Assessing the prevalence of FRNA bacteriophages and their correlation with SARS-CoV-2 RNA in the wastewater of Mumbai city

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) was declared a global pandemic by the World Health Organization (WHO) on 11 March 2020 (WHO 2020). This enveloped virus with a single-stranded RNA genome belongs to the Coronaviridae family and is responsible for mild cough, and cold to acute respiratory syndromes. Although SARS-CoV-2 spreads mainly through the respiratory route, the virus can also replicate in the intestinal tract, shedding through feces (and possibly urine) to subsequently make its way to the wastewater, making them a potential reservoir of the virus by the infectivity of SARS-CoV-2 excreted through feces and their persistence in the dynamic and complex wastewater matrix route is yet to be conclusively established. The presence of increased levels of SARS-CoV-2 in the wastewater could also indirectly indicate the spread of virus infection within the community, especially in areas with high population density (Ahmed et al. 2020; Cerrada-Romero et al. 2022; Chu et al. 2022; Koirala et al. 2023; Termansen & Frische 2023; Gogoi et al. 2024).

Globally, scientists working in the field of public health have utilized wastewater-based epidemiology (WBE) as a tool to monitor levels of SARS-CoV-2 present in wastewater to understand the magnitude of the spread of active infection. Wastewater surveillances, related to SARS-CoV-2 undertaken worldwide, have reported the presence of the viral RNA in raw wastewater samples (Ahmed et al. 2020; Medema et al. 2020; Westhaus et al. 2021). While many studies have reported a reduction in the viral RNA copies to undetectable levels post-treatment of the wastewater, studies have also shown the presence of SARS-CoV-2 RNA in treated wastewater albeit at a very low concentration (Kang et al. 2020; Westhaus et al. 2021; Tandukar et al. 2022; Wang et al. 2022; Koirala et al. 2023; Gogoi et al. 2024). Tracking of this virus in pre- and post-treated wastewater could aid in monitoring the elimination of this virus during wastewater treatment (Ahmed et al. 2020; Medema et al. 2020; Westhaus et al. 2021). This could also help in avoiding any possible public health risks (as shown by the Quantitative Microbial Risk Assessment (QMRA)) reported by a study carried out in the USA, especially in the event of the virus-laden treated wastewater being discharged in freshwater systems (Burch et al. 2021). Studies carried out in the Middle East (Rashed et al. 2022) and Korea (Choo & Kim 2006) have used cell culture-based and quantitative PCR (qPCR)-based approaches for detecting human viruses. These studies show that both approaches are good indicators for assessing the possible health hazards of contaminated water.

Traditionally used bacterial pathogens as indicators for all groups of pathogens have proved to be poor indicators of viral contaminants (Arredondo-Hernandez et al. 2017; Farkas et al. 2020; Tandukar et al. 2020; Kapoor et al. 2021). Scientists have been working on suitable single indicator viruses for better representation of human enteric viruses in wastewater (Stachler et al. 2017; Tandukar et al. 2020, 2022). However, selecting an appropriate viral indicator may not be easy to establish due to variations in sewage treatment processes, geographical differences and largely the human population dynamics. Studies by Tandukar et al. (2020) showed that pepper mild mottle virus (PMMoV) and tobacco mosaic virus (TMV) are the most abundant and frequently detected viruses in sewage. They are distributed globally and more abundant in environmental samples than human enteric viruses, without any pronounced seasonal variation. In addition, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with the human fecal waste (Stachler et al. 2017). This bacteriophage infects the human gut bacterium Bacteroides intestinalis and is found in abundance in the human gut. Studies on the application of crAssphage in stool and environmental water samples and the subsequent development of qPCR assays for its detection are encouraging and worth noting (Bibby et al. 2016).

Despite the tremendous progress made in biological wastewater treatment and microbial ecology research including the availability of concentration, extraction, and precipitation protocols as well as molecular and metagenomic tools for easy detection of human pathogenic viruses, tracking these viruses either using cell culture or specific molecular-based methods, especially for routine monitoring, can be cost- and labor-intensive, particularly for low-resource settings (Arredondo-Hernandez et al. 2017; Runa et al. 2021; Shrestha et al. 2021). The recent COVID-19 pandemic showed that strategies, such as systematic planning and different types of detection methods for diagnosis, surveillance and effective COVID-19 management, can be scientifically balanced for the authenticity of testing and cost-effectiveness in low-income countries (Shrestha et al. 2021; Chong et al. 2023). Hence, exploring cost-effective alternative approaches may be required for monitoring human viral pathogens.

The presence of bacteriophages in wastewaters has been well documented and their role as potential indicators of human viruses transmitted through water and wastewater has also been explored (Lee et al. 2019b; Ballesté et al. 2021). Protocol for phage quantification in water and wastewater has been defined by both ISO and EPA since 2001, and they are part of several national frameworks, including India (US EPA 1601 2001, ISO 2003). These indicator bacteriophages include somatic coliphages that infect enteric bacteria and F+ coliphages that specifically infect the coliform bacteria possessing the F+ pili within the mammalian gut. F+ coliphages are classified into two families. The Inoviridae family consists of filamentous phages having a single-stranded DNA as its genome (M13, fd, and f1) (Székely & Breitbart 2016). The Leviviridae family consists of small, icosahedral phages with a single-stranded RNA genome (MS2, GA, Qβ, and SP). The group of FRNAPH in particular is of suitable interest based on the Microbial Source Tracking (MST) studies carried out for tracking fecal pollution in urban wastewater (Ogorzaly & Gantzer 2006; Lee et al. 2019b).

FRNAPH are small (20–30 nm diameter), non-enveloped bacteriophages with single-stranded RNA genome and infect Escherichia coli cells with the F pili. They have been classified into four groups (GI–GIV) based on their genomes, characteristic occurrence, resistance and stability against different water treatment methodologies (Schaper et al. 2002; Cole et al. 2003). FRNAPH GII and GIII are frequently found in human excreta, while GI and GIV are associated with animal excreta. In urban municipal wastewaters, FRNAPH GI, GII and GIII are predominantly detected while GIV is rarely detected (Cole et al. 2003; Ogorzaly & Gantzer 2006). FRNAPH GI is most resistant to various wastewater disinfection processes during wastewater treatment, followed by FRNAPH GII, GIII and GIV (Haramoto et al. 2015; Lee et al. 2019a). In different studies assessing the use of FRNAPH as an indicator for other RNA viruses (human noroviruses), FRNAPH GII was proposed as a potential indicator for human norovirus contamination in shellfish such as oysters and mussels (Hartard et al. 2018; Gyawali et al. 2021).

Apart from being a potential source of viral disease transmission due to the presence of different human viruses in wastewater, the recent SARS-CoV-2 pandemic has highlighted the need for routine monitoring of these wastewaters to ensure the effective elimination of the virus from the treated wastewater. To make holistic microbiological surveillance a routine, besides bacterial indicators, to assess the presence of viral population in raw and treated wastewater as well, there is a need to have a cost-effective, relatively simple yet robust alternative approach that can be implemented across laboratories. With this aim, and in line with global reports, the present study investigated the presence of F+ coliphages and FRNAPH group (I–IV) genomes in raw and treated wastewater from three WWTPs of Mumbai city. The prevalence patterns of these bacteriophages were subsequently correlated with the presence of SARS-CoV-2 genomes in these waters to provide evidence that FRNA bacteriophages can be used as indicators for assessing the elimination of SARS-CoV-2 during wastewater treatment.

MS2 bacteriophage ATCC 15597-B1 and its corresponding host (E. coli 15597), bacteriophage Qβ DSM 13768 and the corresponding host (E. coli DSM 5210) and bacteriophage Phi6 DSM 21518 and host (Pseudomonas syringae DSM 21482) were procured from American Type Culture Collection (ATCC) and German Collection of Microorganisms and Cell Cultures- Leibniz Institute (DSMZ), respectively, and propagated as per standard instructions of the supplier. Lysates of MS2 and Qβ (representative of FRNAPH groups I and III, respectively) were used as positive controls for one-step RT-PCR assays and also to establish the limit of detection. Bacteriophage Phi6 lysate was used as an internal process control to assess the recovery of the viruses from wastewater samples.

Raw (influent), secondary- and tertiary-treated wastewater (effluent) samples were collected by the grab sampling technique from three different WWTPs located in Mumbai. The technical specifications of the three WWTPs have been summarized in Table 1. A total of 162 wastewater samples comprising raw/influent wastewater (n=63), secondary-treated wastewater (n=63), and tertiary-treated wastewater (n=36) were collected from the three WWTPs from 7th April to 10th June 2021. 1,000 mL wastewater samples were collected in sterile polypropylene bottles and transported in an ice box (4–8 °C) to the laboratory and processed within 3 h of sample collection.

100 mL of collected wastewater sample was clarified by centrifugation at 4,000 × g for 20 min to remove coarse debris (Sapula et al. 2020). This supernatant was then transferred into a sterile screw cap glass bottle containing 8% (w/vol) Polyethylene glycol (PEG) (Sigma Aldrich, USA) and 0.2 M (w/v) Sodium chloride (Sigma Aldrich, USA). Bacteriophage phi6 (DSM 21518), spiked at 5.5 × 10⁵ pfu/100 mL of wastewater was used as an internal process control to assess the recovery of enveloped RNA viruses from wastewater samples. The samples were transferred to an orbital shaker incubator adjusted at 4 °C and 130 RPM for 2 h and then subsequently held overnight at 2–8 °C for virus precipitation (Flood et al. 2021). The samples were centrifuged at 10,000 × g for 30 mins at 4 °C. The concentrated virus pellet was suspended in 2 mL PBS (Thermofisher, USA) and stored at −80 °C for further analysis.

RNA extraction from the preserved viral concentrates was carried out using a QIA amp viral RNA extraction mini kit following the manufacturer's instructions (Qiagen, USA). The eluted RNA was aliquoted, stored at −80 °C and was used within 48 h.

The one-step RT-PCR method, as described by Friedman et al. (2009), was performed to detect the presence of various FRNA phage groups. 12.5 μL single-step PCR reaction mixture was prepared using the Superscript III one-step RT-qPCR kit (Thermofisher, USA) containing 6.25 μL of 2× RT buffer, 1 μL each of forward and reverse primers (Table 2) of respective FRNAPH genogroups and 0.25 μL of the SSIII enzyme mix to which 4 μL of extracted RNA was added. PCR was carried out in Eppendorf Nexus Gradient Thermal cycler (Eppendorf, USA) with the following cycling conditions: 50 °C for 30 min, 95 °C for 15 min followed by 40 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min with a final extension of 72 °C for 10 min. The amplicons were separated on a 2% agarose gel (Sigma Aldrich, USA) containing SYBR™ Safe (Thermofisher, USA). The gel was visualized in a gel doc XR imager (BIORAD, USA). The images were recorded using the quantity one software (v 4.69, BIORAD, USA) and the semi-quantitative densitometric analysis to estimate the FRNAPH RNA copies was carried out as per the software manufacturer's instructions. Based on the densitometric analysis, the RNA copies of FRNAPH GI and FRNAPH GIII were calculated per 100 mL of the wastewater sample and expressed as Log₁₀ FRNAPH GI or GIII RNA copies/100 mL of wastewater. Serially diluted RNA extracted from available FRNAPH positive controls MS-2 (group I) and Q-Beta (group III) were used as controls for semi-quantitation and to assess the limit of detection. The limit of detection was established as 100 gene copies of FRNAPH per 100 mL of wastewater, i.e. 2 Log₁₀ FRNAPH gene copies per 100 mL of wastewater.

The enumeration of F+ coliphages from the virus concentrates was carried out as per (US EPA 1601 2001) using E. coli F_amp HS ATCC 700891 as the host. 100 μL of the virus concentrate was serially diluted and spot-inoculated. FRNAPH MS2 and Qß were used as positive controls. All the plaque-forming units (pfu) were counted and expressed as Log₁₀ F+ coliphage pfu/100 mL of wastewater. The limit of detection for the assay was 1 coliphage/100 ml of wastewater.

The 162 samples collected in this study were also assessed for the presence of SARS-CoV-2 by qualitative and quantitative one-step RT-PCR tests recommended by the Indian Council for Medical Research (https://www.icmr.gov.in/pdf/covid/labs/2_SOP_for_Confirmatory_Assay_for_2019nCoV.pdf accessed on 19 June 2022). Qualitative detection of SARS-CoV-2 in the wastewater samples was performed using the TRUPCR SARS-CoV-2 RT- qPCR kit (V-3.2) (3B BlackBio Biotech India Ltd., India) and the quantitative tests were performed using primers and probes for N gene ORF1b-nsp14 and RdRp gene. Gene copies/reactions obtained from the assay were used to back-calculate gene copies/100 mL of wastewater samples. SARS-CoV-2 quantitative RNA reference standard (ATCC 3276SD) was used as a standard copy control to assess the limit of detection (Wani et al. 2023). The limit of detection for the assay was 300 copies/5 ul of RNA template, i.e. 300 gene copies of SARS-CoV-2 per 100 mL of wastewater (2.47 Log₁₀ gene copies of SARS-CoV-2 per 100 mL of wastewater).

Data were collected and compiled using an excel spreadsheet (MS Office 2016, Microsoft, USA). A pairwise correlation analysis was performed among the F+ coliphages, FRNAPH and SARS-CoV-2 data on IBM SPSS Statistics Software version 23 (IBM, USA). Pearson's correlation coefficient (r) was calculated and data were considered statistically significant for p value<0.05.

FRNAPH genome in wastewaters was detected in 130 samples out of a total of 162 samples (80.25%) tested using one-step RT-PCR. This genome was detected in 61 out of 63 (96.83%) raw, 52 out of 63 (82.54%) secondary-treated and 17 out of 36 (47.22%) tertiary-treated wastewater samples. While looking at the individual FRNAPH groups, FRNAPH GI was detected in 80.25% (130 out of 162) wastewater samples, FRNAPH GII was present in 38.88% (63 out of 162) samples, FRNAPH GIII was detected in 14.19% (23 out of 162) wastewater samples while only three samples (1.85%) were positive for FRNAPH GIV (Table 3).

FRNAPH in wastewater was represented by their occurrence in the raw wastewater across the three WWTPs. FRNAPH were detected in 61 of 63 samples (96.82%). FRNAPH GI was detected in 61 (n= 63, 96.82%) followed by FRNAPH GII, where 49 samples were positive (n= 63, 77.77%) in the raw wastewater samples. FRNAPH GIII was detected in 23 (n=63, 36.50%), while FRNAPH GIV was detected in only 03 (4.76%) raw wastewater samples.

Across various secondary and tertiary treatment stages, FRNAPH GI was detected in 52 (n=63, 80.95%) secondary-treated and 17 (n= 36, 47.22%) tertiary wastewater samples. This was followed by FRNAPH GII, which was detected in 22.22% (14 out of 63) of secondary-treated samples and was undetected in the tertiary-treated samples. FRNAPH GIII and GIV were not detected from secondary- and tertiary-treated wastewater samples. These data suggest that there is an abundance of F+ coliphage and FRNAPH in wastewaters and their varying degrees of incidence in raw, secondary- and tertiary-treated wastewater suggest differences in their susceptibility to various treatment processes. Taken together these data suggest a difference in the prevalence of F+ coliphages and various FRNA phage groups in raw and treated wastewater samples which could also be due to the methods of detection, i.e. molecular versus biological, leading to overestimation and inaccurate estimation of the treatment efficacy.

SARS-CoV-2 was also assessed in these samples using an RT-PCR kit establishing a limit of detection of 6 SARS-CoV-2 RNA copies/reaction. Using this kit, SARS-CoV-2 was detected in 51 (n= 162, 31.48%) wastewater samples and comprised 48 (n= 63, 76.2%) raw and three (n= 63, 4.8%) secondary-treated wastewater samples. All the 36 tertiary-treated samples were negative for SARS-CoV-2 (Wani et al. 2023).

Data on the semi-quantitative estimation of FRNAPH group RNA copies, F+ coliphages and SARS-COV-2 gene copies have been summarized in Table 4.

FRNAPH GI RNA copies/100 mL sewage were 0.5–1.0 Log₁₀ higher than FRNAPH GIII copies. On the other hand, post-treatment FRNAPH GIII RNA copies were reduced to undetectable levels (i.e. <100 copies/reaction) across all the three WWTPs, suggesting elimination of these bacteriophages post-wastewater treatment.

The F+ coliphage pfu (Log₁₀ mean ± SD) in the raw wastewater samples were lowest in WWTP-Z3 (1.76 ± 1.42), followed by WWTP-Z5 (2.93 ± 0.85) and WWTP-Z1 (3.54 ± 0.41). In WWTP- Z5, the F+ coliphages were detected only in the raw wastewater samples and not in secondary- and tertiary-treated wastewater samples, i.e. the counts were below the detection limit of the method.

The positive SARS-CoV-2 samples were quantified using the qRT-PCR method and the results have been summarized in Table 4. The SARS-CoV-2 numbers were lower in WWTP-Z1 than WWTP-Z3 and Z5. SARS-CoV-2 numbers were reduced to below the detection limit (300 copies/reaction) (Wani et al. 2023) in secondary- and tertiary-treated waters of WWTP-Z1 and WWTP-Z5; however, they survived in the secondary-treated waters of WWTP-Z3.

A pairwise correlation analysis revealed a significant correlation between the prevalence of SARS-CoV-2 and F+ coliphages at all three WWTPs (Table 5). When the correlation was assessed between the log SARS-CoV-2 gene copies (N, ORF1b-nsp14 and RdRp) and F+ coliphage pfu counts, a significant correlation was observed between the two in all the WWTPs (Table 6).

Treating domestic sewage and monitoring the performance of the treatment for effectiveness is important to ensure that the targeted chemical and microbial reductions are achieved before releasing the wastewater into the water bodies or for reuse. This specifically becomes important in the case of disease outbreaks such as the recent SARS-CoV-2 pandemic. Assessing the presence of SARS-CoV-2 using WBE would require suitable technical expertise and advanced laboratory infrastructure that may be limited in a low-resource setting. Alternative, suitable approaches may help in overcoming these limitations to routinely assess the presence of SARS-CoV-2 and other similar etiological agents. With this aim, a study was designed to understand the prevalence of FRNA phage groups (FRANPH I to IV) and F+ coliphages in the urban wastewater of Mumbai city using molecular and classical methods, respectively. The two sets of phages were assessed at different stages of treatment in three different WWTPs and were also correlated with the presence of SARS-CoV-2 using a pairwise correlation analysis.

Wastewater concentrated using the PEG/NaCl method (Flood et al. 2021) allowed adequate recoveries of both bacteriophages (Gyawali et al. 2021) and human viruses including SARS-CoV-2 (Medema et al. 2020). Grab sampling of waste waters during peak flow allowed the presence of the maximum number of bacteriophages and SARS-CoV-2 in the samples and also invalidated the absence of provisions of an auto-sampler across all three WWTPs. Viruses adhere to the suspended particulate matter and hence their recoveries from wastewater matrices can be a challenge. Studies have shown poor recoveries of viruses in the presence of suspended particles, compared to the clarified medium. This was reported by Sapula et al. (2020), wherein the addition of a preliminary centrifugation step before the concentration step using PEG/NaCl gave higher recoveries (45%) with better consistency using different qRT-PCR kits, as opposed to PEG/NaCl without the centrifugation step (2.00%) where the recoveries of SARS-CoV-2 were compromised due to the presence of PCR inhibitors in the separated solids of wastewater sample matrix. Thus, the clarification step in this study led to the removal of coarse debris and helped address the problem of accumulating PCR inhibitors in the final virus concentrate and this process could help successfully detect FRNAPH (96.82%), F+ coliphages (92.06%) and SARS-CoV-2 (76.19%) from a total of 63 raw samples.

Clarification of the wastewater by centrifugation can result in loss of the viral population as viruses tend to adsorb onto the suspended solids in the wastewater, thus the quantitative estimations may get impacted. Studies have attributed the differences in surface hydrophobicity of enveloped and non-enveloped viruses influencing their adsorption onto the suspended solids. Ye et al. (2016) reported higher adsorption of enveloped viruses (murine hepatitis virus and Phi6; 26 and 22%, respectively) onto the solids compared to the non-enveloped viruses (MS2 and T3) which had lower adsorption onto the solids (6% and <5%, respectively). Studies have also observed a higher tendency of FRNA phages to adsorb onto the suspended solids and hence lower recoveries in clarified wastewaters resulting in an average loss of approximately 20% (Martínez-Carreras et al. 2021). Thus, the recoveries reported here may not provide an absolute count of the population of FRNAPH, F+ coliphage and SARS-CoV-2 present in the wastewaters assessed in this study which may be one of the limitations. The bacteriophage phi-6 that was used as an internal control was added post the centrifugation step to assess the recovery of the viruses by the PEG/NaCl precipitation method. The observed recoveries (using both molecular and culture-based methods) were in the range of 11–56% which was in line with the Phi6 recoveries reported in the other studies, i.e. Flood et al. (2021) and Jarvie et al. (2023) [10–55%] and by Conway et al. (2023) [10–80%].

Detection of F+ coliphages and FRNAPH in raw wastewater across all three WWTPs mirrors the abundance of these bacteriophages in nature and their ability to survive in wastewater. F+ coliphages, belonging to different FRNA phage groups, are readily observed in wastewater from different sources such as wastewater originating from urban sources, farms and slaughter-houses (Cole et al. 2003; Ogorzaly & Gantzer 2006) and certain FRNA phage groups survive through the wastewater treatment processes (Cole et al. 2003; Haramoto et al. 2015). This characteristic of the FRNA phages can be further explored to evaluate their virus elimination from the wastewater during the treatment process.

This study is the first to report the data on the prevalence of the FRNAPH groups from Indian wastewater. FRNAPH genome was detected in 82.25% of wastewater samples and mainly present in raw (46.92%) wastewater samples followed by secondary (40.00%) and tertiary (13.08%) wastewater. Detection of all four groups of FRNAPH across different treatment stages of wastewater shows that the selected primers (Friedman et al. 2009) demonstrated specificity with no cross-reactivity among the groups which also limited false-positive data.

Low detection of FRNAPH GIV with consistent presence of FRNAPH GI in raw, secondary- and tertiary-treated wastewaters across all three WWTPs in our study correlates with the reported findings of FRNAPH GIV and GI in the raw wastewaters (Cole et al. 2003; Ogorzaly & Gantzer 2006). The presence of FRNAPH GIII in only raw wastewaters and not in the secondary-treated or tertiary-treated wastewaters may have been due to its susceptibility to the physical and chemical conditions encountered during the wastewater treatment process (Schaper et al. 2002) while the presence of FRNAPH GII in raw and secondary-treated wastewaters may indicate the resistance of the group to these conditions in the WWTP. The prevalence of F+ coliphages in the different stages of treatment of wastewaters was similar to prevalence of the FRNAPH GII and GIII RNAs in three WWTPs. The detection of these bacteriophages in its live biological form testifies to the impact of treatment on the virus particle, in its ability to survive, infect and replicate. The absence of FRNAPH GII and GIII and F+ coliphages in tertiary-treated waters and their subsequent elimination may suggest the efficacy of the chemical disinfection carried out at two WWTPs (chlorination at WWTP-Z1 and ozonation at WWTP-Z5), by the specific chemicals exerting its effect on the capsid and the genome of these phages (Brié et al. 2018; Gomes et al. 2019). The settling and the clarification step post-aeration in sequential batch reactors followed by chlorination at WWTP-Z1 and the use of a Rotating media Bio Reactor to separate the sludge biofilm from the wastewater followed by ozonation at WWTP-Z5 may have additionally aided the elimination of FRNAPH GII, GIII and F+ coliphage from the wastewates. The elimination of F+ coliphage from secondary- and tertiary-treated wastewater in WWTP Z5 may also infer that the treatment process followed by ozonation may be a better suitable elimination strategy.

On the other hand, the detection of FRNAPH GI consisting of bacteriophages like MS2 in 40% of the tertiary-treated samples confirms the resistance of these groups to the various disinfection processes which has also been reported earlier (Schaper et al. 2002). Also, resistance of this group of phages to chemical disinfection technologies used in WWTP-Z1 (Chlorination) and WWTP-Z5 (Ozonation) is also well reported (Fang et al. 2014; Lee et al. 2019a, 2019b). Our findings corroborate with the available literature and suggest that FRNAPH GI phage (e.g. MS-2 like) is consistently persistent across all the three stages of treatment in the three WWTPs. Thus, the elimination of this FRNA phage group from the wastewaters will positively imply the elimination of different groups of microorganisms that may have lesser or similar persistence patterns as FRNAPH GI. The proposal of the utility of FRNAPH GI as a tool to monitor the efficiency of wastewater treatment in virus inactivation is in line with the studies presented in the systematic review by Amarasiri et al. (2017).

Wastewater samples collected in this study were also assessed for the presence of SARS-CoV-2 and the complete data and the detailed analysis of the same have been published elsewhere (Wani et al. 2023). The presence of SARS-CoV-2 in the raw wastewater samples and its absence in the secondary- and tertiary-treated wastewater samples suggested that the virus has a similar elimination pattern to that of F+ coliphages and FRNAPH GII and GIII. Therefore, we explored these trends further using a pairwise correlation analysis to assess the potential of these bacteriophages to be used as indicators for the elimination of SARS-CoV-2. SARS-CoV-2 data showed a significant correlation with FRNAPH GII and GIII (qualitative data) and F+ coliphages (qualitative and quantitative data) as well. Like SARS-CoV-2, FRNAPH GII and FRNAPH GIII (a subgroup belonging to the F+ coliphages) are predominantly known to be present in the excrement of human origin (Cole et al. 2003; Ogorzaly & Gantzer 2006). A previous study reported from France has also indicated that FRNAPH GII may be a suitable indicator to assess the presence of SARS-CoV-2 in the wastewater (Serra-Compte et al. 2021) and this FRNA phage group has also been under investigation as an indicator for norovirus; a non-enveloped RNA virus (Hartard et al. 2018; Lowther et al. 2019).

We also carried out semi-quantitative densitometry to estimate the FRNAPH RNA copy numbers in the positive samples to assess the correlation between FRNAPH groups and the quantitative SARS-COV-2 gene copies. This technique allows for a rough estimation of FRNAPH RNA copies when used with its respective copy controls and has been previously utilized in resource-limited settings in veterinary- (Antiabong et al. 2016) and wastewater-based studies (Siagian et al. 2020). Based on ready availability, only the RNA copies of FRNAPH GI (MS-2) and FRNAPH GIII (Qβ) could be estimated.

A pairwise correlation revealed no statistically significant correlation between FRNAPH GI and SARS-CoV-2. This observation was in line with the study by Sala-Comorera et al. (2021) which also indicated that FRNAPH GI (MS2- like phages) persists longer than SARS-CoV-2 in the wastewater and may limit their use in assessing the elimination of SARS-CoV-2 in aquatic environments (Sala-Comorera et al. 2021). We noted that FRNAPH GIII RNA copies followed a similar prevalence pattern to that of SARS-CoV-2 in all the three WWTPs and a significant correlation was also observed between the two groups. To the best of our knowledge, no published data on the correlation between FRNAPH GIII and SARS-CoV-2 have been reported so far and may be a new finding in this study. Thus, taken together, the correlation data suggest that F+ coliphages and the two FRNA phage groups (FRNAPH GII and GIII) could be explored further as potential indicators to assess the presence of SARS-CoV-2 in wastewater-based studies. Thus, extensive studies including lab-scale spiking studies, assessing the effect of seasonal variations on the prevalence of FRNA bacteriophages and SARS-CoV-2 would be an immediate requirement, given that former are a group of non-enveloped viruses as against enveloped SARS-CoV-2 and the two also have differences in their sizes.

Given the difficulties in establishing a sophisticated virology set-up for studying animal viruses, performing the culture method (US EPA 1601 2001) to detect F+ coliphages would be a relatively simpler approach. When co-relating F+ coliphages with FRNA bacteriophages it has been previously reported that 90–95% of the F+ coliphages detected using the bacterial hosts by ISO-10705-1 or the US EPA-1601 methods are FRNA phages (Debartolomeist & Cabelli 1991; Jebri et al. 2019), thereby eliminating the requirement of RNase treatment step (used to differentiate FDNA from FRNA phage) from the methodology. Removal of the RNase treatment would also reduce the cost of performing this method by approximately 10–15% (Jebri et al. 2019). Hence, establishing the use of F+ coliphages for monitoring viral elimination during the wastewater treatment, could be a significant benefit in a limited resource set-up. Some challenges envisaged in the integration of FRNAPH monitoring into the existing public health surveillance would be, the requirement of an upgradation of the laboratory infrastructure and trained manpower at various WWTP sites which would require policy-based intervention. Simultaneously, it will be important to initiate the monitoring of the FRNA bacteriophage in the treated and the untreated wastewater using a specific host as per USEPA 1601 (2001); which is currently is not a strictly implemented policy.

Approaches, such as change in policy, regulatory enforcement, strategic planning by the authorities and cost-effective techniques that balance culture-based and molecular methods, may be effective in implementing routine monitoring of not just SARS-CoV-2 but also other viral pathogens for early warning signals and managing viral infections in the population. Data of FRNA prevalence combined with that on the prevalence of other human viruses and environmental factors can subsequently be used to develop a robust model for predicting the survival of pathogenic viruses post-wastewater treatment.

This study undertaken in Mumbai was the first to generate data on the prevalence of FRNA phage groups (GI to GIV) in urban wastewater of an Indian mega city and observed the prevalence of F+ coliphage, FRNAPH GI, GII and GIII in abundance in raw wastewater. FRNAPH GIV had low incidence in raw wastewater. FRNAPH GIII was present in raw wastewater; FRNAPH GII was present in raw and secondary-treated wastewaters while FRNAPH GI was consistently present in both raw and treated wastewater. Significant correlation between F+ coliphages, FRNAPH GII and GIII with SARS-COV-2 suggests their potential to be studied further for the elimination of human viruses like SARS-CoV-2 during wastewater treatment. Also, FRNAPH GI phages (as highlighted in previously prescribed studies) may be used to monitor the viral reductions and evaluate the biological performance of the wastewater treatment process. These observations can also be validated by conducting similar studies on other human viruses of concern in the wastewater treatment and effluent discharge processes.

This study was funded by the Indo-US Science and Technology Forum (IUSSTF) [Ref No: IUSSTF/VN-COVID/081/2020]. We sincerely thank the authorities of Municipal Corporation of Greater Mumbai (MCGM) as well as the technical team working at all the WWTPs for their assistance. We would like to extend our thanks to Dr Nishita D'Souza, MSU, USA for providing the training on the concentration of viruses. We thank Mr Mayur Shelar and Mr Shivam Nikam for helping with sample collection. We appreciate the assistance of all the BRC staff in this work.

N.D., Z.B., J.R., and S.S. conceptualized the study. D.D., H.W., and S.M. coordinated sample collection and generation of the data. D.D. and N.D. critically analyzed the data. S.M. prepared bacteriophage and host stock cultures. D.D. drafted the manuscript, H.W. and S.M. edited the manuscript draft and N.D. critically reviewed the manuscript. All authors read and approved the manuscript.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.