Occurrence of enteric viruses in reclaimed and surface irrigation water ...

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Occurrence of enteric viruses in reclaimed and surface irrigation water: relationship with microbiological and physicochemical indicators pez-Ga lvez1, P. Truchado1, G. Sa nchez2,3, R. Aznar2,3, M.I. Gil1 and A. Allende1 F. Lo 1 Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Murcia, Spain 2 Department of Biotechnology, IATA-CSIC, Valencia, Spain 3 Department of Microbiology and Ecology, University of Valencia, Valencia, Spain

Keywords Escherichia coli, faecal contamination, fresh produce safety, Norovirus, reclaimed water, wastewater. Correspondence Ana Allende, Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Campus Universitario de Espinardo, 25, 30100 Murcia, Spain. E-mail: [email protected] 2016/1031: received 13 May 2016, revised 21 June 2016 and accepted 23 June 2016 doi:10.1111/jam.13224

Abstract Aims: To assess the prevalence of enteric viruses in different irrigation water sources and in the irrigated produce, and the possible links with microbiological and physicochemical water characteristics. Methods and Results: The prevalence and levels of Escherichia coli, Norovirus (NoV) genogroup I (GI) and II (GII), as well as Hepatitis A virus were assessed in three types of water: surface water (surface-W), reclaimed water subjected to secondary treatment (secondary-W) and reclaimed water subjected to tertiary treatment (tertiary-W), as well as in zucchini irrigated with these irrigation water sources. Chemical oxygen demand (COD), turbidity, total suspended solids, alkalinity and maximum filterable volume (MFV) were also measured in the water. Higher prevalence of NoV in secondary-W (GI 100%, GII 556%) and tertiary-W (GI 917%, GII 667%) compared with surface-W (GI 584%, GII 222%) was observed. Nov GI showed positive correlation with E. coli (Spearman’s correlation coefficient = 068, P < 001), and with some physicochemical parameters such as COD (052, P < 001), turbidity (052, P < 001) and MFV (054, P < 001). Escherichia coli and enteric viruses were not detected in zucchini. Conclusion: There is a potential risk of contamination of crops with NoV when reclaimed water is used for irrigation. Significance and Impact of the Study: Increase the knowledge on the prevalence of enteric viruses in different irrigation water sources, and its consequences for fresh produce safety.

Introduction Fresh produce is an important vector for the transmission of foodborne viruses like Norovirus (NoV) and Hepatitis A virus (HAV) to the human host (Li et al. 2015). Irrigation water is one of the vehicles identified for the contamination of produce during primary production with pathogenic micro-organisms including viruses (Du Plessis et al. 2015). Enteric viruses can get on edible parts of the plants not only by direct contact with irrigation water but also by internalizing through the roots (DiCaprio et al. 2012; Hirneisen et al. 2012; Hirneisen and Kniel 2013). 1180

Wastewater is used, mainly in arid and semi-arid regions of the world, to increase water resources available for irrigation (Pachepsky et al. 2011). Foodborne outbreaks caused by enteric viruses linked to irrigation of fresh produce with sewage have been reported (Li et al. 2015). Noroviruses are frequently present in influent and effluent wastewater (Hewitt et al. 2011). Wastewater treatment is able to reduce but not remove or inactivate viruses completely (Rusi~ nol et al. 2015). At the wastewater treatment plants, some reduction in the amount of viruses is achieved by activated sludge systems (Hata et al. 2013) and tertiary treatments (Montemayor et al.

Journal of Applied Microbiology 121, 1180--1188 © 2016 The Society for Applied Microbiology

pez-G F. Lo alvez et al.

2008). Common wastewater treatment systems can eliminate 20–80% of enteric viruses while stronger wastewater treatments can achieve bigger reductions (Ottoson et al. 2006; Barker-Reid et al. 2010; Mok et al. 2014). Although enteric viruses are more common in reclaimed water, they can also be found in surface water (Perez-Sautu et al. 2012). Mitigation strategies can be applied to lower the risk associated with the presence of enteric viruses in irrigation water, for example, the use of good quality water in the last period before harvest combined with less risky irrigation systems such as drip instead of overhead irrigation (Hamilton et al. 2006). Microbiological safety of environmental waters and treated wastewater is currently assessed through the use of bacterial indicators. Although some of them perform adequately for pathogenic bacteria, they are not always good predictors of the fate of viral pathogens in irrigation water and wastewater (Harwood et al. 2005; Perez-Sautu et al. 2012). Low numbers of bacterial indicators do not guarantee the absence of enteric viruses (Payment and Locas 2011; Vieira et al. 2012). Escherichia coli has been described as a suitable indicator to predict prevalence of pathogenic bacteria in irrigation water (Castro-Iba~ nez et al. 2015; Ceuppens et al. 2015). However, several authors highlighted that it would not be a good indicator of the wastewater treatment efficacy in viruses’ reduction, mostly because it is less resistant than human pathogenic viruses to such treatments (Nasser et al. 1993; Steele and Odumeru 2004; Hijnen et al. 2006; Gerba et al. 2013). According to this, La Rosa et al. (2010) reported that after treatment in a wastewater treatment plant the removal of 989, 784 and 626% of E. coli, Norovirus genogroup I (NoV GI) and II (NoV GII), respectively, was achieved. More research is needed to estimate the prevalence of enteric viruses and their relationship with indicator micro-organisms such as E. coli in reclaimed water used for irrigation. In the present study, occurrence and levels of enteric viruses (NoV GI, NoV GII and HAV) and E. coli in reclaimed and surface water were assessed. Additionally, different water physicochemical parameters were measured. Potential links between presence and levels of enteric viruses and E. coli, and between enteric viruses and physicochemical parameters were evaluated. Materials and methods Experimental design Irrigation water samples were taken at the irrigation head of a commercial greenhouse hydroponic system located in Balsicas (Murcia, Spain) from December 2014 until March 2015. Data acquisition including climatological

Viruses in irrigation water and fresh produce

data, types of irrigation water, organization of the irrigation head and fertilization was as previously reported by Lopez-Galvez et al. (2014). Two samples (2 l) of each type of water were taken two times per week during 9 weeks from 12th January to 11th March, 2015, for a total of 108 samples in 18 sampling dates. Three different types of water were analysed: tertiary treatment effluent from the urban wastewater treatment plant of RoldanBalsicas (tertiary-W), secondary treatment effluent from the same treatment plant (secondary-W) and surface water from an Irrigation Community (surface-W). Secondary treatment consisted in activated sludge systems followed by coagulation–flocculation. Tertiary treatment effluent was obtained after the secondary reclaimed water was sand-filtrated followed by UV disinfection. Zucchini plants (Cucurbita pepo L.) were grown using coconut fibre (Pelemix, Alhama de Murcia, Spain) as the substrate for the hydroponic growing system. Three replicates of 15 zucchini plants for a total of 45 plants per irrigation water were grown. Physicochemical analysis of water Alkalinity, chemical oxygen demand (COD), total suspended solids (TSS), turbidity and maximum filterable volume (MFV) were measured weekly in the three types of irrigation water. Alkalinity was determined by potentiometric titration until pH 43 with HCl and a pH meter (Crison, Barcelona, Spain). COD was determined by the standard photometric method (APHA 1998) using a photometer (Spectroquant NOVA 60; Merck, Darmstadt, Germany). Total suspended solids (TSS) were evaluated using the APHA Standard method 2540 D (APHA 1998). Turbidity was measured by means of the turbidimeter Turbiquant 3000 IR (Merck). MFV was measured as the volume that pass through a 045-lm membrane filter in 1 min applying a suction of 80 kPa by means of a vacuum pump. Microbiological analysis of water The presence of E. coli, NoV and HAV was assessed in different irrigation water types and zucchini. Assessment of E. coli in water samples was made by plating filtered and nonfiltered samples in Chromocult coliform agar (Merck). Plates were incubated for 24 h at 37°C before interpretation of the results. Dark blue-violet colonies were considered positives for E. coli. Recovery of viruses from water was performed as in Helmi et al. (2011). MgCl2 was added to each water sample to a concentration of 005 mol dm 3, adjusting the pH to 35 with HCl 1 mol dm 3. Samples of 200 ml were filtered through 045-lm membrane filters (Sartorius, G€ ottingen,


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Germany). Filters were transferred to sterile 15-ml tubes. Elution buffer (5 ml) made of 1% beef extract, 3% Tween-80 and 05 mol dm 3 NaCl were added and pH adjusted to 95 using NaOH 10 mol dm 3. Tubes were shaken for 1 min in a vortex, kept for 4 min in an ultrasonic bath and shaken again in a horizontal orbital shaker at 250 rev min 1 for 10 min and then, pH was adjusted to 7 using HCl (10%). After that, samples were kept at 70°C until analysis. For RNA extraction, 120 ll of the concentrated sample was mixed with 600 ll of lysis buffer from the NucleoSpinâ RNA virus kit (MachereyNagel, D€ uren, Germany) and subjected to pulse-vortexing for 1 min. Afterwards, the homogenate was centrifuged for 5 min at 10 000 g to remove the debris. The supernatant was subsequently processed using the NucleoSpinâ RNA virus kit according to the manufacturer’s instructions. Finally, RNA was resuspended in 50 ll of RNasefree H2O. Mengovirus (kindly provided by Dr Albert Bosch, University of Barcelona, Spain) was added in each sample before RNA extraction to monitor the efficiency of nucleic acid extraction from water samples, as described elsewhere (Costafreda et al. 2006). RNA samples were analysed in duplicate by RT-qPCR using the RNA UltraSense One-Step quantitative RT-PCR system (Invitrogen, Waltham, MA) and the LightCycler 480 instrument (Roche Diagnostics, Basel, Switzerland). The set of primers and probes for NoV GI and GII, HAV and mengovirus were previously validated (Loisy et al. 2005; Costafreda et al. 2006; Da Silva et al. 2007) and included in the ISO procedures for detection of NoV and HAV in selected foodstuff (CEN/ISO TS 15216-1 and CEN/ISO TS 15216-2). Microbiological analysis of zucchini Zucchini samples were harvested weekly from January to March 2015 for a total of n = 135 samples taken at nine different sampling dates. At each sampling date, five samples were harvested from plants irrigated with each irrigation water type. Zucchini grade US No. 1 or grade US No 2 (USDA 1984), were randomly picked from the plants and transferred aseptically into sterile bags (2–3 fruits per sample). For E. coli enumeration, zucchini samples (35 g) were diluted 1 : 5 in buffered peptone water (20 g l 1) and homogenized in a stomacher for 1 min. Homogenate was plated in Chromocult coliform agar and incubated at 37°C for 24 h before interpretation of results. For viruses, zucchini samples (25 g) were prepared as described by Coudray et al. (2013). Samples were placed in sterile stomacher bags containing a filter compartment and were soaked in 40 ml of elution buffer (Tris–HCl 100 mmol dm 3, glycine 50 mmol dm 3, 1% beef extract (TGBE), pH 95) covering the sample, for 1182

20 min at room temperature with constant shaking. The fluid was recovered from the filter compartment of the bag and was centrifuged at 8500 g for 30 min at 4°C to pellet the particles. Then, pH of the decanted supernatant was adjusted to 72 02 using 5 mol dm 3 HCl. Polyethylene glycol (PEG) 6000 (Acros Organics, Geel, Belgium) and NaCl (Panreac Quımica, Barcelona, Spain) were added to the neutralized supernatant to a concentration of 10% (w/v) and 03 mol dm 3 respectively. Subsequently, fluid was incubated for 2 h at 4°C. Viruses were concentrated by centrifugation of the solution at 8500 g for 30 min at 4°C. The supernatant was discarded and additional centrifugation was carried out at 8500 g for 5 min at 4°C to compact the pellet. Pellet was finally resuspended in 3 ml of phosphate-buffered saline and stored at 75°C before RNA extraction. RNA extraction and RT-qPCR reactions from zucchini samples were performed as described for water samples. Statistical analysis Microbial populations were log-transformed and introduced in Excel spreadsheet (Microsoft Excel, 2010). Results were compiled and graphs were made using SIGMA PLOT 12.0 Systat Software, Inc. (Addilink Software Scientific, S.L. Barcelona, Spain). IBM SPSS STATISTICS 20 (IBM, Armonk, NY, USA) was used for statistical analysis. The Kolmogorov–Smirnov test and Levene’s test were used to assess normality and equality of variance respectively. When normality could be assumed, ANOVA was performed, with Tukey’s HSD or Dunnett’s as post hoc tests depending on the homogeneity of the variances. When data were not following a normal distribution, nonparametric tests (Kruskal–Wallis and Wilcoxon) were applied. To compare prevalence of positive samples for viruses between different types of irrigation water, the chi-square (v2) test was used. Spearman’s rho correlation coefficient was calculated to evaluate correlation between microbiological data, between physicochemical data and also between microbiological and physicochemical data. For correlation calculations only positive samples (i.e. with numbers above the detection limit) were included. Binary logistic regression was used to evaluate the presence of enteric virus and E. coli and physicochemical parameters. Results Physicochemical characteristics of irrigation water Physicochemical characteristics of the different types of irrigation water are shown in Table 1. Alkalinity was significantly (P < 005) lower in tertiary-W compared with secondary-W and surface-W. COD was significantly




Table 1 Physicochemical characteristics of irrigation water Water type

Alkalinity (mg CaCO3 l 1)

COD (mg O2 l 1)

TSS (mg l 1)

Turbidity (NTU)

MFV (ml)

Surface-W Secondary-W Tertiary-W

579 155a 571 74a 352 203b

273 139a 708 268b 705 369b

64 29 73 43 90 30ns

16 15 31 20 48 35ns

303 204 254 157 206 87ns

COD, chemical oxygen demand; TSS, total suspended solids; MFV, maximum filterable volume; ns, no significant differences. Different letters in the same column indicate statistically significant differences (P < 005).

Microbiological characteristics of irrigation water and zucchini Among the water samples, 981% were positive for E. coli (Table 2; limit of detection 1 CFU per 100 ml). There were only two negative samples which corresponded to surface-W. Levels in surface-W were in the range of 0– 2 log CFU per 100 ml, while they were approximately between 1 and 4 log CFU per 100 ml in secondary-W and tertiary-W. Escherichia coli levels in surface-W were significantly lower (P < 001) than those detected in secondary and tertiary water types. When enteric viruses were examined, a higher % of water samples were positive for the presence of NoV GI (824%) compared with NoV GII (481%) and HAV (83%) (Table 1). Of them, a higher prevalence was detected in secondary-W and tertiary-W, with almost all the samples positive, while only about half of the surfaceW samples were positive (Table 2). Additionally, NoV GI levels in the positive samples of secondary-W and tertiaryW samples were significantly (P < 001) higher than the levels in surface-W ones. Regarding NoV GII, 52 of 108 water samples were positive (481%). As in the case of NoV GI, a higher amount of samples was positive for NoV GII in secondary-W and tertiary-W than in surface-W. However, there were no significant differences (P > 001)

between water types regarding NoV GII concentration in positive samples. A significantly (P < 001) higher concentration of NoV GI compared with NoV GII was observed in secondary-W and tertiary-W but there were no significant differences (P > 001) between the level of NoV GI and GII in surface-W. Only nine water samples were positive for HAV, among which only three of them were within the quantification range (ranging between 263 and 388 copies per 100 ml), for which calculation of the median, minimum and maximum values was not possible (Table 2). Regarding the influence of outdoor temperature, the prevalence of NoV GI and GII was higher in samples taken in periods with higher temperature (Fig. 1).

100 80 Prevalence (%)

(P < 005) higher in tertiary-W and secondary-W compared with surface-W. There were no significant differences between the three types of water in TSS content, MFV and turbidity.

60 40 20 0

7–8

9–10

11–12

13–14

Mean outside temperature (°C) Figure 1 Prevalence of NoV GI ( ), NoV GII ( ) and HAV ( ) in water samples grouped in function of the mean outside temperature.

Table 2 Prevalence of Escherichia coli and enteric viruses in irrigation water. Norovirus genogroup I (NoV GI) and II (NoV GII), and Hepatitis A virus (HAV) Surface water

Secondary water

Tertiary water

Micro-organism

%*

Median

Minimum

Maximum

%

Median

Minimum

Maximum

%

Median

Minimum

Maximum

NoV GI NoV GII HAV† E. coli

584 222 83 944

409 333

254 299

491 423

534 361

360 293

624 417

431 309

615 462

0

192

291

159

405

917 667 55 100

523 361

097

100 556 55 100

234

165

392

*Proportion of positive samples from each type of water (n = 36). Data are expressed as log CFU per 100 ml for E. coli, and log genome copies per 100 ml for enteric viruses. †The number of positive samples obtained for HAV was very low (4/108) to calculate median, minimum and maximum values.


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However, such differences were only statistically significant (P < 005) for NoV GII in samples taken when mean outside temperature was 7–8°C or 9–10°C compared with those taken at 11–12°C. In the case of NoV GI and HAV, prevalence was higher in secondary-W compared with tertiary-W, while in the case of NoV GII prevalence was higher in tertiary-W compared with secondary-W. As it could be expected, all the analysed samples were positive for the presence of mengovirus used as extraction process control. The estimated limit of detection for enteric viruses in water was 3 9 102 genome copies per 100 ml of water. No positive samples were recorded for the presence of E. coli and enteric viruses in zucchini (n = 135) with an estimated limit of detection of 07 log CFU g 1 and 16 genome copies per g of zucchini, respectively (data not shown). As in the case of water, all samples were positive for the presence of mengovirus (used as extraction process control). Correlation between enteric viruses, Escherichia coli and physicochemical parameters in surface and reclaimed irrigation water NoV GI showed moderate correlation with COD, turbidity and MFV (Table 3). Binary logistic regression results

suggested a significant but weak link between the presence of NoV GII and higher COD and turbidity levels in water samples (Table 4). Correlations between HAV and physicochemical parameters were not calculated, due to the low number of positive samples for HAV. However, a good correlation was observed between E. coli and COD and E. coli and turbidity. NoV GI enumeration data showed a significantly strong (068) correlation with E. coli (Table 3 and Fig. 2). Additionally, binary logistic regression showed a relationship between higher E. coli levels and presence of NoV GI in the samples (Table 4). Regarding the relationship of E. coli counts with NoV GII counts, no significant correlation was observed. Binary logistic regression results showed a weak significant association of higher E. coli counts with the presence of NoV GII in the samples (Table 4). There was no significant correlation between the NoV GI and GII (P > 001) (Table 3). Additionally, according to the results of binary logistic regression, the presence of a NoV genogroup, that is, GI was not linked to the concentration of the other genogroup, that is, GII in the same sample. Correlations between HAV and NoV, and between HAV and E. coli, were not calculated, due to the low number of positive samples for HAV. Furthermore, no relationship was detected between HAV

Table 3 Spearman’s correlation coefficients between microbiological and physicochemical parameters in irrigation water NoV GI NoV GI Coefficient N NoV GII Coefficient N E. coli Coefficient N Alkalinity Coefficient N COD Coefficient N TSS Coefficient N Turbidity Coefficient N MFV Coefficient N

NoV GII

Escherichia coli

Alkalinity

COD

TSS

Turbidity

MFV

024 50

068** 89

038 44

052** 44

034 44

052** 44

054** 44

031 51

019 24

017 24

041 24

038 24

013 24

003 54

055** 54

023 54

047** 54

029 54

039** 54

024 54

034 54

004 54

063** 54

080** 54

006 54

089** 54

009 54

024 50 068** 89

031 51

038 44

019 24

003 54

052** 44

017 24

055** 54

039** 54

034 44

041 24

023 54

024 54

063** 54

052** 44

038 24

047** 54

034 54

080** 54

089** 54

054** 44

013 24

029 54

004 54

006 54

009 54

004 54 004 54

NoV GI, Norovirus genogroup I; NoV GII, Norovirus genogroup II; COD, chemical oxygen demand; TSS, total suspended solids; MFV, maximum filterable volume. **P < 001.

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Table 4 Results of binary logistic regression between the presence of enteric viruses vs Escherichia coli and enteric viruses vs physicochemical data NoV GI

E. coli Alkalinity COD TSS Turbidity MFV

NoV GII

HAV

Nagelkerke’s r2

Odds ratio

Nagelkerke’s r2

Odds ratio

Nagelkerke’s r2

Odds ratio

044** 000 024 008 021 002

596 099 104 107 161 098

018** 002 023** 011 024** 001

217 099 103 117 138 101

001 002 009 001 000 002

085 102 097 105 096 097

NoV GI (log genome copies per 100 ml)

NoV GI, Norovirus genogroup I; NoV GII, Norovirus genogroup II; HAV, Hepatitis A virus; COD, chemical oxygen demand; TSS, total suspended solids; MFV, maximum filterable volume. **P < 001.

7 6 5 4 3 2 1 0

0

1

2 3 4 E. coli (log CFU per 100 ml)

5

Figure 2 Scattergram of Norovirus GI vs Escherichia coli. Data from samples positive for NoV GI from the three types of irrigation water are included in the graph (n = 89). Limit of detection for NoV GI in water samples (25 log genome copies per 100 ml).

presence and E. coli or NoV concentration by binary logistic regression. Alkalinity showed a significant and moderate negative correlation with COD ( 039) (Table 3). As expected, COD showed a significant and strong correlation with TSS (063) and a very strong correlation with turbidity (080). TSS showed a very strong (089) significant correlation with turbidity and MFV was not significantly correlated with any of the physicochemical parameters analysed. Discussion Reclaimed water is expected to bear higher concentrations of enteric viruses than other irrigation water sources (Suslow 2010). In the study by Perez-Sautu et al. (2012), and in accordance with our results, higher concentration of

NoV was detected in reclaimed water when compared with surface water. Accordingly, Kitajima et al. (2014) detected NoV GI and GII in most of the effluent samples from an activated sludge system. However, the efficacy of the water treatments applied in wastewater treatment plants varies. This fact has been highlighted by La Rosa et al. (2010) who reported lower prevalence of NoV GI and GII in effluents from a wastewater treatment plant than that observed in our study. Several studies also reported higher occurrence of NoV GI compared to GII in treated wastewater effluents (Da Silva et al. 2007; La Rosa et al. 2010), while others reported higher prevalence of NoV GII (Sales-Ortells et al. 2014). Regarding surface water and similarly to our results, Liang et al. (2015) reported the presence of NoV GI and GII in 20 and 48%, respectively, while E. coli was present in 100% of the samples. In agreement with our results, Katayama et al. (2008) and Sales-Ortells et al. (2014) showed that there were no significant differences in NoV concentration between secondary and tertiary treatment effluents. This lack of difference is attributable to the fact that RT-PCR detects both infective and inactivated viruses (Sobsey et al. 1998). Hewitt et al. (2011) observed similar amount of NoV GI and GII in influent and effluent waters from wastewater treatment plants using RT-qPCR for detection. Furthermore, it has been reported that viruses are very resistant to UV light (Gerba and Smith 2005; Hijnen et al. 2006), which was the disinfection treatment applied to tertiary water in our study. However, although enteric viruses are more resistant than E. coli to the tertiary treatment, some inactivation has been already reported using the selected water treatments (Montemayor et al. 2008). In the present study, infectivity of viruses was not assessed and this is a limitation when trying to get conclusions because the number of infective virus particles may be lower than the virus levels detected by RT-qPCR (Lodder and De Roda Husman 2005).


1185



Several authors associate the decay or survival of virus with different physicochemical characteristics in irrigation water (John and Rose 2005; Gerba 2007). Correlation between COD levels and levels of NoV GI could be explained by the fact that the survival of viruses in aquatic environments is enhanced by the presence of organic matter (Gerba 2007). Higher water turbidity could be linked to higher protection of micro-organisms from environmental stress (Harris et al. 2012). In the present study, a correlation between the levels of NoV GI and water turbidity was observed. In the case of NoV GII, no correlation with turbidity was observed, maybe due to the lower prevalence compared with NoV GI. In contrast, Lee et al. (2013) and H€ orman et al. (2004) found no correlation between turbidity and the presence of enteric viruses in aquatic environments. Solid particles can protect pathogenic micro-organisms, and their presence can be indicated by a lower maximum filterable volume because of the presence of particles bigger than 045 lm. Association between MFV and NoV GI was established in our study. A trend to a higher prevalence of NoV in periods with higher environmental temperature was observed. However, it has been reported that virus decay rate in aquatic environments is normally decreased at lower temperatures (Gerba 2007). Ngazoa et al. (2008) observed a slow NoV genome degradation in water at low temperatures. Regarding the correlation between enteric viruses and E. coli, a higher probability of E. coli occurrence has been shown in surface water samples positive for viral or protozoan pathogens (Haramoto et al. 2012). Accordingly, correlation between E. coli and NoV was observed in surface water (H€ orman et al. 2004), although in well water E. coli was a poor indicator for the presence of enteric viruses detected by RT-PCR and a good indicator of the absence of culturable enteroviruses (Borchardt et al. 2004). In our study, a positive correlation was found between the levels of E. coli levels and NoV GI. However, the number of positive samples for NoV GII and HAV was low and therefore, no correlations with E. coli or physicochemical parameters could be found. Previous studies showed no correlation between faecal coliforms and HAV occurrence has been observed in urban river water (Jiang and Chu 2004) while high levels of E. coli in environmental samples positive for HAV have been reported (Kittigul et al. 2006). In summary, available information regarding relationships between enteric viruses and E. coli, and between enteric viruses and physicochemical parameters is inconsistent. Results obtained in thisstudy also show inconsistencies, but in general, correlations between physical parameters as well as E. coli levels and enteric viruses were observed for NoV GI, which showed the higher 1186

prevalence and levels in the tested samples, while in the cases of NoV GII and HAV, and probably due to a low prevalence, no correlation was observed. Therefore, suitability of E. coli or physicochemical parameters as enteric viruses indicators is still not consistent. Due to the lack of good reliable indicators, Dalla Vecchia et al. (2015) suggested that searching for the target viruses is the only way to check faecal contamination by viruses. Lack of detection of E. coli and enteric viruses in zucchini, despite their presence in irrigation water, could be due to several factors such as the limited sampling size (n = 135) and the type of irrigation system used (drip irrigation) which prevents the direct contact of water with the fruit among others. However, reclaimed water showed higher occurrence of enteric viruses, and therefore, a higher potential risk of crop contamination. The risk linked to the use of this water for irrigation could be minimized by selecting the type of crop and the irrigation system, and also using mitigation strategies such as submitting the water to additional treatments. Acknowledgements Authors are thankful for the financial support from MINECO (Project AGL2013-48529-R and INIA RTA2014-00024-C04-03), and to the project IRIS ‘Intelligent Reclaim Irrigation System’ (EUROSTAR EUS20110154). Francisco L opez Galvez is indebted to CSIC and ESF (JAE-Doc-2011 contract co-funded by the European Social Fund). Gloria Sanchez is supported by the ‘Ram on y Cajal’ Young Investigator Program. Conflict of Interest No conflict of interest declared.

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