Somatic Phages Research Articles

Quantification and fate of plasmid-specific bacteriophages in wastewater: Beyond the F-coliphages

Plasmid-specific bacteriophages specifically infect bacteria carrying conjugal plasmids. While wastewater has been used as isolation source for such phages, to date, only the distribution and ecology of RNA phages specific to the F plasmid have been described, because they serve as a water quality indicator. Yet, several other plasmid classes have higher clinical and ecological relevance, and the distribution, fate, and ecology of the phages that target them remain uncharacterized. We aimed to (i) provide an experimental platform to quantify the abundance of plasmid-specific phages applicable to several different conjugal plasmid classes, (ii) describe the distribution of such phages in wastewater systems, and (iii) relate their abundance to plasmid abundance and to municipal wastewater treatment processes. We introduced four model conjugal plasmids, belonging to incompatibility groups IncP-1, IncN, IncHI1, or IncF into an avirulent Salmonella enterica strain, for which somatic phages are at low abundance in wastewater. These strains were used in double layer agar assays with waters from contrasting sources. Plasmid-specific phages were common in wastewater but rare in river water. Hospital wastewater contained significantly more IncP-1-, but fewer IncF- and IncN- specific phages than domestic wastewater. This pattern did not match that of plasmid abundance estimated by Inc group targeting high-throughput quantitative PCR. The comparison between influent and effluent of wastewater treatment plants revealed a reduction in phage concentration by ca. 2 log, without significant contribution of primary settling. Overall, the ubiquity of these phages hints at their importance for plasmid ecology, and can provide opportunities in water quality monitoring and in ecological management of mobile resistance genes.

Biosólidos como fertilizante en el cultivo de tomate

The sludge produced in wastewater treatment plants constitutes a potential alternative to replace traditional fertilizers and reduce costs in agricultural activities. The objective of this work was to compare the fertilizing effect of the sludge produced in the wastewater treatment plant of Sotaquirá-Colombia, with the fertilizers traditionally used on the tomato crop (Solanum lycopersicum L). For this, the sludge was previously stabilized with two different treatments: dehydration and the addition of CaO. Subsequently, four treatments were applied to the tomato seedlings, 135 g.kg-1 of dehydrated biosolid, 135 g.kg-1 biosolid stabilized with CaO, 135 g.kg-1 of ABIMGRA®, 135 g.kg-1 of naturcomplet®-G, and greenhouse soil without biosolids. The height of the plant, the fresh and dry mass, foliar area, and fruits per plant, were measured at 0, 30, 60 and 90 days after sowing. In tomato fruits, the concentrations of heavy metals, coliforms, helminth eggs, somatic phages, and Salmonella sp., were determined. The dehydrated biosolids had a significant effect on the size, the fresh mass, foliar area, and the number of fruits per plant, compared to the alkaline biosolids. The dry mass of the plants (120 g. plant-1) was similar to traditional fertilizers and biosolids. Tomatoes produced with biosolids had low levels of heavy metals and an absence of pathogenic microorganisms. In conclusion, the biosolid obtained by dehydration in Sotaquirá can be used as a potential fertilizer in tomato cultivation.

A model to predict the kinetics of direct (endogenous) virus inactivation by sunlight at different latitudes and seasons, based on the equivalent monochromatic wavelength approach

Sunlight plays an important role in the inactivation of pathogenic microorganisms such as bacteria and viruses in water. Here we present a model that is able to predict the kinetics of direct virus inactivation (i.e. inactivation triggered by sunlight absorption by the virion, without the role played by photochemically produced reactive intermediates generated by water-dissolved photosensitizers) on a global scale (from 60 °S to 60 °N latitude) and for the different months of the year. The model is based on the equivalent monochromatic wavelength (EMW) approach that was introduced recently, and which largely simplifies complex polychromatic calculations by approximating them with a monochromatic equation at the proper wavelength, the EMW. The EMW equation was initially established for mid-July conditions at a mid-latitude, and was then extended to different seasons and to the latitude belt where the day-night cycle is always observed throughout the year. By so doing, the first-order rate constant of direct virus photoinactivation can be predicted on a global scale, with the use of a relatively simple equation plus tables of pre-calculated input data, as a function of latitude, month, and key water parameters. The model was here applied to the virus organism phiX174, a somatic phage that is often used as proxy for pathogenic viruses undergoing fast direct inactivation, and for which a wide array of published inactivation data is available. Model predictions are validated by comparison with field data of inactivation of somatic phages by sunlight.

Colombian water sources polluted by car-washgenerated sludge

The fact that Colombian hydric sources, despite their great importance, can contain microorganisms, led this study to categorize sludge from car wash, because this activity results in high volume of residual water that fluctuates, according to wash demand, between 0.5 and 1.0lt/sec. Detergents, fats, oil, fuels and sediment, among many others, produce very high pollution levels. A three-period experimental design carried out taking random samples of sludge found presence of Salmonella, somatic phages, helminth eggs and fecal coliforms coming from three car wash locations around metropolitan Bucaramanga, Colombia. Sludge analyzed was classified as category B, apt for different uses in forest plantations, as precursor material in production of construction materials, and energy-valorization processes.

Assessment of two sludge stabilization methods in a wastewater treatment plant in Sotaquirá, Colombia

Wastewater sludge is a by-product of waste water treatment; it is often employed in agricultural processes following appropriate microbiological stabilization. In this study, we evaluated two methods of sludge stabilization in the waste water treatment plant of Sotaquirá, Colombia. The two assessed stabilization methods were dewatering on drying beds and alkaline stabilization. The stabilization through dewatering on drying bed was carried out for five months. Alkaline stabilization was conducted for 96 hours with three concentrations of calcium oxide, 7%, 9%, and 13% w/w. Humidity, pH, organic carbon, phosphorus, organic nitrogen, fecal coliforms, somatic phages, total helminth eggs, and Salmonella sp. were monitored monthly for the sludge under dewatering treatment and at 0, 12, 24 hours for sludge under alkaline stabilization treatment. Both treatments succeded in eliminating Salmonella sp. Helminth eggs were eliminated by alkaline stabilization, whereas it was reduced to one or zero helminth eggs with the dewatering treatment. Somatic phages were eliminated with alkaline stabilization but were only reduced to 3.52 log CFU/g with the dewatering method. Dewatering on drying beds produced biosolids that can be used for soil restoration. Whereas alkaline stabilization produced biosolids that can be used for agricultural purposes. Alkaline stabilization with 9% and 13 % calcium oxide ostensibly reduced nitrogen and phosphorus contents in the sludge, whereas 7 % calcium affected less the phosphorus concentration of the sludge. These results indicate that sludge dewatering on drying beds is an effective sludge sanitation protocol to be implemented in small waste water treatment plants, such that in Sotaquirá, Colombia.

Somatic and F-specific bacteriophages in waters of the small, municipal Rusałka Lake in Szczecin

Investigations of bacteriophages showed that in the environment are mostly in water, but in small municipal lakes sporadically been studied. Previous studies of bacteriophages in the aqueous environment related to the determination of the quantity and diversity and use them as indicators of water pollution. In the last study probably of the most important are the F-specific and somatic bacteriophages specific to E. coli. In our study examined them in a small municipal lake in Szczecin (Poland) in context of occurrence of F-specific and somatic bacteriophages specific to E. coli with used of SAL method. We proved that count of bacteriophages in this water tank is lower in context of different waters, but the relation between the amounts of this viruses and environments condition (air and water temperature) were similar to results observed in waters of seas, rivers and lakes.

Comparison of microbial removal between slow dead-end versus tangential sand filtration.

Both River-Bank Filtration and dead-end sand-filtration are becoming increasingly applied in rural areas to improve quality of fecally contaminated water. To evaluate the capacity of both treatments to remove E. coli, fecal streptococci, somatic and K13 phages, we have studied their concentrations in diluted wastewater after short-distance tangential sand-filtration and after dead-end sand-filtration.Bacteria were almost undetectable in both systems after sixty cm depth and at a pore water velocity of 1 m/d. Both phages underwent removal of 2.5 logs by tangential filtration, whereas dead-end filtration removed 5.1 logs and 3.9 logs of K13 and somatic phages, respectively. After discounting removal by the schmutzdecke, observed only in the dead-end filtration, both systems removed phages similarly.We conclude that short-distance river bank filtration alone does not meet WHO requirements for drinking water. However, the reduction of suspended solids renders the filtered water amenable to further treatment steps.

Microbiological assessment of fresh, minimally processed vegetables from open air markets and supermarkets in Luzon, Philippines, for food safety

A wide-range investigation of the microbiological quality of fresh produce from different open air markets and supermarkets in Luzon, Philippines was performed. Different vegetables which are usually consumed raw (bell pepper, carrot, lettuce, mung bean sprout, and tomato) had been screened for the presence and amount of contamination of enteric microorganisms, namely Escherichia coli, Salmonella spp., and somatic bacteriophages (or viruses). Phenotypic characterization was employed using selective and differential media such as eosin methylene blue agar and xylose lysine deoxycholate agar. Viable plate count (colony-forming units or CFU) and most probable number (MPN) methods were used to estimate the cell numbers of E. coli and Salmonella spp., respectively. Double agar assay was used to detect and quantify somatic phages as plaque-forming units (PFU). Polymerase chain reaction was also done to confirm the identity of the isolates using species-specific primers. From a total of 410 vegetable samples collected, 44 bacterial (prevalence of 5.85% E. coli and 5.12% of Salmonella spp.) and 21 (prevalence of 5.12%) viral isolates were obtained. It is alarming to note that samples contaminated with Salmonella spp. were significantly higher in counts (3.751 ± 0.60 log MPN/g) as compared to E. coli (1.472 ± 0.25 log CFU/g), while samples with coliphage (0.316 ± 0.12 log PFU/g) were the least in numbers. The degree of contamination in the samples can cause great risks on the health of individuals especially in developing countries such as the Philippines. Thus, monitoring on the handling of different produce is important to improve the country’s food quality and safety.

Enumeration of Somatic and F-RNA Phages as an Indicator of Fecal Contamination in Potable Water from Rural Areas of the North West Province.

Bacteriophages are regarded as enteric viral indicators in faecally contaminated water systems and may indicate the presence of human viral pollution. They are relatively resistant to inactivation by natural and treatment processes. In this study, the presence of somatic coliphages and F-RNA coliphages was investigated in potable water from rural areas in the North West province. Water samples were aseptically collected from boreholes and tap water from some rural communities in the North West Province. Physical parameters of the water, such as the temperature, pH and turbidity, were measured before sample collection. Double-agar layer assay was performed using ISO, (1995, 2000) standard methods. Bottled water was used as a negative control and the strains фX174 and MS2 as positive controls. Of the 16 water samples collected, 15 were positive for somatic bacteriophages while F-RNA coliphages were detected in only two samples. Amongst the positive samples 189 and three plaque forming units were obtained for both somatic and F-RNA coliphages, respectively. No coliphage was detected in water from Masamane tap 1. The rest of the samples obtained from various rural areas were positive and did not comply with national and international standards for potable water. This was a cause for concern and should be further investigated.

Assessment of indicators of fecal contamination in soils treated with biosolids for growing grasses

Developing countries currently face challenges related to the management and disposal of biosolids that result from wastewater treatment. One of the most important issues they must deal with involves the maximum level of pathogens permitted and the effect they may have on the environment and human health once they are used for agricultural purposes. In order to learn about the risk generated by these kinds of practices, we assessed the behavior of indicators of fecal contamination in grasses used to feed dairy cattle. The study area was divided into two plots: an experimental plot in which diluted biosolids were applied and a control plot without the application of biosolids. We collected samples from soil, grass, surface water and groundwater and analyzed the presence of fecal coliforms, Salmonella, somatic phages and helminth eggs. Experimental and control plots showed a decrease in the concentration of bacterial indicators assessed in soil, and low concentrations in surface water, groundwater and grass. Concentrations of viral and parasite indicators tend to decrease more slowly. Our results show that this way of biosolid management allows a better use of its benefits as an organic amendment by lowering the sanitary risk due to the presence of pathogens.

Virus removal vs. subsurface water velocity during slow sand filtration.

In an attempt to obtain a conservative estimate of virus removal during slow sand and river bank filtration, a somatic phage was isolated with slow decay and poor adsorption to coarse sand. We continuously fed a phage suspension to a 7-m infiltration path and measured the phage removal. In a second set of experiments, we fed the phage suspension to 1-m long columns run at different pore water velocities. Using the data obtained, a mathematical model was constructed describing removal vs. pore water velocity (PWV), assuming different statistical distributions of the adsorption coefficient λ. The bimodal distribution best fit the results for PWVs higher than 1 m/d. It predicted a removal of approximately 4 log10 after 50 days infiltration at 1 m/d. At PWVs below 1 m/d the model underestimated removal. Sand-bound phages dissociated slowly into the liquid phase, with a detachment constant kdet of 2.6 × 10⁻⁵. This low kdet suggests that river bank filtration plants should be intermittently operated when viral overload is suspected, e.g. during flooding events or at high water-marks in rivers, in order for viruses to become soil-associated during the periods of standstill. Resuming filtration will allow only a very slow virus release from the soil.

Microbiological quality of fresh produce from open air markets and supermarkets in the Philippines.

This study is the first in the Philippines to conduct a comprehensive assessment of the prevalence of bacterial pathogens and somatic phages in retailed fresh produce used in salad preparation, namely, bell pepper, cabbage, carrot, lettuce, and tomato, using culture and molecular methods. Out of 300 samples from open air and supermarkets, 16.7% tested positive for thermotolerant Escherichia coli, 24.7% for Salmonella spp., and 47% for somatic phages. Results show that counts range from 0.30 to 4.03 log10 CFU/g for E. coli, 0.66 to ≥2.34 log10 MPN/g for Salmonella spp., and 1.30 to ≥3.00 log10 PFU/g for somatic phages. Statistical analyses show that there was no significant difference in the microbial counts between open air and supermarkets (α = 0.05). TaqMan and AccuPower Plus DualStar real-time polymerase chain reaction (RT-PCR) was used to confirm the presence of these organisms. The relatively high prevalence of microorganisms observed in produce surveyed signifies reduction in shelf-life and a potential hazard to food safety. This information may benefit farmers, consumers, merchants, and policy makers for foodborne disease detection and prevention.

Fluctuation of densities of bacteriophages and Escherichia coli present in natural biofilms and water of a main channel and a small tributary

Fluctuation of bacteriophage and Escherichia coli densities in naturally developed riverbed biofilms were investigated for a 1-year period. E. coli ranged from 1,500 to 15,500 most probable number (MPN)/100 mL and from 580 to 18,500 MPN/cm(2) in the main channel in the river water and biofilms, respectively. However, the fluctuations were much greater in the tributary, ranging from 0.8 to 100 MPN/100 mL and from 0.3 to 185 MPN/cm(2) in water and biofilms, respectively. The fluctuations of coliphages were also greater in the tributary than in the main channel. FRNA phage serotyping results indicated no significant differences in the source type of the fecal contamination in the main channel and tributary sampling stations. Significant correlations between phage groups in biofilms and water were found at both main channel and tributary. It was assumed that natural biofilms developed in the streambed captured and retained somatic phages in the biofilms for a certain period of time in the main channel site. At the location receiving constant and heavy contamination, the usage of phage indicators may provide additional information on the presence of viruses. In the small tributary it may be possible to estimate the virus concentration by monitoring the E. coli indicator.

Removal and relationships of microbial indicators in a water treatment and reclamation facility

A wastewater treatment and reclamation facility in north-east Spain was monitored over 1 year to determine the occurrence and concentrations of different microbial indicators (Escherichia coli, fecal enterococci, somatic bacteriophages and spores of sulfite-reducing clostridia). The removal of the indicators and its relationships through the wastewater treatment and reclamation trains were evaluated. The results obtained show that the reclamation treatments evaluated present a different efficiency in indicator microorganisms' removal depending on the type of microorganism. The E. coli and enterococci present an average reduction slightly higher than the other indicators, followed by somatic bacteriophages and spores of sulfite-reducing clostridia. The Spearman's correlations indicate that it is not suitable to use any of the bacterial indicators evaluated to predict the content of virus or spores of sulfite-reducing clostridia. Therefore, in order to evaluate the microbiological risk of the reclaimed effluent use, it is necessary to monitor the three types of indicator microorganisms (bacteria, virus and protozoa).

Determination of fecal contamination origin in reclaimed water open-air ponds using biochemical fingerprinting of enterococci and fecal coliforms

Low levels of fecal indicator bacteria (FIB) were recently detected in two reclaimed water open-air ponds used to irrigate a golf course located in Northeastern Spain. The aim of this study was to evaluate the feasibility of a biochemical fingerprinting method to track the origin of fecal contamination in water with low FIB levels, as in the aforementioned ponds. We also aimed to determine whether FIB presence was due to regrowth of the reclaimed water populations or to a contribution of fecal matter whose source was in the golf facility. Three hundred and fifty enterococcal strains and 308 fecal coliform strains were isolated from the ponds and reclamation plant, and they were biochemically phenotyped. In addition, the inactivation of several microbial fecal pollution indicators (fecal coliforms, total bifidobacteria, sorbitol-fermenting bifidobacteria, somatic bacteriophages, and bacteriophages infecting Bacteroides thetaiotaomicron) was studied using a mesocosm in situ in order to obtain information about their decay rate. Although FIB concentration was low, the biochemical fingerprinting provided evidence that the origin of the fecal contamination in the ponds was not related to the reclaimed water. Biochemical fingerprinting thus proved to be a successful approach, since other microbial source-tracking methods perform poorly when dealing with low fecal load matrices. Furthermore, the mesocosm assays indicated that none of the microbial fecal indicators was able to regrow in the ponds. Finally, the study highlights the fact that reclaimed water may be recontaminated in open-air reservoirs, and therefore, its microbial quality should be monitored throughout its use.

Leaching and ponding of viral contaminants following land application of biosolids on sandy-loam soil

Much of the land available for application of biosolids is cropland near urban areas. Biosolids are often applied on hay or grassland during the growing season or on corn ground before planting or after harvest in the fall. In this study, mesophilic anaerobic digested (MAD) biosolids were applied at 56,000 L/ha on a sandy-loam soil over large containment lysimeters seeded to perennial covers of orchardgrass (Dactylis glomerata L.), switchgrass (Panicum virgatum), or planted annually to maize (Zea mays L.). Portable rainfall simulators were to maintain the lysimeters under a nearly saturated (90%, volumetric basis) conditions. Lysimeter leachate and surface ponded water samples were collected and analyzed for somatic phage, adenoviruses, and anionic (chloride) and microbial (P-22 bacteriophage) tracers. Neither adenovirus nor somatic phage was recovered from the leachate samples. P-22 bacteriophage was found in the leachate of three lysimeters (removal rates ranged from 1.8 to 3.2 log10/m). Although the peak of the anionic tracer breakthrough occurred at a similar pore volume in each lysimeter (around 0.3 pore volume) the peak of P-22 breakthrough varied between lysimeters (<0.1, 0.3 and 0.7 pore volume). The early time to peak breakthrough of anionic and microbial tracers indicated preferential flow paths, presumably from soil cracks, root channels, worm holes or other natural phenomena. The concentration of viral contaminants collected in ponded surface water ranged from 1 to 10% of the initial concentration in the applied biosolids. The die off of somatic phage and P-22 in the surface water was fit to a first order decay model and somatic phage reached background level at about day ten. In conclusion, sandy-loam soils can effectively remove/adsorb the indigenous viruses leached from the land-applied biosolids, but there is a potential of viral pollution from runoff following significant rainfall events when biosolids remain on the soil surface.

Comparing extraction buffers to identify optimal method to extract somatic coliphages from sewage sludges

Somatic coliphages are present in high numbers in sewage sludge. Since they are conservative indicators of viruses during wastewater treatment processes, they are being used to evaluate the effectiveness of sludge treatment processes. However, efficient methods to extract them from sludge are lacking. The objective was to compare different virus extraction procedures and develop a method to extract coliphages from sewage sludge. Twelve different extraction buffers and procedures varying in composition, pH, and sonication were compared in their ability to recover indigenous phages from sludges. The 3% buffered beef extract (BBE) (pH 9.0), the 10% BBE (pH 9.0), and the 10% BBE (pH 7.0) with sonication were short-listed and their recovery efficiency was determined using coliphage-spiked samples. The highest recovery was 16% for the extraction that involved 10% BBE at pH 9.0. There is a need to develop methods to extract somatic phages from sludges for monitoring sludge treatment processes.

Using temperature and time criteria to control the effectiveness of continuous thermal sanitation of piggery effluent in terms of set microbial indicators

To determine the minimal conditions (temperature-time), necessary to achieve set sanitation targets for selected microbial indicators during the continuous thermal treatment of pig slurry. The effectiveness of thermal treatment between 55 and 96°C was studied using Escherichia coli, enterococci, sulfite-reducing Clostridia (SRC), mesophilic culturable bacteria (MCB), F+-specific and somatic phages. Identification of SRC and MCB was performed using 16S rRNA gene analysis. Ten minutes at 70°C or 1 h at 60°C was sufficient to reduce the vegetative bacteria by 4-5 log(10), but it had little effect on somatic phages nor on spore formers, dominated by Clostridium sp. At 96°C, somatic phages were still detected, but there was a reduction of 3.1 log(10) for SRC and of 1.4 log(10) for MCB. At 96°C, Clostridium botulinum was identified among the thermotolerant MCB. Only those hygienic risks relating to mesophilic vegetative bacteria can be totally eliminated from pig slurry treated at 60°C (60 min) or 70°C (<10 min). Hygiene standards based on the removal of the indicators E. coli and enterococci can easily be met by treatment as low as 60°C (enabling, a low-cost treatment using heat recovery). However, even at 96°C, certain pathogens may persist.

The removal of microorganisms and organic micropollutants from wastewater during infiltration to aquifers after irrigation of farmland in the Tula Valley, Mexico

The Tula Valley receives untreated wastewater from Mexico City for agricultural irrigation, half of which infiltrates to aquifers from where drinking water is extracted. Samples of wastewater and infiltrated water from three areas of the valley were analyzed for microorganisms, organic micropollutants, and some basic parameters. Concentrations of microorganisms in the infiltrated water were generally very low but the incidence of fecal coliforms (present in 68% of samples), somatic bacteriophages (36%), Giardia spp. (14%), and helminth eggs (8%) suggested a health risk. Organic micropollutants, often present at high concentrations in the wastewater, were generally absent from the infiltrated water except carbamazepine which was in 55% of samples (up to 193 ng/L). There was no correlation between carbamazepine concentrations and the presence of microorganisms but highest concentrations of carbamazepine and boron coincided. A treatment such as nanofiltration would be necessary for the infiltrated water to be a safe potable supply.

Nationwide Groundwater Surveillance of Noroviruses in South Korea, 2008

To inspect the norovirus contamination of groundwater in South Korea, a nationwide study was performed in the summer (June to August) and winter (October to December) of 2008. Three-hundred sites designated by the government ministry were inspected. Water samples were collected for analysis of water quality, microorganism content, and viral content. Water quality was assessed by temperature, pH, turbidity, residual chlorine, and nitrite nitrogen content. Microorganism contents were analyzed bacteria, total coliforms, Escherichia coli, and bacteriophage. Virus analyses included panenterovirus and norovirus. Two primer sets were used for the detection of norovirus genotypes GI and GII, respectively. Of 300 samples, 65 (21.7%) were norovirus positive in the summer and in 52 (17.3%) were norovirus positive in the winter. The genogroup GI noroviruses that were identified were GI-1, GI-2, GI-3, GI-4, GI-5, GI-6, and GI-8 genotypes; those in the GII genogroup were GII-4 and GII-Yuri genotypes. The analytic data showed correlative relationships between the norovirus detection rate and the following parameters: water temperature and turbidity in physical-chemical parameters and somatic phage in microbial parameters. It is necessary to periodically monitor waterborne viruses that frequently cause epidemic food poisoning in South Korea for better public health and sanitary conditions.