A real-time polymerase chain reaction assay for quantitative detection of the human-specific enterococci surface protein marker in sewage and environmental waters

A real-time polymerase chain reaction (PCR) assay using SYBR Green I dye was developed to quantify the Enterococcus faecium enterococci surface protein (esp) marker in sewage (n = 16) and environmental waters (n = 16). The concentration of culturable enterococci in raw sewage samples ranged between 1.3 x 10(5) and 5.6 x 10(5) colony-forming units (cfu) per 100 ml. The real-time PCR detected 9.8 x 10(3)-3.8 x 10(4) gene copies of the esp marker per 100 ml of sewage. However, the concentration of culturable enterococci and the esp marker in secondary effluent was two orders of magnitude lower than raw sewage. Surface water samples were collected from a non-sewered catchment after storm events and the real-time PCR was applied to quantify the esp marker. Of the 16 samples tested, 6 (38%) were PCR-positive and the concentration of the esp marker ranged between 1.1 x 10(2) and 5.3 x 10(2) gene copies per 100 ml of water samples. The newly developed real-time PCR method was successfully used to quantify the esp marker in samples collected from sewage and environmental waters. The presence of the esp marker in water samples immediately after storm events not only indicated human faecal pollution but also provided evidence of the degree of human faecal pollution. To our knowledge, this is the first study that reports the use of a real-time PCR assay to quantify the esp marker in sewage and surface waters. Such study would provide valuable information for managers for the improved management of water quality.     

Detection and quantification of group C rotaviruses in communal sewage

Group C rotaviruses have been recognized as a cause of acute gastroenteritis in humans, cattle, and swine, although the true epidemiologic and clinical importance of this virus in these hosts has not yet been fully established. A real-time PCR assay based on a broadly reactive primer pair was developed and used to quantitatively determine the viral load of group C rotaviruses in environmental samples. A total of 35 raw and 35 treated sewage samples collected at the same sampling time in four Hungarian sewage treatment plants during a survey in 2005 were tested for the presence of group C rotaviruses. The overall detection rates were 91% (32 of 35) for the influent and 57% (20 of 35) for the effluent samples. Molecular characterization of the amplified partial VP6 gene revealed the cocirculation of human and animal (i.e., bovine and porcine) strains that were easily distinguishable by melting curve analysis. Human strains yielded relatively high viral loads (mean, 1.2 x 10(7); median, 6.9 x 10(5) genome equivalents per liter influent sewage) and appeared to display seasonal activity over the study period, whereas animal strains appeared to circulate throughout the year at much lower average titers (bovine strains mean, 9.9 x 10(4); median, 3.0 x 10(4); porcine strains mean, 3.9 x 10(4); median, 3.1 x 10(4) genome equivalents per liter influent sewage). Our findings suggest that monitoring of communal sewage may provide a good surrogate for investigating the epidemiology and ecology of group C rotaviruses in humans and animals.     

Isolation of enterovirus and reovirus from sewage and treated effluents in selected Puerto Rican communities

Sewage treatment plant effluents were surveyed for viral contributions to gastroenteritis outbreaks in Puerto Rico. Of the 15 sewage treatment plants studied, all discharged their effluents upstream from water treatment plant intakes. No base-line data on the degree of viral challenge to these sewage treatment plants or the subsequent reduction of viruses before discharge existed. Enterovirus counts were generally much higher than those found in the continental United States. At four plants, viruses in the incoming sewage exceeded 100,000 PFU/liter, and one of these, a trickling filter plant, was discharging 24,000 PFU/liter to receiving waters. Virus identification showed that more than 80% of the enterovirus isolates were coxsackievirus B5. These overwhelming viral numbers pointed to defects in the sewage treatment processes. Without reasonable barriers to protect receiving waters, several of the downstream communities were using raw waters that posed extraordinary demands on the ability of their water treatment plants to supply virologically safe drinking water.     

Isolation of enterovirus and reovirus from sewage and treated effluents in selected Puerto Rican communities.

Sewage treatment plant effluents were surveyed for viral contributions to gastroenteritis outbreaks in Puerto Rico. Of the 15 sewage treatment plants studied, all discharged their effluents upstream from water treatment plant intakes. No base-line data on the degree of viral challenge to these sewage treatment plants or the subsequent reduction of viruses before discharge existed. Enterovirus counts were generally much higher than those found in the continental United States. At four plants, viruses in the incoming sewage exceeded 100,000 PFU/liter, and one of these, a trickling filter plant, was discharging 24,000 PFU/liter to receiving waters. Virus identification showed that more than 80% of the enterovirus isolates were coxsackievirus B5. These overwhelming viral numbers pointed to defects in the sewage treatment processes. Without reasonable barriers to protect receiving waters, several of the downstream communities were using raw waters that posed extraordinary demands on the ability of their water treatment plants to supply virologically safe drinking water.     

Genogroup distribution of F-specific coliphages in wastewater and river water in the Kofu basin in Japan

Aims: To determine the genogroup distribution of F‐specific coliphages in aquatic environments using the plaque isolation procedure combined with genogroup‐specific real‐time PCR. Methods and Results: Thirty water samples were collected from a wastewater treatment plant and a river in the Kofu basin in Japan on fine weather days. F‐specific coliphages were detected in all tested samples, 187 (82%) of 227 phage plaques isolated were classified into one of the 4 F‐specific RNA (F‐RNA) coliphage genogroups and 24 (11%) plaques were F‐specific DNA coliphages. Human genogroups II and III F‐RNA coliphages were more abundant in raw sewage than animal genogroups I and IV, excluding one sample that was suspected to be heavily contaminated with sporadic heavy animal faeces. The secondary‐treated sewage samples were highly contaminated with genogroup I F‐RNA coliphages, probably because of different behaviours among the coliphage genogroups during wastewater treatment. The river water samples were expected to be mainly contaminated with human faeces, independent of rainfall effects. Conclusions: A wide range of F‐specific coliphage genogroups were successfully identified in wastewater and river water samples. Significance and Impact of the Study: Our results clearly show the usefulness of the genogroup‐specific real‐time PCR for determining the genogroups of F‐specific coliphages present in aquatic environments.     

Viral pollution of surface waters due to chlorinated primary effluents.

The role of chlorinated primary effluents in viral pollution of the Ottawa River (Ontario) was assessed by examining 282 field samples of wastewaters from two different sewage treatment plants over a 2-year period. The talc-Celite technique was used for sample concentration, and BS-C-1 cells were employed for virus detection. Viruses were detected in 80% (75/94) of raw sewage, 72% (68/94) of primary effluent, and 56% (53/94) of chlorinated effluent samples. Both raw sewage and primary effluent samples contained about 100 viral infective units (VIU) per 100 ml. Chlorination produced a 10- to 50-fold reduction in VIU and gave nearly 2.7 VIU/100 ml of chlorinated primary effluent. With a combined daily chlorinated primary effluent output of approximately 3.7 x 10(8) liters, these two plants were discharging 1.0 x 10(10) VIU per day. Because the river has a mean annual flow of 8.0 x 10(10) liters per day, these two sources alone produced a virus loading of 1.0 VIU/8 liters of the river water. This river also receives at least 9.0 x 10(7) liters of raw sewage per day and undetermined but substantial amounts of storm waters and agricultural wastes. It is used for recreation and acts as a source of potable water for some 6.0 x 10(5) people. In view of the potential of water for disease transmission, discharge of such wastes into the water environment needs to be minimized.     

Quantification and Genotyping of Torque Teno Virus at a Wastewater Treatment Plant in Japan

Torque teno virus (TTV) DNA was quantitatively detected in influent and effluent samples collected from a wastewater treatment plant in Japan, with the highest concentration being 4.8 × 10⁴ copies/liter. Genogroup-specific nested PCR demonstrated that TTV of genogroup 3 was the most abundant in wastewater among the five genogroups tested.     

Detection and distribution of rotavirus in raw sewage and creeks in São Paulo, Brazil.

Rotaviruses were concentrated from 8-liter samples of raw domestic sewage and sewage-polluted creek water by adsorption to and elution from positively charged microporous filters (Zeta Plus 60S), followed by ultracentrifugation of the filter eluates. Indirect immunofluorescence and direct immunoperoxidase methods allowed detection and enumeration of rotavirus in 6 (20.6%) of 29 sewage samples and in 19 (34.5%) of 55 creek water samples. Levels of rotaviruses ranged from < 3 to 63 focus-forming units (FFU)/liter, and the geometric means were 2.2 FFU/liter in sewage, 2.9 FFU/liter at creek Tremembé, and 2.6 FFU/liter at creek Pirajussara. Wastewater samples examined during autumn and winter months showed a higher rate positivity for rotavirus than those collected in spring and summer, corresponding to the seasonal variation of rotaviral diarrhea in the city of São Paulo.     

F-specific RNA bacteriophages are adequate model organisms for enteric viruses in fresh water.

Culturable enteroviruses were detected by applying concentration techniques and by inoculating the concentrates on the BGM cell line. Samples were obtained from a wide variety of environments, including raw sewage, secondary effluent, coagulated effluent, chlorinated and UV-irradiated effluents, river water, coagulated river water, and lake water. The virus concentrations varied widely between 0.001 and 570/liter. The same cell line also supported growth of reoviruses, which were abundant in winter (up to 95% of the viruses detected) and scarce in summer (less than 15%). The concentrations of three groups of model organisms in relation to virus concentrations were also studied. The concentrations of bacteria (thermotolerant coliforms and fecal streptococci) were significantly correlated with virus concentrations in river water and coagulated secondary effluent, but were relatively low in disinfected effluents and relatively high in surface water open to nonhuman fecal pollution. The concentrations of F-specific RNA bacteriophages (FRNA phages) were highly correlated with virus concentrations in all environments studied except raw and biologically treated sewage. Numerical relationships were consistent over the whole range of environments; the regression equations for FRNA phages on viruses in river water and lake water were statistically equivalent. These relationships support the possibility that enteric virus concentrations can be predicted from FRNA phage data.     

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm² to 645.0 cm²) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm². The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm²) to 1.2 to 33.6% for toilet seat surfaces (700 cm²), with detection limits of 3.5 log₁₀ and 4.0 log₁₀ RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.     

Deciphering the Diversities of Astroviruses and Noroviruses in Wastewater Treatment Plant Effluents by a High-Throughput Sequencing Method

Although clinical epidemiology lists human enteric viruses to be among the primary causes of acute gastroenteritis in the human population, their circulation in the environment remains poorly investigated. These viruses are excreted by the human population into sewers and may be released into rivers through the effluents of wastewater treatment plants (WWTPs). In order to evaluate the viral diversity and loads in WWTP effluents of the Paris, France, urban area, which includes about 9 million inhabitants (approximately 15% of the French population), the seasonal occurrence of astroviruses and noroviruses in 100 WWTP effluent samples was investigated over 1 year. The coupling of these measurements with a high-throughput sequencing approach allowed the specific estimation of the diversity of human astroviruses (human astrovirus genotype 1 [HAstV-1], HAstV-2, HAstV-5, and HAstV-6), 7 genotypes of noroviruses (NoVs) of genogroup I (NoV GI.1 to NoV GI.6 and NoV GI.8), and 16 genotypes of NoVs of genogroup II (NoV GII.1 to NoV GII.7, NoV GII.9, NoV GII.12 to NoV GII.17, NoV GII.20, and NoV GII.21) in effluent samples. Comparison of the viral diversity in WWTP effluents to the viral diversity found by analysis of clinical data obtained throughout France underlined the consistency between the identified genotypes. However, some genotypes were locally present in effluents and were not found in the analysis of the clinical data. These findings could highlight an underestimation of the diversity of enteric viruses circulating in the human population. Consequently, analysis of WWTP effluents could allow the exploration of viral diversity not only in environmental waters but also in a human population linked to a sewerage network in order to better comprehend viral epidemiology and to forecast seasonal outbreaks.     

Enumeration of Pseudomonas species and Pseudomonas aeruginosa bacteriophages in domestic sewage

Domestic sewage in Kuwait is mainly treated by an activated sludge process. Pseudomonas species were enumerated at all steps of sewage treatment. About 98-99% reduction in the number of these bacterial species were found in the treated effluent compared with raw sewage, which indicates a rather efficient removal of Pseudomonas from sewage. Spherical tail-less phages of Pseudomonas aeruginosa were found in all sewage samples. About 25-85% of the total phages encountered with the raw sewage were retained in the treated effluents. Seasonal variations of Pseudomonas spp and P. aeruginosa phages in two treatment stations are reported.     

Development and application of a real-time PCR approach for quantification of uncultured bacteria in the central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.     

Distribution of Coliphages in Hong Kong Sewage

Coliphage content of sewage collected from 11 different localities in Hong Kong was determined. The number of plaque-forming units (PFU) ranged from 0.036 x 10(3) to 15.9 x 10(3) per ml. In general, urban sewage tended to be richer than rural sewage both in PFU count as well as plaque morphological variation. Seventy-seven isolates were subjected to a host range study. Fifty per cent of these were able to grow on Escherichia coli K-12 as well as E. coli B. Approximately 32% were found to be male specific, and the remaining 18% were K-12 specific although sex-indifferent.