and amplified urban, pastures and agricultural runoffs having human and animal excreta were the possible sources of contamination. The questionnaire survey revealed that majority of the local people using 10-20years old drinking water supply schemes at the rate of 73% well supply, 13% hand pump supply, 11% spring supply and 3% river/streams supply, which spreads high prevalence of water borne diseases including hepatitis, intestinal infections and diarrhea, dysentery, cholera, typhoid fever, jaundice and skin diseases in children followed by older and younger adults.

Faecal contamination of echinoderms: first report of heavy Escherichia coli loading of sea urchins from a natural growing area.

UNLABELLED: Although little evidence existed to support that view, European countries and in particular France, have regarded echinoderms, including sea urchins, as low risk in terms of feacal contamination. It is hypothesized that the sea urchins mode of feeding, which is based on grazing and differs from bivalve molluscs, would prevent it from concentrating high levels of Escherichia coli. Here, we monitored E. coli levels in sea urchins (Paracentrotus lividus) and in filter-feeder mussels (Mytillus galloprovincialis), collected concurrently from the same natural area over a 1-year period to verify this assumption. Sea urchins were collected on the seafloor, whereas mussels were collected from the water column at a depth of 4 m. Our results showed heavy bacterial loading of sea urchins in a natural growing environment. Moreover, we highlighted that E. coli contamination of sea urchins could, in certain conditions, be higher than those detected in filter-feeding mussels collected at the same location. Finally, the results showed a significant correlation between rainfall and E. coli concentrations in sea urchins, suggesting that the bacterial safety of sea urchin could be linked to the quality of the surrounding water. SIGNIFICANCE AND IMPACT OF THE STUDY: The European regulation requires competent authorities to monitor the sanitary status of shellfish, including live echinoderms, through faecal indicator organisms. In the French Mediterranean, sea urchin production is significant. Until now, as no data showed significant E. coli contamination levels, no monitoring programs focused on this species. This study demonstrates that sea urchins are more vulnerable to faecal contamination than previously hypothesized, especially during heavy rainfall. In consequence, the European authority general approach to microbiological management of shellfish should be applied to sea urchins.

Coliform detection in cheese is associated with specific cheese characteristics, but no association was found with pathogen detection.

Coliform detection in finished products, including cheese, has traditionally been used to indicate whether a given product has been manufactured under unsanitary conditions. As our understanding of the diversity of coliforms has improved, it is necessary to assess whether coliforms are a good indicator organism and whether coliform detection in cheese is associated with the presence of pathogens. The objective of this study was (1) to evaluate cheese available on the market for presence of coliforms and key pathogens, and (2) to characterize the coliforms present to assess their likely sources and public health relevance. A total of 273 cheese samples were tested for presence of coliforms and for Salmonella, Staphylococcus aureus, Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and other Listeria species. Among all tested cheese samples, 27% (75/273) tested positive for coliforms in concentrations >10cfu/g. Pasteurization, pH, water activity, milk type, and rind type were factors significantly associated with detection of coliforms in cheese; for example, a higher coliform prevalence was detected in raw milk cheeses (42% with >10cfu/g) compared with pasteurized milk cheese (21%). For cheese samples contaminated with coliforms, only water activity was significantly associated with coliform concentration. Coliforms isolated from cheese samples were classified into 13 different genera, including the environmental coliform genera Hafnia, Raoultella, and Serratia, which represent the 3 genera most frequently isolated across all cheeses. Escherichia, Hafnia, and Enterobacter were significantly more common among raw milk cheeses. Based on sequencing of the housekeeping gene clpX, most Escherichia isolates were confirmed as members of fecal commensal clades of E. coli. All cheese samples tested negative for Salmonella, Staph. aureus, and Shiga toxin-producing E. coli. Listeria spp. were found in 12 cheese samples, including 5 samples positive for L. monocytogenes. Although no association was found between coliform and Listeria spp. detection, Listeria spp. were significantly more likely to be detected in cheese with the washed type of rind. Our data provide information on specific risk factors for pathogen detection in cheese, which will facilitate development of risk-based strategies to control microbial food safety hazards in cheese, and suggest that generic coliform testing cannot be used to assess the safety of natural cheese.

Microbiological and hydrogeological assessment of groundwater in southern Italy.

This study represents the first investigation of microbiological groundwater pollution as a function of aquifer type and season for the Apulia region of southern Italy. Two hundred and seven wells were randomly selected from those monitored by the Regional Agency for Environmental Protection for emergency use. Both compulsory (Escherichia coli, Total Coliform, and Enterococci) and optional (Pseudomonas aeruginosa, Salmonella spp., Heterotrophic Plate Count at 37 and 22 °C) microbiological parameters were assessed regularly at these wells. Groundwater from only 18 of the 207 (8.7 %) wells was potable; these all draw from karst-fissured aquifers. The remaining 189 wells draw from karst-fissured (66.1 %) or porous (33.9 %) aquifers. Of these, 82 (43.4 %) tested negative for Salmonella spp. and P. aeruginosa, while 107 (56.6 %) tested positive for P. aeruginosa (75.7 %), Salmonella spp. (10.3 %), or for both Salmonella spp. and P. aeruginosa (14 %). A logistic regression model shows that the probability of potable groundwater depends on both season and aquifer type. Typically, water samples were more likely to be potable in autumn-winter than in spring-summer periods (odds ratio, OR = 2.1; 95 % confidence interval, 95 % CI = 1.6-2.7) and from karst-fissured rather than porous aquifers (OR = 5.8; 95 % CI = 4.4-7.8). Optional parameters only showed a seasonal pattern (OR = 2.6; 95 % CI = 1.7-3.9). Clearly, further investigation of groundwater microbiological aspects should be carried out to identify the risks of fecal contamination and to establish appropriate protection methods, which take into account the hydrogeological and climatic characteristics of this region.

Surgical outcomes and prognostic factors of emergency surgery for colonic perforation: would fecal contamination increase morbidity and mortality?

PURPOSE: Complications resulting from colonic perforation are related to secondary peritonitis due to bacterial or fecal contamination. We investigated outcomes of emergency surgery for colonic perforation associated with fecal contamination with regard to early and late postoperative complication rates and mortality rates, and investigated prognostic factors influencing those outcomes. METHODS: A retrospective analysis of prospectively collected data on factors influencing complications and mortality rates was conducted on data from 152 patients who had undergone emergent operations for colonic perforation between January 2005 and December 2011. Patients were categorized into two groups: those with and without gross fecal contamination at the time of operation. RESULTS: Forty-one (26.9 %) patients had gross fecal contamination. Patients who had fetal contamination had a higher Mannheim peritonitis index (31.3 ± 5.1 vs. 21.9 ± 7.2, p < 0.001), higher organ failure rate (53.7 vs. 24.3 %, p = 0.001), and longer operating time (168.8 ± 49.9 vs. 144.8 ± 66.1 min, p = 0.036) than patients without fecal contamination. Early complications (<30 days) occurred more frequently in the fecal contamination group (82.9 vs. 49.5 %, p = 0.001), although late complications (46.2 vs. 39.3 %, p = 0.942) and mortality (17.1 vs. 8.1 %, p = 0.110) did not differ. In multivariate analysis, fecal contamination significantly predicted early complications (odds ratio, 2.78; p = 0.037) but not late complications or mortality. CONCLUSIONS: The frequency of early complications can increase if fecal contamination exists. However, when early complications are well managed, fecal contamination does not significantly influence occurrences late complications or mortality.

Interference-resistant gold nanoparticle assay for detecting Enterococcus in fresh and marine waters.

Enterococci are common indicators of fecal contamination and are used to assess the quality of fresh and marine water, sand, soil, and sediment. However, samples collected from these environments contain various cells and other factors that can interfere with the assays used to detect enterococci. We developed a novel assay for the sensitive and specific detection of enterococci that is resistant to interference from other cells and environmental factors. Our interference-resistant assay used 30-nm gold nanoparticles (AuNPs), streptavidin, and a biotinylated Enterococcus antibody. Enterococci inhibited the interaction between streptavidin and biotin and led to the disaggregation of AuNPs. The absence of enterococci led to the aggregation of AuNPs, and this difference was easily detected by spectrophotometry. This interference-resistant AuNP assay was able to detect whole cells of Enterococcus in the range of 10 to 107 CFU/mL within 3 h, had high specificity for enterococci, and was unaffected by the presence of other intestinal bacteria, such as Escherichia coli. Our examination of fresh and marine water samples demonstrated no interference from other cells or environmental factors. The interference-resistant AuNP assay described here has the potential to be used as a rapid, simple, and effective method for monitoring enterococci in diverse environmental samples.

Identification of Cryptosporidium parvum and Blastocystis hominis subtype ST3 in Cholga mussel and treated sewage: Preliminary evidence of fecal contamination in harvesting area.

Cryptosporidium parvum and Blastocystis hominis are foodborne parasites known for causing diarrhea. They accumulate in mussels grown on contaminated water bodies, due to the discharge of treated sewage from sewage treatment plants (STP). Despite this, some countries like Chile do not include these parasites in the control or monitoring of sewage water. The objective of this research was to evaluate the contamination of C. parvum. and B. hominis from treated sewage (disinfected by chlorination) and Cholga mussels in a touristic rural cove from the bay of Concepción. Cholga mussels from commercial stores and a treated sewage sample were analyzed. Cryptosporidium spp. was identified by Ziehl-Neelsen-Staining (ZNS) and C. parvum by direct-immunofluorescence assay (IFA) from ZNS-positive samples. Blastocystis hominis was identified by PCR using locus SSU rDNA. C. parvum and B. hominis subtype ST3 were found in 40% and 45% of Cholga mussel samples, respectively, and both parasites were identified in the treated sewage. Blastocystis hominis SSU rDNA gene alignment from Cholga mussels and treated sewage showed 89% of similarity, indicating that could be the same parasite in both samples. We describe the first evidence of possible contamination with these parasites from treated sewage to Cholga mussel suggesting an environmental contamination with high human risk. Based on these results, further studies will consider all the rural coves and STP from the bay to prevent possible contamination of these parasites.

Assessment of crAssphage as a human fecal source tracking marker in the lower Great Lakes.

CrAssphage or crAss-like phage ranks as the most abundant phage in the human gut and is present in human feces-contaminated environments. Due to its high human specificity and sensitivity, crAssphage is a potentially robust source tracking indicator that can distinguish human fecal contamination from agricultural or wildlife sources. Its suitability in the Great Lakes area, one of the world's most important water systems, has not been well tested. In this study, we tested a qPCR-based quantification method using two crAssphage marker genes (ORF18-mod and CPQ_064) at Toronto recreational beaches along with their adjacent river mouths. Our results showed a 71.4 % (CPQ_064) and 100 % (ORF18-mod) human sensitivity for CPQ_064 and ORF18-mod, and a 100 % human specificity for both marker genes. CrAssphage was present in 57.7 % or 71.2 % of environmental water samples, with concentrations ranging from 1.45 to 5.14 log10 gene copies per 100 mL water. Though concentrations of the two marker genes were strongly correlated, ORF18-mod features a higher human sensitivity and higher positive detection rates in environmental samples. Quantifiable crAssphage was mostly present in samples collected in June and July 2021 associated with higher rainfall. In addition, rivers had more frequent crAssphage presence and higher concentrations than their associated beaches, indicating more frequent and greater human fecal contamination in the rivers. However, crAssphage was more correlated with E. coli and Enterococcus at the beaches than in the rivers, suggesting human fecal sources may be more predominant in driving the increases in E. coli and Enterococcus at the beaches when impacted by river plumes.