Occurrence of F-Specific Bacteriophages in Untreated and Treated Wastewaters in Mumbai.

F + coliphages are considered as potential enteric viral indicators in water systems as a tool for on-site validation of wastewater treatment processes. The present study evaluated the occurrence of F + coliphages in wastewaters collected from three wastewater treatment plants (WWTPs) in Mumbai city, to assess this potential. The detection and enumeration of F + coliphages was carried out from WWTPs Z1, Z3 and Z5 using the ISO 10705-1 and U.S EPA 1601 methods. F + coliphages were majorly detected in untreated wastewater samples followed by a few secondary treated samples in WWTP-Z1 and Z3 and one tertiary treated sample from Z1, these differences were found to be statistically significant. The difference in F + coliphage levels between the treatment stages highlight their potential as indicators for monitoring the efficiency of wastewater treatment. The overall positivity of F + coliphage was 35.09% for Salmonella. typhimurium WG49 host (as per ISO 10705-1), was higher by 10.52% for Escherichia coli Famp HS host (as per U.S EPA 1601) (45.61%), highlighting the efficiency of the latter host over the former in F + coliphage detection. Significant difference in F + coliphage counts using the two bacterial hosts were observed in WWTP-Z3 (p = 0.001) and WWTP-Z1 (p = 0.047) but not in WWTP-Z5 (p = 0.332). Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01181-7.

Foodborne Viruses and Somatic Coliphages Occurrence in Fresh Produce at Retail from Northern Mexico.

Foodborne disease outbreaks linked to consumption of vegetables have been often attributed to human enteric viruses, such as Norovirus (NoV), Hepatitis A virus (HAV), and Rotavirus (RoV). Information about the occurrence of these viruses is scarce in many fresh-producing countries. Viral contamination detection of indicators, such as somatic coliphages, could indirectly reflect the presence of viral pathogens, being a valuable tool for better viral risk assessment in food industry. This study aimed to establish the occurrence and correlation of foodborne viruses and somatic coliphages in leafy greens in northern Mexico. A total of 320 vegetable samples were collected, resulting in 80 composite rinses, 40 of lettuce and 40 of parsley. Somatic coliphages were determined using the EPA 1602 method, while foodborne viruses (HAV, RoV, NoV GI, and GII) were determined by qPCR. The occurrence of RoV was 22.5% (9/40, mean 2.11 log gc/g) in lettuce and 20% (8/40, mean 1.91 log gc/g) in parsley. NoV and HAV were not detected in any samples. Somatic coliphages were present in all lettuce and parsley samples, with mean levels of 1.85 log PFU/100 ml and 2.28 log PFU/100 ml, respectively. Spearman analysis established the correlation of somatic coliphages and genomic copies of RoV, resulting in an r2 value of - 0.026 in lettuce and 0.349 in parsley. Although NoV or HAV were undetected in the samples, the presence of RoV is a matter of concern as leafy greens are usually eaten raw, which poses a potential risk of infection.

Somatic Coliphages as an Operational Tool to Assess Loss of Bathing Water Quality after Heavy Rain Events.

Rapid population growth and coastal development has led to increased fecal contamination of coastal surface waters worldwide, enhancing the potential risk of waterborne human pathogens in bathing areas. More frequent heavy rainfall events, attributed to global warming, have further exacerbated the problem by causing sometimes sewer overflows into recreational waters. As traditional bacterial indicators have limited accuracy for predicting health risks associated with waterborne viruses, the additional use of viral indicators such as coliphages is recommended. In this study, we compared the behavior of bacterial and viral indicators of water quality at 10 Barcelona beaches during three bathing seasons, in dry conditions, and after four rainstorms that caused specific pollution events due to rain runoff with combined sewer overflows (CSO). Levels of all target indicators increased after the rainstorms, but compared to Escherichia coli and intestinal enterococci, somatic coliphages exhibited a slower decline and higher environmental persistence following a rain event. Daily continuous sampling carried out during the days following a rainstorm allowed not only the determination of the decay kinetics of each target indicator but also the day when the water quality recovered the values established in the current European regulation in approximately 2 -3 days after each CSO. These observations indicate that the combined use of bacterial and viral indicators can enhance the surveillance of microbial quality of bathing waters. Moreover, coliphages can swiftly provide insights into transient fecal pollution linked to rainfall episodes, thanks to available analytical techniques that enable same-day recommendations. The management of urban wastewater and recreational water regulations should consistently employ microbial indicators to address rainwater runoff or sewer overflows resulting from heavy rainfall.

A new understanding of somatic coliphages belonging to the Microviridae family in urban wastewater.

Somatic coliphages (SC) and F-specific RNA coliphages (FRNAPH) have been included in regulations or guidelines by several developed countries as a way of monitoring water safety and the microbiological quality of shellfish harvesting waters. SC are highly diverse in their morphology, size and genome. The Microviridae family contains three genera of phages (Alphatrevirus, Gequatrovirus, and Sinsheimervirus), all having a capsid of similar morphology (icosahedral) and size (25-30 nm in diameter) to that of common pathogenic enteric viruses. Three PCR assays specific for each genus of Microviridae were designed to study these phages in raw and treated wastewater (WW) in order to gain knowledge about the diversity and prevalence of Microviridae among SC, as well as their inactivation and removal during WW treatments. Among the four wastewater treatment plants (WWTPs) monitored here, two WWTPs applied disinfection by UV light as tertiary treatment. First, we noticed that Microviridae represented 10 to 30 % of infectious SC in both raw and treated WW. Microviridae appeared to behave in the same way as all SC during these WW treatments. As expected, the highest inactivation, at least 4 log10, was achieved for infectious Microviridae and SC in both WWTPs using UV disinfection. PCR assays showed that the highest removal of Microviridae reached about 4 log10, but the phage removal can vary greatly between WWTPs using similar treatments. This work forms the basis for a broader evaluation of Microviridae as a viral indicator of water treatment efficiency and WW reuse.

Diverse infective and lytic machineries identified in genome analysis of tailed coliphages against broad spectrum multidrug-resistant Escherichia coli / Diversas maquinarias infecciosas y líticas identificadas en el análisis del genoma de colifagos con cola contra Escherichia coli multirresistente de amplio espectro

The emergence of multidrug-resistant (MDR) E. coli with deleterious consequences to the health of humans and animals has been attributed to the inappropriate use of antibiotics. Without effective antimicrobials, the success of modern medicine in treating infections would be at an increased risk. Bacteriophages could be used as an alternative to antibiotics for controlling the dissemination of MDR bacteria. However, before their use, the bacteriophages have to be assessed for the safety aspect. In this study, three broad host range highly virulent coliphage genomes were sequenced, characterized for infective and lytic potential, and checked for the presence of virulence and resistance genes. The genome sequencing indicated that coliphages ϕEC-S-21 and ϕEC-OE-11 belonged to Myoviridae, whereas coliphage ϕEC-S-24 belonged to the Autographiviridae family derived from the Podoviridae family. The genome size of the three coliphages ranged between 24 and 145 kb, with G + C content ranging between 37 and 51%. Coding sequences (CDS) ranged between 30 and 251 amino acids. The CDS were annotated and the proteins were categorized into different modules, viz., phage structural proteins, proteins associated with DNA replication, DNA modification, bacterial cell lysis, phage packaging, and uncharacterized proteins. The presence of tRNAs was detected only in coliphage ϕEC-OE-11. All three coliphages possessed diverse infective and lytic mechanisms, viz., lytic murein transglycosylase, peptidoglycan transglycosylase, n-acetylmuramoyl-l-alanine amidase, and putative lysozyme. Furthermore, the three coliphage genomes showed neither the presence of antibiotic resistance genes nor virulence genes, which makes them desirable candidates for use in phage therapy-based applications.(AU)

Mathematical model of interaction Escherichia coli and Coliphages.

We propose a mathematical model based in ordinary differential equations between bacterial pathogen and Bacteriophages to describe the infection dynamics of these populations, for which we use a nonlinear function with an inhibitory effect. We study the stability of the model using the Lyapunov theory and the second additive compound matrix and perform a global sensitivity analysis to elucidate the most influential parameters in the model, besides we make a parameter estimation using growth data of Escherichia coli (E.coli) bacteria in presence of Coliphages (bacteriophages that infect E.coli) with different multiplicity of infection. We found a threshold that indicates whether the bacteriophage concentration will coexist with the bacterium (the coexistence equilibrium) or become extinct (phages extinction equilibrium), the first equilibrium is locally asymptotically stable while the other is globally asymptotically stable depending on the magnitude of this threshold. Beside we found that the dynamics of the model is particularly affected by infection rate of bacteria and Half-saturation phages density. Parameter estimation show that all multiplicities of infection are effective in eliminating infected bacteria but the smaller one leaves a higher number of bacteriophages at the end of this elimination.

Detection and Level Evaluation of Antibodies Specific to Environmental Bacteriophage I11mO19 and Related Coliphages in Non-Immunized Human Sera.

Bacteriophages (phages) are viruses infecting bacteria. They are widely present in the environment, food, and normal microflora. The human microbiome is a mutually interdependent network of bacteria, bacteriophages, and human cells. The stability of these tri-kingdom interactions may be essential for maintaining immunologic and metabolic health. Phages, as with each other's antigens, may evoke an immune response during a human's lifetime and induce specific antibody generation. In this manuscript, we labeled these antibodies as naturally generated. Naturally generated antibodies may be one of the most important factors limiting the efficacy of phage therapy. Herein, we attempted to determine the physiological level of these antibodies specific to a population bacteriophage named I11mO19 in human sera, using an ELISA-based assay. First, we purified the phage particles and assessed the immunoreactivity of phage proteins. Then, affinity chromatography was performed on columns with immobilized phage proteins to obtain a fraction of human polyclonal anti-phage antibodies. These antibodies were used as a reference to elaborate an immunoenzymatic test that was used to determine the level of natural anti-phage antibodies. We estimated the average level of anti-I11mO19 phage antibodies at 190 µg per one milliliter of human serum. However, immunoblotting revealed that cross-reactivity occurs between some proteins of I11mO19 and two other coliphages: T4 and ΦK1E. The antigens probably share common epitopes, suggesting that the determined level of anti-I11mO19 phage might be overestimated and reflects a group of antibodies reactive to a broad range of other E. coli phages. Anti-I11mO19 antibodies did not react with Pseudomonas bacteriophage F8, confirming specificity to the coliphage group. In this work, we wanted to show whether it is possible to determine the presence and level of anti-phage antibodies in nontargeted-immunized sera, using an immunoenzymatic assay. The conclusion is that it is possible, and specific antibodies can be determined. However, the specificity refers to a broader coliphage group of phages, not only the single phage strain.

Coliphage cocktails for controlling antimicrobial-resistant Escherichia coli: emphasizing polyphage and multihost interactions at different levels of multiplicity of infection.

AIM: This study elucidates the in-vitro bactericidal effectiveness of polyphage cocktail combinations of 2, 4, 6, 8, and 10 individual coliphages against a cocktail of 20 AMR Escherichia coli. METHODS AND RESULTS: Different polyphage cocktails viz., 45 two-phage combinations, 28 four-phage combinations, 15 six-phage combinations, 6 eight-phage combinations, and 1 ten-phage combination were formulated using a pool of ten coliphages that were isolated from two different geographical locations (East and West coasts of India). The different polyphage cocktails were tested at four different levels of Multiplicity of Infection (MOI) viz., MOI-1, MOI-10, MOI-100, and MOI-1000. All the 2, 4, 6, 8, and 10-phage cocktails were found to be effective in controlling the growth of a cocktail of 20 AMR bacteria when tested at MOI-1000 and MOI-100 but variations in antibacterial activity were observed at lower MOIs of 10 and 1. The ten coliphage cocktail showed lytic activity against 100% of AMR E. coli from farmed brackish water shrimp, 96% of laboratory collection of AMR E. coli, 92% of AMR E. coli from farmed freshwater fish, and 85% of AMR E. coli from market shrimp. CONCLUSION: Polyphage cocktails of 2, 4, 6, 8, and 10 coliphages applied at an MOI of 1000 effectively suppressed the growth of antimicrobial-resistant E. coli. The results indicated phage-phage synergy in the lytic activity of several coliphage combinations at higher MOIs of 1000 and 100 while phage-phage antagonism was evidenced at lower MOIs of 10 and 1.

Antibiotic resistance genes, mobile elements, virulence genes, and phages in cultivated ESBL-producing Escherichia coli of poultry origin in Kwara State, North Central Nigeria.

The paucity of information on the genomic diversity of drug-resistant bacteria in most food-producing animals, including poultry in Nigeria, has led to poor hazard characterization and the lack of critical control points to safeguard public health. Hence, this study used whole genome sequencing (WGS) to assess the presence and the diversity of antibiotic resistance genes, mobile genetic elements, virulence genes, and phages in Extended Spectrum Beta Lactamase producing Escherichia coli (ESBL - E. coli) isolates obtained from poultry via the EURL guideline of 2017 in Ilorin, Nigeria. The prevalence of ESBL - E. coli in poultry was 10.5 % (n = 37/354). The phenotypic antibiotic susceptibility testing showed that all the ESBL- E. coli isolates were multi-drug resistant (MDR). The in-silico analysis of the WGS raw-read data from 11 purposively selected isolates showed that the isolates had a wide array of ARGs that conferred resistance to beta-lactam antibiotics, and 8 other classes of antibiotics (fluoroquinolones, foliate pathway antagonists, aminoglycoside, phenicol, tetracycline, epoxide, macrolides, and rifamycin). All the ARGs were in the bacterial chromosome except in two isolates where plasmid-mediated quinolone resistance (PMQR) was detected. Two isolates carried the gyrAp.S83L mutation which confers resistance to certain fluoroquinolones. The mobilome consisted of several Col-plasmids and the predominant IncF plasmids belonged to the IncF64:A-:B27 sequence type. The virulome consisted of genes that function as adhesins, iron acquisition genes, toxins, and protectins. Intact phages were found in 8 of the 11 isolates and the phageome consisted of representatives of four families of viruses: Myoviridae (62.5 %, n = 5/8), Siphoviridae (37.5 %, n = 3/8), Inoviridae (12.5 %, n = 1), and Podoviridae (12.5 %, n = 1/8). ESBL - E. coli isolates harboured 1-5 intact phages and no ARGs were identified on any of the phages. Although five of the isolates belonged to phylogroup A, the isolates were diverse as they belonged to different serotype and sequence types. Our findings demonstrate the high genomic diversity of ESBL - E. coli of poultry origin in Ilorin, Nigeria. These diverse isolates harbor clinically relevant ARGs, mobile elements, virulence genes, and phages that may have detrimental zoonotic potentials on human health.

Risk of Gastroenteritis from Swimming at a Wastewater-Impacted Tropical Beach Varies across Localized Scales.

Population growth and changing climate are expected to increase human exposure to pathogens in tropical coastal waters. We examined microbiological water quality in three rivers within 2.3 km of each other that impact a Costa Rican beach and in the ocean outside their plumes during the rainy and dry seasons. We performed quantitative microbial risk assessment (QMRA) to predict the risk of gastroenteritis associated with swimming and the amount of pathogen reduction needed to achieve safe conditions. Recreational water quality criteria based on enterococci were exceeded in >90% of river samples but in only 13% of ocean samples. Multivariate analysis grouped microbial observations by subwatershed and season in river samples but only by subwatershed in the ocean. The modeled median risk from all pathogens in river samples was between 0.345 and 0.577, 10-fold above the U.S. Environmental Protection Agency (U.S. EPA) benchmark of 0.036 (36 illnesses/1,000 swimmers). Norovirus genogroup I (NoVGI) contributed most to risk, but adenoviruses raised risk above the threshold in the two most urban subwatersheds. The risk was greater in the dry compared to the rainy season, due largely to the greater frequency of NoVGI detection (100% versus 41%). Viral log10 reduction needed to ensure safe swimming conditions varied by subwatershed and season and was greatest in the dry season (3.8 to 4.1 dry; 2.7 to 3.2 rainy). QMRA that accounts for seasonal and local variability of water quality contributes to understanding the complex influences of hydrology, land use, and environment on human health risk in tropical coastal areas and can contribute to improved beach management. IMPORTANCE This holistic investigation of sanitary water quality at a Costa Rican beach assessed microbial source tracking (MST) marker genes, pathogens, and indicators of sewage. Such studies are still rare in tropical climates. Quantitative microbial risk assessment (QMRA) found that rivers impacting the beach consistently exceeded the U.S. EPA risk threshold for gastroenteritis of 36/1,000 swimmers. The study improves upon many QMRA studies by measuring specific pathogens, rather than relying on surrogates (indicator organisms or MST markers) or estimating pathogen concentrations from the literature. By analyzing microbial levels and estimating the risk of gastrointestinal illness in each river, we were able to discern differences in pathogen levels and human health risks even though all rivers were highly polluted by wastewater and were located less than 2.5 km from one another. This variability on a localized scale has not, to our knowledge, previously been demonstrated.

Diverse infective and lytic machineries identified in genome analysis of tailed coliphages against broad spectrum multidrug-resistant Escherichia coli.

The emergence of multidrug-resistant (MDR) E. coli with deleterious consequences to the health of humans and animals has been attributed to the inappropriate use of antibiotics. Without effective antimicrobials, the success of modern medicine in treating infections would be at an increased risk. Bacteriophages could be used as an alternative to antibiotics for controlling the dissemination of MDR bacteria. However, before their use, the bacteriophages have to be assessed for the safety aspect. In this study, three broad host range highly virulent coliphage genomes were sequenced, characterized for infective and lytic potential, and checked for the presence of virulence and resistance genes. The genome sequencing indicated that coliphages ϕEC-S-21 and ϕEC-OE-11 belonged to Myoviridae, whereas coliphage ϕEC-S-24 belonged to the Autographiviridae family derived from the Podoviridae family. The genome size of the three coliphages ranged between 24 and 145 kb, with G + C content ranging between 37 and 51%. Coding sequences (CDS) ranged between 30 and 251 amino acids. The CDS were annotated and the proteins were categorized into different modules, viz., phage structural proteins, proteins associated with DNA replication, DNA modification, bacterial cell lysis, phage packaging, and uncharacterized proteins. The presence of tRNAs was detected only in coliphage ϕEC-OE-11. All three coliphages possessed diverse infective and lytic mechanisms, viz., lytic murein transglycosylase, peptidoglycan transglycosylase, n-acetylmuramoyl-l-alanine amidase, and putative lysozyme. Furthermore, the three coliphage genomes showed neither the presence of antibiotic resistance genes nor virulence genes, which makes them desirable candidates for use in phage therapy-based applications.

Effects of temperature and light exposure on the decay characteristics of fecal indicators, norovirus, and Legionella in mesocosms simulating subarctic river water.

Knowledge of the decay characteristics of health-related microbes in surface waters is important for modeling the transportation of waterborne pathogens and for assessing their public health risks. Although water temperature and light exposure are major factors determining the decay characteristics of enteric microbes in surface waters, such effects have not been well studied in subarctic surface waters. This study comprehensively evaluated the effect of temperature and light on the decay characteristics of health-related microbes [Escherichia coli, enterococci, microbial source tracking markers (GenBac3 & HF183 assays), coliphages (F-specific and somatic), noroviruses GII and Legionella spp.] under simulated subarctic river water conditions. The experiments were conducted in four different laboratory settings (4 °C/dark, 15 °C/dark, 15 °C/light, and 22 °C/light). The T90 values (time required for a 90 % reduction in the population of a target) of all targets were higher under cold and dark (2.6-51.3 days depending upon targets) than under warm and light conditions (0.6-3.5 days). Under 4 °C/dark (simulated winter) water conditions, F-specific coliphages had 27.2 times higher, and coliform bacteria had 3.3 times higher T90 value than under 22 °C/light (simulated summer) water conditions. Bacterial molecular markers also displayed high variation in T90 values, with the greatest difference between 4 °C/dark and 22 °C/light recorded for HF183 DNA (20.6 times) and the lowest difference for EC23S857 RNA (6.6 times). E. coli, intestinal enterococci, and somatic coliphages were relatively more sensitive to light than water temperature, but F-specific coliphages, norovirus, and all bacterial rDNA and rRNA markers were relatively more sensitive to temperature than light exposure. Due to the slow microbial decay in winter under subarctic conditions, the microbial quality of river water might remain low for a long time after a sewage spill. This increased risk associated with fecal pollution during winter may deserve more attention, especially when river waters are used for drinking water production.

Comparison of methods for the enumeration of coliphages in 100 mL water samples.

In the last decade coliphages have been included in many water quality regulations as viral faecal indicators. However, the standardised methods used to detect and quantify coliphages differ in bacterial host strains, culture media and techniques. In this comparative study, 100 mL samples of mineral drinking water, river water and wastewater were analysed with International Organization for Standardization (ISO) standard methods, with United States-Environmental Protection Agency (U.S. EPA) based methods as well as commercial kits combining a single agar layer (SAL) assay with ISO bacterial host strains. The three methods gave similar counts (p-value>0.05) for somatic and total coliphages in the matrices with less than 100 PFU/100 mL, whereas for F-specific coliphages, the U.S. EPA method provided statistically significant lower numbers (p-value<0.05) than the other two protocols, possibly because it uses a different bacterial host strain (Escherichia coli HS (pFamp) R vs. the ISO strain Salmonella enterica serovar Typhimurium WG49). In samples with more than 100 PFU/100 mL, the ISO method yielded higher counts of somatic coliphages than the other two protocols (p-value<0.05). As the three methods provided similar results in clean water, the approach combining a SAL assay with the ISO bacterial host strain could be a useful option for coliphage analysis in this type of sample, as it does not require a concentration step.

Monitoring coliphages to reduce waterborne infectious disease transmission in the One Water framework.

Coastal waters, surface waters, and groundwater are impacted by wastewater and stormwater discharges, as well as agricultural flows containing animal waste and nutrients. A One Water approach posits that components of the water system have overlapping and interactive impacts on other aspects of the system, for which a comprehensive approach to water management is needed to further inform public health decisions. Current frameworks for monitoring wastewater effluent and recreational surface waters include the measurement of fecal indicator bacteria. Although viral pathogens are likely to be transported further and can survive longer than bacterial pathogens, virus monitoring is not required for recreational waters. A scientific consensus is emerging that the use of bacterial indicators alone does not account for nor represent the health risks associated with viral pathogens due to the differences in the fate and transport of bacterial versus viral pathogens in wastewater treatment, surface water, and groundwater. Furthermore, it is likely that the public health risk associated with these waterborne pathogens is variable and diverse. For example, under drought conditions, effluents of urban water systems can comprise most of the dry weather flow in downstream waters, which are often used as sources of drinking water. This de facto reuse could increase viral risk for the end users of this water. A One Water approach will aid in protecting the health of the public from waterborne pathogens, regardless of where those pathogens entered the water system. In this review, we assert that monitoring for fecal indicator viruses can complement the monitoring of bacterial indicators, thereby improving public health protections. Bacteriophages have the strongest research foundation and correlation with viral pathogens along with some prediction power for risk to human health. Methods for detecting and quantifying coliphages are briefly summarized, as are challenges in the implementation of testing. Key knowledge gaps and research priorities are discussed so that the potential value and limitations of coliphage monitoring can be better addressed and understood.

Modeling human pollution in water bodies using somatic coliphages and bacteriophages that infect Bacteroides thetaiotaomicron strain GA17.

The ability to detect human fecal pollution in water is of great importance when assessing the associated health risks. Many microbial source tracking (MST) markers have been proposed to determine the origin of fecal pollution, but their application remains challenging. A range of factors, not yet sufficiently analyzed, may affect MST markers in the environment, such as dilution and inactivation processes. In this work, a statistical framework based on Monte Carlo simulations and non-linear regression was used to develop a classification procedure for use in MST studies. The predictive model tested uses only two parameters: somatic coliphages (SOMCPH), as an index of general fecal pollution, and human host-specific bacteriophages that infect Bacteroides thetaiotaomicron strain GA17 (GA17PH). Taking into account bacteriophage dilution and differential inactivation, the threshold concentration of SOMCPH was calculated to be around 500 PFU/100 mL for a limit of detection of 10 PFU/100 mL. However, this threshold can be lowered by increasing the analyzed volume sample, which in turn lowers the limit of detection. The resulting model is sufficiently accurate for application in practical cases involving MST and could be easily used with markers other than those tested here.

Somatic coliphages are conservative indicators of SARS-CoV-2 inactivation during heat and alkaline pH treatments.

High concentrations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described in wastewater and sewage sludge. It raises the question of the security of land sludge disposal practices during a pandemic. This study aimed to compare SARS-CoV-2's resistance to the main inactivating factors in sludge treatments, pH and heat, to that of native wastewater somatic coliphages. The latest can be easily used as an indicator of treatment efficiency in the field. The effects of heat treatment and pH on the survival of SARS-CoV-2 and somatic coliphages were investigated in simple media. The T90 value (time required for a 90% reduction in the virus or a 1 × log10 decline) at 50 °C was about 4 min for infectious SARS-CoV-2, and around 133 min for infectious somatic coliphages, with no decrease in SARS-CoV-2 genome. For infectious SARS-CoV-2, a slight decrease (<1 log10 unit) was observed at pH 9 or 10 for 10 min; the decrease was over 5 log10 units at pH 11. However, both SARS-CoV-2 genome and infectious somatic coliphages decreased by less than 1 log10 unit at pH 12. All thermal or pH-based treatments that can remove or significantly reduce infectious somatic coliphages (>4 log10) can be considered efficient treatments for infectious SARS-CoV-2. We concluded that somatic coliphages can be considered highly conservative and easy to use indicators of the inactivation of SARS-CoV-2 during treatments based on heat and alkaline pH.

Development and evaluation of DEAE silica gel columns for simultaneous concentration of coliphages and rotavirus from natural water samples.

Enteric viruses are commonly present in water bodies in regions with poor sanitation. Although the occurrence of these viruses poses a health risk they are difficult to quantify due to their low concentration and they may remain undetected in the absence of adequate preconcentration. The present study reports the synthesis and utilization of DEAE silica gel (DSiG) as an adsorbent for virus concentration. Two coliphages, MS2 and SUSP2, and an enteric virus, rotavirus A (RVA) were chosen for examining the preconcentration efficiency of DSiG columns. Studies conducted at a low flow rate of 5 mL/min yielded good removal of viruses through adsorption. Studies at a higher flow rate of 50 mL/min followed by elution with optimized eluents yielded a high recovery of MS2 and RVA even when they were present at low concentration (0.01 copy/mL). The eluent Na(1.5 M)-Tw(2%)-G3X (glycine 3X broth, 1.5 M NaCl, 2% Tween, pH 10.2) showed maximum elution of RVA and MS2. Optimal SUSP2 recovery was observed on employing an eluent composed of 1.5 M NaCl, 3% Tween, 0.05 M KH2PO4 at pH 9.2. Subsequently, both the eluents were successively applied for elution of the adsorbed viruses. This method was applied for virus preconcentration from lake water in the monsoon and winter seasons. The DSiG column could achieve adequate preconcentration for all the three viruses, i.e., SUSP2, MS2, and RVA, even when they were present at very low concentration and the recovery achieved was comparable to that achieved with ultracentrifugation while the processing time required for handling large volumes of water was considerably lower.

Occurrence of male-specific and somatic coliphages and relationship with rainfall in privately-owned wells from peri­urban and rural households.

Privately-owned drinking water wells serving fewer than 25 people (private wells) are prevalent and understudied across most of the US. Private wells primarily serve rural households located outside of municipal drinking water and sewerage service coverage areas. These wells are not regulated by United States Environmental Protection Agency (EPA) under the Safe Drinking Water Act, are not regularly monitored by any public agency or utility, and generally do not undergo disinfection treatment. Coliphages are a group of viruses that infect coliform bacteria and are useful viral surrogates for fecal contamination in water systems in much the same way that fecal indicator bacteria (FIB), such as E. coli and to a lesser extent total coliforms, are used to quantify fecal contamination. Coliphages are approved by the EPA for regulatory monitoring in groundwater wells in the USA, but are not routinely used for this purpose. The present study characterizes the occurrence of male-specific and somatic coliphages, along with FIB, in private wells (n = 122) across two different counties in North Carolina. While occurrences of E. coli were rare and frequency of total coliform was generally low (~20%), male-specific and somatic coliphages were detectable in 66% and 54% of samples, respectively. Concentrations of male-specific coliphages were higher than somatics at each county and on a monthly basis. Rainfall appears to be partly influencing higher coliphage concentrations in December, January and February. This research underscores the need for increased surveillance in private wells and consideration of using coliphages in order to better characterize occurrence of fecal contamination at the time of sampling, especially during rainier months.

Monitoring of human enteric virus and coliphages throughout water reuse system of wastewater treatment plants to irrigation endpoint of leafy greens.

One solution to current water scarcity is the reuse of treated wastewater. Water reuse systems have to be examined as a whole, including the efficacy of water-reclamation treatments and the operation steps from the wastewater inlet into the WWTP to the irrigation endpoint, including the irrigated crop. In this study, the monitoring of human enteric viruses and coliphages were assessed in two water reused systems. The presence of hepatitis A virus (HAV) and human noroviruses genogroups I and II (GI and GII) were analyzed by real-time RT-PCR (RT-qPCR) in water (n = 475) and leafy green samples (n = 95). Total coliphages were analyzed by the double-layer agar plaque technique. The prevalence of HAV in water samples was very low (c.a. 2%), mostly linked to raw sewage, while for leafy green samples, none was positive for HAV. In leafy greens, prevalence of norovirus was low (less than 5-6%). The highest reductions for norovirus were observed in samples taken from the water reservoirs used by the growers near the growing field. The virus die-off during water storage due to solar radiation could be considered as an additional improvement. Reclamation treatments significantly reduced the prevalence and the counts of noroviruses GI and GII and coliphages in reclaimed water. However, the coliphage reductions (c.a. 5 log) do not comply with the specifications included in the new European regulation on reclaimed water (≥6.0 log). Correlations between noroviruses GI and GII and coliphages were found only in positive samples with high concentrations (>4.5 log PFU/100 mL). A high percentage of samples (20-25%) negative for total coliphages showed moderate norovirus counts (1-3 logs), indicating that coliphages are not the most suitable indicator for the possible presence of human enteric viruses.

Caracterização da exposição humana a lodo de esgoto sanitário na cadeia: tratamento, uso agrícola e consumo de hortaliças / Characterization of human exposure to sewage sludge in the chain: treatment, agricultural use and consumption of vegetables

RESUMO Objetivou-se caracterizar a exposição humana à infecção por bactérias e vírus via ingestão, considerando os cenários: tratamento do lodo (cenário 1); uso do lodo como adubo agrícola (cenário 2) e consumo de hortaliças adubadas com lodo (cenário 3). A exposição foi caracterizada pelas concentrações de Escherichia coli, vírus entéricos cultiváveis (VEC) e colifagos somáticos (CS) nas amostras: lodo de esgoto em tratamento (LET), água de lavagem das mãos (ALM) no tratamento e no plantio, mistura solo + lodo (MSL) e hortaliças alface e cenoura. No cenário 1, em torno de 50-60 dias, as amostras LET apresentaram umidade < 10% e padrão classe A. Nas amostras LET, concentrações de CS variaram entre 9,1 × 102 e 1,9 × 105 UFP.g-1 ST e de VEC entre 2,42 e 7,15 UFP.g-1 ST. No cenário 2, CS foram detectados em 72% das amostras MSL com concentrações entre 10 e 330 UFP.mL-1. Concentrações de E. coli ≤ 102 NMP.100 mL-1 foram detectadas nas amostras ALM-tratamento (63,7%). Para vírus, todas as ALM-tratamento foram negativas e 17,4% das ALM-plantio apresentaram VEC entre 3 e 409,2 UFP.mL-1. Todas as amostras de alface foram negativas para CS e VEC. Considerando-se as características dos cenários avaliados, trabalhadores do serviço de saneamento, trabalhadores rurais e consumidores de hortaliças estiveram expostos a baixas concentrações de E. coli e VEC. CS foram resistentes ao tratamento térmico e ao plantio, o que sugere seu potencial como indicadores de VEC.

ABSTRACT The aim of this study was to characterize human exposure to microbiological contamination (bacteria and viruses) via ingestion, considering the following scenarios: treatment of sludge (scenario 1); use of sludge as fertilizer (scenario 2) and consumption of vegetables fertilized with sludge (scenario 3). Exposure was characterized by the levels of Escherichia coli, cultivable enteric viruses (CEV) and somatic coliphages (SC) in the samples: sewage sludge under treatment (SST); handwashing water (HW) in treatment and planting; mix soil + sludge (MSL) and the vegetables lettuce and carrots. In scenario 1, SST samples presented < 10% humidity and class A pattern around 50-60 days; SC concentrations ranged from 9.1 × 102 to 1.9 × 105 PFU.g-1 TS, and CEV ranged from 2.42 to 7.15 PFU.g-1 TS. In scenario 2, CS were detected in 72% of HW samples, and concentrations ranged from 10 to 330 PFU.mL-1. E. coli concentrations ≤ 102 NMP.100 mL-1 were detected in HW-treatment samples (63.7%). Viruses were not detected in 100% of HW-treatments samples, and 17.4% of HW-plantings had CEV concentrations between 3 and 409.2 PFU.mL-1. All the vegetable samples were negative for SC and CEV. Considering the characteristics of the scenarios evaluated, sewage workers, rural workers and consumers of vegetables were exposed to low concentrations of E. coli and CEV. Somatic coliphages were resistant to heat treatment and planting, which supports their potential as indicators of CEV.