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

Abstract

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 F_amp 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.

Introduction

Enteric bacteria have conventionally been used as indicators of faecal contamination in source, drinking and recreational waters [1]. Tracking the sources of contamination is critical for pollution prevention and pathogen control measures, however, bacteria may not be adequate indicator of non-bacterial pathogens. Hence, the need for a reliable indicator of pathogens other than bacteria mainly the enteric viruses viz. rotavirus, hepatitis A virus, norovirus and coxsackie virus is widely recognized [2]. Somatic and F-specific coliphages have been proposed as indicators for viral pathogens in water by the U.S Environmental Protection Agency [3] and the Indian standard IS 10500: 2012. Of these, the F + RNA coliphages are of specific importance as they are often used as identifiers for source of faecal contamination in ground and surface waters. F + coliphages have been proposed by the USEPA as surrogate for human viruses, also because they have similar or higher resistance to decay in the environment as compared to human enteric viruses including the poliovirus [4]. Of the F + coliphage, MS2, which is a F-RNA phage, has been proposed as a tool for on-site validation of wastewater treatment processes [5], due to its similarity to human enteroviruses and ease of detection. Once prevalence of these recommended bacteriophages is confirmed, they can be scheduled for routine monitoring of water/wastewater. Major advantages of using bacteriophage over the actual viral pathogen would be in terms of their ease of detection, cost and time. Bacteriophages can be cultivated in most labs having basic microbiology setting, unlike animal viruses whose detection requires cultivation in cell lines and or using molecular methods like qPCR which are costlier and time consuming. Additionally, bacteriophages are non-pathogenic, thus safety concerns while working with them are less as against working with viral pathogens.

The Indian standard for drinking water (IS 10500: 2012) mandates absence of MS2, an F-RNA group I phage, in 1L of water. However, there is a gap in the availability of baseline data on prevalence and concentrations of F + coliphages in water and wastewater system in Indian setting. As these bacteriophages have been reported globally to be prevalent in high numbers in human and animal faeces, it is important to establish a baseline of F + coliphages in wastewaters of inland for justifying their inclusion in the national regulatory framework.

Methods for detection of F + coliphages are well established. Two standards methods commonly used for direct quantitative plaque assays are; ISO 10705–1 and USEPA 1601 [6]. However, as the study of this nature was being conducted for the first time in this part of the country, the two methodologies were required to be compared to see if there are any significant differences in bacteriophage recovery with respect to geographic location and wastewater treatment, and also ease of method execution for routine purposes.

Hence, the present study is conceptualized with two objectives, i.e., (i) to estimate the concentrations of F + coliphages in raw and treated effluents in Mumbai, and (ii) to perform an assessment of the methodologies used for the enumeration of phages. This will help in assessing the usefulness of F + coliphages as a wastewater surveillance tool in tracing possible drinking water contamination by wastewater, and evaluation of the two methodologies to detect and enumerate F + coliphages in raw as well as treated wastewaters, that can be recommended for routine monitoring purposes.

Materials & Methods

Study Sites and Sample Collection

Wastewater samples were collected by grab sampling technique from three different sewage treatment plants (STPs) WWTP-Z1 (n = 42), WWTP-Z3 (n = 54) and WWTP- Z5 (n = 75) of Mumbai City mainly receiving domestic sewage. The technical details of the three treatment plants are presented in Supplementary Table S1.

A total of 171 wastewater samples were collected from the three plants during the study period from 7th April to 10th June 2021 on weekdays (Monday to Thursday/Friday) between 9:00 AM–10:00 AM, which comprised of raw/influent wastewater (n = 66), secondary treated wastewater (n = 66) and tertiary treated wastewaters (n = 39) [from WWTP-Z1 and WWTP-Z5]. 1000 mL wastewater samples were collected in sterile plastic polypropylene bottles and transported at 2–8˚C to the laboratory and processed within 3 h of sample collection.

Bacteriophage Concentration by PEG/NaCl Method

Samples were processed on the same day within 3 h of collection. Coliphages were concentrated by PEG/NaCl precipitation as described by Wani H. et al., 2023[7]. 100 ml of wastewater sample was centrifuged at 4000 × g for 20 min (Eppendorf Centrifuge 5804R) to remove debris and was added to sterile glass bottles containing 8.0 g (8%, w/v) of Polyethylene glycol (PEG) 8000 [Sigma Aldrich, USA] and 1.17 g (0.2 M, w/v) of sodium chloride (NaCl) [Sigma Aldrich, USA], mixed and transferred to orbital shaker incubator (130 RPM) at 4 °C for two hours for dissolution of PEG and NaCl. Subsequently, the bottles were transferred to the refrigerator (2–8 °C), kept overnight for phage precipitation. The phage precipitates were then centrifuged at 10000 × g for 30 min at 4 °C to obtain the viral pellet. The supernatant was discarded and final pellet obtained was re-suspended in 2 mL of phosphate buffered saline (PBS) [Thermofisher, USA] and vortexed to mix the pellet. The concentrate was then filtered through 0.22μ filter to remove any organisms or debris and the filtrate stored at − 80 °C until further analysis.

Bacteriophages and Bacterial Host

Standard strains of F + coliphages MS2 (ATCC 15597-B1) and Qβ (DSM 13768) were used as reference phages in all the assays. These phages were cultured overnight at 37 °C in their respective hosts, E. coli 15597 and E. coli DSM 5217 for phage MS2 and Qβ respectively. The bacteriophage lysates were clarified by centrifugation at 10,000 × g for 10 min, filtered through a 0.22µ syringe filter and titered by plaque assay using the double agar overlay method to obtain plaque forming units (PFU) per mL, aliquoted and stored at 4 °C until further use.

For detection and enumeration of F + coliphages, bacterial host strains Salmonella enterica serovar typhimurium WG49 (ATCC 700730) and E. coli F_amp HS (ATCC 700891) were procured from American Type Culture Collection. The Salmonella enterica serovar typhimurium WG49 strain was propagated in Tryptic soy broth (TSB) [HiMedia, India] supplemented with Kanamycin (20 µg/mL) and Nalidixic acid (100 µg/mL) [HiMedia, India], while the medium for E. coli F_amp HS was supplemented with Ampicillin and Streptomycin (150 µg/mL each) [HiMedia, India]. For use in the assays, the two bacterial hosts were cultured overnight in TSB supplemented with the appropriate antibiotic at 37 °C and used as inoculum to prepare log-phase cultures.

Enumeration of F + Coliphages from the Concentrates

The detection and enumeration of F + coliphages was carried out by the double agar overlay technique using two FRNA bacteriophage host as per the two standard, ISO 10705–1 (1995) and USEPA 1601(2001) methods with minor modifications. The respective bacterial hosts were cultured to a mid-log phase, O. D₆₀₀ 0.2 to 0.4 in growth media supplemented with antibiotics as mentioned in Sect. "Bacteriophages and Bacterial Host". 100 µL of freshly prepared host culture and 100 µL of virus concentrate were added to Tryptone soya broth (TSB) supplemented with 0.5% agar [HiMedia, India] and the required antibiotics. This was mixed well and poured on Tryptone soya agar (TSA) [HiMedia, India] bottom agar plates, mixed and allowed to solidify. The plates were then inverted and incubated at 37 °C for 24 h. The assay was repeated twice and the data represents mean ± SD of two independent experiments. F-specific coliphages, phage MS2 and Qß were used as positive controls.

Data Analysis

One-way Analysis of variance (ANOVA) and Independent samples t-test was applied to determine differences in F + coliphage levels at different treatment stages. The differences in F + coliphages detection using the two methods were tested for significance by Paired t-test. All statistical analysis was performed using the SPSS software version 23.0.

Results and Discussion

The present study was conceptualized to draw an understanding on the occurrence and level of F + coliphages in the Indian wastewater treatment systems in line with observations from other countries, as no local data is available in public domain in this regard. Thus, the current study is first of its kind reported from Mumbai, that assessed active F + coliphages in the wastewaters. Methods for F + coliphage detection were also evaluated.

Detection and Enumeration of F + Coliphages in Wastewaters

A total of 171 wastewater samples were collected from three different WWTPs in Mumbai and occurrence of F-specific bacteriophages was assessed using both ISO 10705–1 and USEPA 1601 methods. Grab samples during the morning peak hour wastewater outflow were analysed, due to the lack of provision of composite sampling, using auto-sampler at all three WWTPs. Using the host S. typhimurium WG49 (ISO 10705–1), F + coliphages were detected in 59 samples (34.50%) as against E. coli F_amp HS ATCC 700891 (USEPA 1601) host that could capture the phages in 78 (45.16%) samples. Distribution trends of the F + coliphages at different stages were similar for the two hosts, with maximum numbers obtained from the raw sewage, followed by secondary treated and least numbers recovered from the tertiary treated samples (Table 1).

Table 1.

Detection and Enumeration of F + coliphages in untreated and treated wastewater samples

WWTP	F + coliphage Detection and Enumeration host	n	Untreated sewage		Secondary treated sewage		Tertiary treated sewage
			No. of positives (%)	Mean Log10/100 mL sewage (SD)	No. of positives (%)	Mean Log10/100 mL sewage (SD)	No. of positives (%)	Mean Log10/100 mL sewage (SD)
WWTP-Z1	S. typhimurium WG49	14	13 (92.9)	2.94 (0.90)	5 (35.7)	0.77 (1.09)	1 (7.1%)	0.16 (0.61)
	E. coli Famp HS		14 (100)	3.54 (0.41)	10 (71.4)	1.63 (1.29)	1 (7.1%)	0.16 (0.61)
WWTP-Z3	S. typhimurium WG49	27	16 (59.3)	1.27 (1.29)	3 (11.1)	0.27 (0.80)	NA
	E. coli Famp HS		23 (85.2)	1.76 (1.42)	6 (22.2)	0.57 (1.08)	NA
WWT P-Z5	S. typhimurium WG49	25	21 (84)	2.62 (1.20)	0 (0)	–	0 (0%)	–
	E. coli Famp HS		24 (96)	3.00 (0.77)	0 (0)	–	0 (0%)	–

n number of samples tested, SD standard deviation, % = percentage, NA not applicable

Conducting the F + coliphage evaluation at different stages of wastewater treatment would help us know the baseline level of F + coliphages and their fate during wastewater treatment using current technologies.

Their detection would also serve another important purpose as surrogate to evaluate wastewater treatment efficiency and their effectiveness against human enteric viruses like hepatitis A, E, astrovirus, entero and noroviruses [8].

Comparison of F + Coliphage Levels at Different Stages of Wastewater Treatment

In WWTP Z1, coliphages were detected during all treatment stages viz. primary, secondary and tertiary, One-way ANOVA was performed and an overall statistically significant difference in F + coliphage counts at different treatment levels (F_2,39 = 10.15, p = 0.000) using Salmonella typhimurium WG49 and (F_2,39 = 6.51, p = 0.004) using E. coli Famp HS as hosts was found. Post-hoc comparison using Dunnett’s T3 revealed significant pairwise difference between mean F + coliphage counts between primary (M = 1.85E + 03, SD = 2122.11) with secondary (M = 64.28, SD = 115.87) and tertiary levels (M = 14.28, SD = 53.45) (p < 0.05), using Salmonella typhimurium WG49 as host. The mean F + coliphage counts differed significantly between secondary (M = 3.07, SD = 327.57) and tertiary treatment levels (M = 14.29, SD = 53.45) (p < 0.05), however, no difference was observed between primary and secondary treatment levels when E. coli F_amp HS was used as host. F + coliphage levels at WWTP Z3 and Z5 was compared by the independent samples t-test, since Z5 has only the primary and secondary treatment stages and for WWTP Z3, coliphages were detected only in the untreated samples and none in any of the treated samples. Here too, F + coliphage levels differed significantly between the raw and secondary treatment stages for WWTPs Z3 & Z5 (Salmonella typhimurium WG49, p = 0.009 and 0.000, respectively and for E. coli F_amp HS as host, p = 0.000 and 0.037 respectively). Taken together, these results indicate that the concentration of active F + coliphages significantly decreased during the treatment process and were efficiently eliminated from the wastewater post treatment. The log reduction trend observed in the current study was in line with that reported by Debartolomeis and Cabelli, 1991[9], wherein 1–2 log difference was reported between raw and secondary treated sewage (Fig. 1).

Fig. 1.

F-specific coliphage counts in the untreated and treated wastewater samples from the three WWTPs. Data represents Mean counts ± S.D

As the three WWTPs under study employ different technologies for wastewater treatment, review and correlation of the same with regards to coliphage reduction was done. WWTPs Z1 and Z5 are upgraded up to tertiary treatment level, whereas Z3 has facility only up to the secondary treatment level (Supplementary Table S1). A significant reduction in F + coliphage numbers at each treatment level were observed at each WWTP despite the difference in technology used.

In the Rotating Media Bioreactor (RMBR) technology used at WWTP-Z5; the rotating drums allow biofilm formation on the plastic media, which sloughs off once the thickness reaches a threshold and is collected as sludge. This technology has proven to be important in recent years for high load industrial wastewater treatment, pharmaceutical wastewater treatment and agricultural waste bioremediation [10]. This plant further uses ozonation for disinfection, which inactivates the residual microbes.

The Sequential Batch Reactor (SBR) technology for secondary wastewater treatment utilized at WWTP-Z1involves sequential treatment of wastewater in batches using aeration, sedimentation, and supernatant decantation, followed by chlorination at the tertiary level. The sedimentation step allows for biomass separation (as sludge), significantly reducing the number of coliphages in the suspended liquid phase. Coliphages too, like other viruses, adsorb onto surfaces of suspended solid particles present in the wastewater, thus resulting in their significant reduction during sedimentation and decantation step after secondary treatment [11].

Both the tertiary treatment technologies, chlorination and ozonation used at WWTP-Z1 and Z5 respectively, were found to be effective in reducing the number of F + coliphages significantly. Although the running cost involved for ozonation is higher as compared to chlorination, it is much safer than chlorination from an environmental perspective [12].

WWTP-Z3 caters to an overall larger volumes of wastewater per day (Supplementary Table S1) and uses aerated lagoons system for secondary treatment, wherein the reduction in F + coliphage counts from raw to secondary was found to be statistically significant, but lower as compared to the other two WWTPs (Fig. 1). Aerated lagoons efficiency in microbial reduction depends on a number of factors like temperature, air & wind and consistency of aerator performance resulting in variation in mixing and aeration, which can remarkably reduce the oxygen diffusion in water [13] thereby affecting its treatment efficacy. Gomila et al., 2008 [14] in Mallorca, Spain evaluated the effectiveness of different tertiary treatment technologies on bacterial and viral indicators, and demonstrated that lagoon system was effective in reducing the numbers of viruses, specifically the F + coliphages in addition to bacterial indicators from wastewaters.

The above observations, wherein differences in numbers of F + coliphages were found irrespective of the treatment technologies helps in establishing suitability of F + coliphage as a parameter for evaluating virus reduction, and routine monitoring of wastewater in the national regulatory framework. As the probability of wastewater contaminating drinking water always exists, selection of MS2, an important representative of the F + coliphage group by Bureau of Indian Standard as an indicator of pathogen virus in drinking water is also validated.

Comparison between the Two Standard Methods in F + Coliphage Enumeration from Wastewaters

As F + coliphage detection and enumeration study from wastewater was being done for the first time from this part of the country, both ISO & USEPA methods which recommends two different hosts were used to understand if there were any major or significant differences in target phage recovery and method execution.

Overall, the number of samples positive for F + coliphage and their counts obtained using the Salmonella WG49 strain was found to be significantly lower than those using the E. coli F _amp HS strain (Table 1). Further, when F + coliphage counts in raw (untreated) wastewaters were compared using a paired t-test, a statistically significant difference was observed in the F + coliphages detected using the two hosts, in wastewater samples from WWTP-Z1 (p = 0.047) and WWTP-Z3 (p = 0.001) but not in WWTP-Z5 (p = 0.332). A similar observation was made by Bonadonna et al., 1993 [15] when they compared the same Salmonella host with E. coli ATCC 9723 C, wherein the latter strain was found to support multiplication of a wide range of bacteriophages present in sewage. Though S. typhimurium WG49, a hybrid strain, developed specifically to plaque F + coliphages [16] is allowing the coliphages to grow, an E. coli host would be preferable, considering that E. coli is the native host for the coliphages [9]. Amongst the E.coli hosts, Debartolomeis and Cabelli, 1991 [9], have reported the plaquing efficiency of E. coli F_amp HS to be superior to E. coli K-12 Hfr strain further justifying suitability of this host. Besides the differences in the efficiency of the two bacterial host strains, another observation made in our study, also reported earlier by Jofre et al., 2016 [17], was that, E.coli F_amp HS strain is more stable than Salmonella typhimurium WG49 under the laboratory conditions, as WG49 strain easily lost the ability of detecting F + coliphages if not maintained in the required antibiotic (kanamycin and nalidixic acid) pressure. Based on the above observations, USEPA method using E. coli F_amp HS host is recommended for routine monitoring of F + coliphages in waste and drinking waters.

Thus, the study reported herein was a first of its kind to assess the presence of F + coliphages in the wastewater samples in three WWTPs across Mumbai city, India, using two bacterial host systems, including comparison of two standard methods, across different treatment technologies. Summing the major observations of the study, it can be concluded that F + coliphages can be used as a potential candidate to monitor the efficiency of wastewater treatment processes across different levels and can also be further explored as indicators for human RNA viruses in wastewater.

The study has estimated the total F + coliphage population in wastewater which can be further extended to individual FRNA sub-groups comprising; Group I, II, III and IV; of which FRNAPH-I and IV are excreted through animals and FRNAPH-II & III are excreted through human faeces [18].

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

ND, ZB and SS conceptualized the study, SM, DD and HW coordinated sample collection, SM and HW carried out sample processing, SM, DD and SS analysed the data, SM wrote the draft manuscript and DD and SS edited the draft manuscript, all authors have read and approved the manuscript.

Declarations

Conflict of interest

The authors declare they have no conflict of interest.