Towards the selection of the best discriminating parameters of

microbiological water quality: a case study of an urban recreational

water resource involving a dam complex in Córdoba, Argentina
J.V. Pavan1,3*, G. Masachessi1*, C.A. Mateos1, P.A. Barril1, V.E. Prez1, L.C. Martínez1, M.O.
Giordano1, L.J. Ferreyra1,3, M.B Isa1, A. Welter2, M. Martinez Wassaf2, V. Ré1-2 and S.V. Nates1#
1Virology Institute Dr J.M. Vanella, School of Medical Sciences, National University of Cordoba, Enfermera
Gordillo s/n, Ciudad Universitaria 5016 Córdoba, Argentina
2 Chemistry Faculty, Catholic University of Cordoba, Obispo Trejo 323, 5000 Córdoba, Argentina
3 Medical Bacteriology and Virology School of Medical Sciences, National University of Córdoba, Santa Rosa

1095, 5000 Córdoba. National University of La Rioja, Av Luis de la Fuente s/n, 5300 La Rioja, Argentina
(*) Both authors have contributed equally
(#) Corresponding author: e-mail snates@fcm.unc.edu.ar

Abstract

Data from a year-long monitoring campaign of a dam complex, located in the province of Córdoba, Argentina
were analyzed to characterize the best bacterial and viral groups that can reflect the microbiological water quality.
The approach has been to monitor fecal coliforms, E. coli and enterococci together with the virus genome
detection for rotavirus group A, astrovirus, norovirus, JC polyomavirus and picobirnavirus and infectious human
enterovirus. Our results support that the presence of viral genomes as well as an infectious virus in areas where
fecal contamination was not demonstrated by bacterial indicators suggests prolonged virus persistence in aquatic
environments and emphasizes the enteric virus groups as the most reliable for microbiological water quality
assessing. The combined detection of infectious human enterovirus and the genome of picobirnavirus could be
promising indicators of microbiological contamination derived from human and animal feces in the surface water.
In addition, they could give information about the presence of the main viruses responsible for human acute
gastroenteritis.

Keywords: Microbiological water quality, faecal indicator bacteria, viral genome, infectious virus

1. General remarks
Viruses, along with bacteria and protozoa, are excreted in high concentration in the faeces of humans and other
infected animals and can enter the environmental water through the discharge of treated, insufficiently treated or
untreated sewages. Over 100 types of viruses, many of them recognized as potentially pathogenic agents, are
excreted in human and animal waste. As a result, sewage contamination of water bodies creates a risk to human
health via waterborne virus. Currently, the water microbiological quality is determined by faecal bacterial
indicators (FIB) which do not accurately predict the presence of other pathogens, such as viruses. In an attempt to
mitigate the risks of waterborne viruses to the human health by faecal contaminated recreational water, the
assessing and managing of the microbiological quality of surface water is needed. The aim of this work was to
select the best bacterial and viral groups that can reflect the microbiological water quality and also to recognize
the animal species source of faecal pollution. This study was conducted in an urban recreational water resource
involving a dam complex in Córdoba, Argentina. Water samples from a year-long monitoring campaign were
analysed by traditional FIB together with the viral genome detection and infectious virus of six viral groups. The
viral contamination and the level of FIB results in the dam water were analysed to statically correlate
microbiological variables. The findings of this study could provide promising viral indicators for the virological
quality in the surface water.

2. Material and methods

2.1. Study area and sampling sites

The study was conducted at the San Roque Dam ( 31º22’S-64º28’W; 608 m a.s.l), located in a mountain range in
the Punilla valley, next to the city of Carlos Paz, 40 km west of Córdoba city in province of Córdoba, Argentina
(data depicted in Fig 1 a). The San Roque Dam is a human-made reservoir constructed more than 70 years ago
for flood control, water supply, recreation and sport fishing. The Dam has two main tributaries: the San Antonio
River (with an estimated annual mean flow of 4.0 m3/second) and the Cosquin River (with an estimated annual

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mean flow of 16.5 m3/second). Both rivers are subject to a seasonal fluctuation in water flow. The surface area of
this reservoir covers around 16 km2 and attracts a good deal of tourists to the area. Fishing, swimming and sailing
are some of the activities practised in and around its water, thus promoting urbanization in the surroundings of the
dam, especially on the coasts of the San Antonio river mouth (Carlos Paz city).
Four monitoring points located in the San Roque Dam were selected. One central monitoring site named centre
(C), one at the east side of the dam near the spillway and the dock walls named Dam (D) and two monitoring
stations at the mouths of the tributaries: one monitoring site at the San Antonio river mouth (named SA) and the
other one at the Cosquin river mouth (named CQ) (data depicted in Fig. 1b).

a) b)
Fig. 1. Geographical representation of the area under study: (a) San Roque Dam located in the province of Córdoba, Argentina.
(b) Monitoring points located in the San Roque Dam: one central (C), one near the Dam (D) and two in the mouths of the
tributaries: San Antonio river (SA) and Cosquin river (CQ).

2.2 Sample collection

A total of 48 water samples were collected once a month from the four monitoring points previously mentioned,
from January 2012 through December 2012. All water samples (2 L each) were collected during the daytime and
were typically gathered in the morning between the 12 th and 17th days of the respective month. Samples were
taken at a depth of 0.20 m using sterile bottles, and were transported within 12 hours at 4°C to 8°C to the laboratory
for further processing and analysis.

2.3 Bacteriological analysis

Faecal indicator bacteria were quantified using the standard membrane filtration techniques [1]. Fecal coliforms
were quantified by membrane filtration using modified fecal coliform (mFC) agar. Enterococci were quantified
by the EPA method 1600 using modified mE medium (mEI) containing the chromogenic substrate indoxyl-beta-
D-glucoside [2]. E. coli were quantified by EPA method 1603 using modified m-TEC media [3]. Negative controls
were included in each membrane filtration analysis. Samples were filtered in dilutions to obtain counts in the 30–
300 colony forming units (CFU)/100 mL range.
Argentina has adopted setting limits for microbiological contamination in recreational water according to the
guideline levels established by the United States Environmental Protection Agency (USEPA), i.e., a limit of 89
CFU/100-1 ml for enterococci, 298 CFU/100-1 ml for E. coli and 200 CFU/100-1 ml for fecal coliforms, all of
them for secondary contact [4].

2.4 Water concentration for viral analysis

A total of 1.5 liter water samples were concentrated 100-fold to 15 ml by high-speed centrifugation, elution and
PEG precipitation, according to the method previously described by Lewis and Metcalf [5] with modifications
described by Huang et al. [6].

2.5 Infectious human enterovirus (iHEV)

Enterovirus infectivity was evaluated by cell culture in HEp-2 cell line [7]. After inoculation, possible
cytopathogenic effects were identified and iHEV was confirmed by direct immunofluorescence assay.
Monoclonal antibody blend used for iHEV detection consisted of coxsackievirus type A9 monoclonal antibody;
coxsackievirus type B monoclonal antibody blend: B1, B2, B3, B4, B5 and B6; echovirus monoclonal antibody
blend: serotypes 4, 6, 9, 11, 30 and 34; poliovirus monoclonal antibody blend: serotypes 1, 2 and 3; and enterovirus
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monoclonal antibody blend: serotypes 60, 71 and Cox A16. The monoclonal antibody reagents were commercially
prepared and were purchased from Chemicon International (Temecula, CA).

2.6 Viral genomic detection

2.6.1 Rotavirus group A (RVA), human astrovirus (HAstV), norovirus (NoV) and picobirnavirus
(PBV) genomic detection
Viral RNA was extracted from 140 μL of the concentrated sample using the commercial QIAamp Viral RNA kit
(Qiagen Inc., Hilden, Germany). The manufacturer's protocol was followed, and the purified viral RNA was eluted
in 30 μL of elution buffer. Extracted RNA was reverse-transcribed into cDNA using random hexamer primers and
AMV reverse transcriptase (Invitrogen, CA, USA). Primer characteristics and references for the amplification
conditions of PCR protocol used for nucleic acid detection of PBV and heminested protocols used for RVA,
HAstV and NoV detection were all described previously [8-11]. It was considered as a positive signal of RVA or
HAstV or NoV any RVA or HAsV genotype as NoV genogroup that was detected.

2.6.2 JC Polyomavirus (JCPyV) genomic detection

Viral DNA was extracted from 200 μL of the concentrated sample using the commercial AxyPrep Body Fluid
Viral DNA/RNA Miniprep kit (Axigen Scientific Inc, USA). The manufacturer's protocol was followed, and the
purified viral DNA was eluted in 100 μL of the elution buffer. Primers characteristics and reference for
amplification conditions of Nested-PCR used for nucleic acid detection of JC has been described previously [12].
Positive and negative controls were included in all PCR runs. The PCR products were resolved on 10%
polyacrylamide gel electrophoresis [13] followed by silver staining [14], to achieve high resolution of the products
obtained.

2.6.7 Data analysis

The bacterial load was depicted as Log10 CFU/100ml. A linear regression test was used to analyse the relationship
between the densities of FIB. The 95% confidence intervals (CI) for viral proportion were estimated by the Wilson
score interval. Statistical analyses were performed with Minitab 17.

3. Results and discussion

3.1 Occurrence and densities of faecal indicators bacteria

The FIB level exceeding the guide limits considered as microbiologically acceptable for recreational water quality
of fecal coliforms and enterococci were 20.8% ( 95% CI, 11.7-34.3) for fecal coliforms, 16.7% ( 95% CI, 8.7-
29.6) for E.coli and 6.3% ( 95%CI 2.1-16.8) for enterococci. The frequency of FIB exceeding the adopted
guideline levels was significantly higher in the mouths of the tributaries as compared with the monitoring C and
D sites (p=0.01, X2 Test) (Fig. 2). The temporal trend indicated a higher mean concentration of FIB in the months
that correspond to the summer season in Argentina (months of December, January and February). This result
could be linked to human activities (a significantly higher number of people settle nearby the course of San
Antonio and Cosquin rivers during the summer season) and natural processes (increase in rainfall and floods
would lead to the washing out of fecal matter from latrines on land and then into surface water through
contamination of boreholes and reservoirs). Other authors [15] have also reported this notion. A lineal regression
approach used to analyze the relationship between the densities of FIB showed the highest positive correlation
between the densities of E. Coli and fecal coliforms (R2=98). In addition, an internal consistency analysis by
means of Cronbach’s Alpha reveals the inter-relatedness of the items E. coli and fecal coliforms (0.957). Yet,
fecal coliforms resulted slightly more sensitive than E. coli as FIB. According to the results obtained, we propose
to use only fecal coliforms as a FIB in the San Roque Dam to evaluate the level of fecal contamination.

3.2 Profile of virus genome detection for RVA, PBV, iHEV, HAstV, NoV and JCPyV
The overall viral frequency detection was 25/48 (52.1%, 95% CI, 38.3-65.5) for RVA, 39/48 (81,25%, 95%CI,
68.1-89.8) for PBV, 30/48 (62.5%, 95%CI, 48.4-74.8) for iHEV, 24/48 (50%, 95%CI,36.4-63.6) for HAstV, 30/48
(62.5%, 95%CI, 48.4-74.8) for NoV and 26/48 (54,2%, 95%CI, 40.3-67.4) for JCPyV. The monthly frequency of
the six viruses studied in the surface water from four collection sites at the San Roque Dam is depicted in Fig 3.

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Genomes of PBV, NoV and JCPyV were detected monthly in at least one of the four monitoring sites, being PBV
the viral group more frequently detected by month as compared with NoV and JCPyV genomic detection.
Conversely, a temporal trend for HAstV and RVA detection was observed, that is, negative signals for HAstV
were more frequently detected during the summer months (November, January and February) and for RVA during
the spring months (September to November). Therefore, in this setting, PBV seems to be a suitable indicator of
fecal contamination in water. The high frecuency of PBV detection in water samples could be linked to the natural
history of PBV infection, i.e., PBV establishes persistent infections in a wide range of vertebrate species, including
humans, with periods of viral excretion intermingled with periods of silence [16]. The only report regarding PBV
detection in water samples reports a lower PBV detection in water samples [17]. It was noteworthy that infectious
human enterovirus showed a steady frequency of detection during the whole period studied. Viable virus was
detected in at least two of the four monitoring sites by month. The probability of PBV and/or iHEV detection is
94% (CI 87.5-100%).

Fig.2. Charts of individual values of enterococci,

E. coli and fecal coliforms at each monitoring
site: Dam (D); center (C); Cosquin river mouth
(CQ) and San Antonio river mouth (SA) at San
Roque Dam. Bacterial load is depicted as Log10
CFU/100ml.

a)

b) c)

3.3 Relationship between FIB and genomic and infectious virus in surface water samples
The total water samples with values under the level guide of FIB fecal coliforms were positive for the detection
of one or more of the viral groups analyzed, all through the year (Table 1). This results are supported by previously
reported data [18].

Fig.3. Monthly frequency of PBV, iHEV, RVA,

HAstV, NoV and JCPyV detection during the
period January- December 2012 in four
monitoring sites at the San Roque Dam.
Viral detection in 4/4 monitoring sites
Viral detection in 3/4 monitoring sites
Viral detection in 2/4 monitoring sites
Viral detection in 1/4 monitoring sites

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Table 1 Virus detection in water and its correlation with FIB parameters and confidence intervals

Virus Virus detection in water Confidence Virus detection in water Confidence interval
within FIB acceptable interval 95% with no FIB acceptable 95%
limits (%) limits (%)
Picobirnavirus 76.3 60.8-87 100 72.2-100
Enterovirus 63.2 47.3-76.6 70 39.7-89.2
Rotavirus 50 34.8-65.2 60 31.3-83.2
Astrovirus 60.5 44.7-74.4 10 1.8-40.4
Norovirus 71.1 55.2-83 30 10.8-60.3
JC polyomavirus 52.6 37.3-67.5 50 23.7-76.3

4. Conclusion
The presence of a viral genome as well as an infectious virus in areas where faecal contamination was not
demonstrated by FIB suggests prolonged virus persistence in aquatic environments and emphasizes the enteric
virus groups as the most reliable for microbiological water quality monitoring. The combined detection of
infectious human enterovirus and the genome of picobirnavirus in the surface water could be promising indicators
of microbiological contamination derived from human and animal feces. Also, they could give information about
the presence of the main viruses responsible for human acute gastroenteritis.

Acknowledgements. This work was supported by the Council of Science and Technology of the National University of
Cordoba, Argentina (SeCyT 05/H387, Res 1565/2014), the National Agency of Scientific and Technical Promotion, Argentina
(ANPCyT- FONCyT; PICT-2012 Nº0998) and the Ministry of Science and Technology, Government of the Province of
Cordoba, Argentina (MinCyT-Cordoba PIO 170/11).

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