Wastewater treatment and reclamation systems using membrane technologies such as membrane bioreactor (MBR) are becoming increasingly employed in mitigating the shortage of clean water sources [1
]. However, usage of reclaimed wastewater may increase the exposure risk of humans to pathogenic microorganisms, if the wastewater treatment system is not capable of effectively removing these microorganisms [3
Indicator microorganisms are available to assess and guarantee the microbiological quality of water, because the presence of such indicator microorganisms points to the possible existence of similar pathogens and represents a failure in the treatment system which affects the final effluent [4
]. Fecal indicator bacteria (FIB) (total coliforms, fecal coliforms, Escherichia coli
, fecal streptococci and spores of sulphite-reducing clostridia
) have been used to assess the water quality and treatment performance for decades [5
]. However, FIB could not identify the sources of the contamination and there are many complexities related to the extra-enteric ecology of FIBs including environmental persistence and particle association [6
]. It is unclear how to estimate the contribution of different sources of feces when sources are mixed, which would further hinder the water quality management and health risk evaluation.
As an alternative, specific microbial source-tracking (MST) markers have been suggested as suitable indicators for evaluating the contamination and treatment performance. crAssphage is one of the suggested human specific contamination markers and found to have geographical and temporal differences [8
which contain host-specific microorganisms are also suggested as alternative indicators [10
]. Some studies have suggested waterborne gastroenteritis viruses as MST markers due to their prevalence in host feces and stringent host specificity [11
] which provides information on pathogen status that is not provided by indicator bacteria and bacteriophages [6
Even though usage of gastroenteritis viruses as MST markers in evaluating the fecal contamination has been documented, studies in evaluating the suitability of viral indicators to evaluate treatment unit performance are scarce. Especially, in systems like MBR which use size separation as one major virus removal mechanism, microbes with larger diameter sizes (>1 µm), including bacteria (FIB included) and protozoa, can be effectively removed with microfiltration while viral pathogens which are smaller than bacterial pathogens (< 100 nm) could easily pass through the MBR facilities if they are not attached to larger particles, and are much more environmentally resistant than the indicator bacteria [15
]. It is further evinced by the absence of correlations between FIB and enteric viruses in MBR effluents [19
]. Therefore, it is necessary to identify waterborne gastroenteritis viruses circulating in membrane-based wastewater reclamation systems which can be used as indicators to evaluate the treatment unit performance to ensure that reclaimed wastewater is microbiologically safe and not posing infectious risks.
In this study, phylogenetic analysis of multiple waterborne gastroenteritis viruses was applied to estimate contamination sources in a wastewater treatment and reclamation system with a hybrid process of anaerobic/anoxic/oxic (A2
O) combined with a membrane bioreactor (MBR). Noroviruses and rotaviruses were selected because they were of great significance in disease transmission [21
]. The extent of the viral pollution in the system was evaluated by the frequency of positive samples for viral genes from the wastewater samples. The genetic diversity of these viruses was determined by nucleotide sequencing and phylogenetic analysis in order to identify prevalent genotypes and their persistence, which were the underlying evidence for estimating the contamination sources of these gastroenteritis viruses. To the best of our knowledge, a comprehensive study of this kind, by the inclusion of human viruses in wastewater, has rarely before been performed in northwestern China.
We confirmed the presence of human noroviruses (GI and GII) and rotaviruses in the influent wastewater, fine screen effluent, A2
O treatment effluent, and the lake water receiving the wastewater effluents. The lower virus detection rate observed after the A2
O treatment process compared to raw sewage may be owing to the attachment to wastewater solids and the presence of antiviral components in the activated sludge [24
]. Gastroenteritis viruses were not detectable in the samples of MBR effluent after free chlorine disinfection. MBR combined with chlorine treatment may have significantly contributed to the reduction of virus particles, or at least the MBR with chlorine treatment may decrease the virus quantity to a very low extent which was below the detection limit [28
]. However, 54% of the lake water samples were positive for viruses, implying that the MBR effluent disinfected with free chlorine may not be the source of virus contamination in the lake water.
The results of phylogenetic analysis revealed that the artificial lake was contaminated by multiple human viruses. In this case, sewage pipe leakage and overflows are not likely to cause such contamination due to the adequately designed capacity and the proper maintenance of the water distribution system. Secondary contamination of lake water may occur from unidentified nonpoint sources. As the lakes are open water bodies in the local water system, they were vulnerable to contamination generating from natural processes (such as surface runoff, water air transfer and wild animals) or human activities [29
]. As non-point sources of gastroenteritis viruses, rain water inflow and aerosol blowing into the lakes may be considered as possible reasons. Furthermore, it would be of particular concern because the microbial aerosols containing viral particles could be formed during water reclamation, and exposure to reclaimed water can pose a potential health risk [31
]. On the other hand, onshore winds around 4 m/s can contain 5.3 ± 1.2 × 104
of viruses [32
]. These results underscore the possible impact of viral exposure by reclaimed water consumption, and by being exposed to winds containing aerosols and suggests that the control of non-point viral sources, and storage and safe use of reclaimed water should be the focus of wide attention.
The sequence diversity of human noroviruses, especially for the capsid region, from environmental samples has been reported in several studies [33
]. The isolation of both GI and GII strains in this study would indicate the co-existence of extensive recessive infections for both genogroups which may not be included and documented in previous epidemiological surveys. However, results similar to our present study have been obtained in some environmental studies [36
]. Thus it might indicate a distinct genogroup prevalent bias between clinical samples and environmental samples [38
]. It has been demonstrated that the viral loads of GI in fecal samples was reported less than one percent of that of GII and GI is generally more resistant to wastewater treatment and disinfection than GII [38
], suggesting the differences in environmental occurrence and persistence of GI and GII strains [40
]. Although there was no documentation about the viral infection in the studying area, the report of Xi’an Center for Disease Control and Prevention showed that HuNoV GII was more prevalent than HuNoV GI in clinical samples (data not shown). However, human norovirus strains detected in wastewater may reflect more accurate actual circulation among population rather than clinical survey, because wastewater receive viruses shed from patients with both symptomatic and asymptomatic infections. Thus, the findings indicate the possibility that norovirus GI strains might be more widely spread among humans than previously thought. Other explanations such as seasonal or geographic variation in viral RNA levels could not be excluded either.
Number of rotavirus A genotypes (G1, G2, G3, and G9) were detected during the sampling period and G9 was predominant. Previous surveys confirmed the circulation of multiple rotavirus A genotypes in the same area in the same year [22
] even though the predominant rotavirus genotype varied in different geographical regions [41
]. The phylogenetic analysis of rotavirus also suggests that the viruses detected in this study might originate from infant, children or healthy carriers, and thus their contamination sources or transport routes could be different from those of fecal indicators usually originating from adults.
It has been recognized that enteric viruses are more stable than indicator bacteria in water and sewage, constituting not only a potential hazard but also a good tracer for fecal pollution source tracking [14
]. Wastewater treatment plants (WWTPs) have played an important role in microbiological reduction, minimizing the risks associated with pathogen circulation into the environment [3
]. However, little is known about the comparative persistence or survival of source-specific markers and strains, and the available data for markers ranging from E. coli
and phage markers indicate strongly that survival is not proportional [46
]. The general trend is that the dominance of environmental strains differ from strains in the host. Due to the inherent difficulty in finding a correlation between environmental contamination and cases of infection, microbiological monitoring of the environment might be more helpful for source tracking and water safety control rather than risk assessment [47
]. In addition, limited waterborne viral outbreaks usually occurred at distance from the original source of contamination. This study provides novel evidence of the prevalence and genetic diversity of waterborne gastroenteritis viruses and the potential of human noroviruses for microbial source tracking due to its host-specificity and higher sensitivity of (semi-)nested PCR (detection about 100
]. Attention should be paid to the emerging health threat due to the different predominant types of the targeting viruses observed in the study.
Furthermore, although direct sequencing analysis with well-purified PCR amplicons could be useful for providing information on viral identification in wastewater [37
], the potential that the results may have a bias in interpreting the genetic diversity of the viral types might not be neglected. This might be resulted from the inhibition effect as the recovery rate of water concentration [3
] and the affinity selection of PCR reaction might be type and strain different for viruses [51
]. This more comprehensive analysis of the relative abundance and occurrence of viruses in wastewaters may allow for the development of more conservative viral tracers and complementary indicators to further ensure the microbial safety of wastewater reclamation systems.