Single Agar Layer Research Articles

Comparison of methods for the enumeration of coliphages in 100 mL water samples

In the last decade coliphages have been included in many water quality regulations as viral faecal indicators. However, the standardised methods used to detect and quantify coliphages differ in bacterial host strains, culture media and techniques. In this comparative study, 100 mL samples of mineral drinking water, river water and wastewater were analysed with International Organization for Standardization (ISO) standard methods, with United States-Environmental Protection Agency (U.S. EPA) based methods as well as commercial kits combining a single agar layer (SAL) assay with ISO bacterial host strains. The three methods gave similar counts (p-value>0.05) for somatic and total coliphages in the matrices with less than 100 PFU/100 mL, whereas for F-specific coliphages, the U.S. EPA method provided statistically significant lower numbers (p-value<0.05) than the other two protocols, possibly because it uses a different bacterial host strain (Escherichia coli HS (pFamp) R vs. the ISO strain Salmonella enterica serovar Typhimurium WG49). In samples with more than 100 PFU/100 mL, the ISO method yielded higher counts of somatic coliphages than the other two protocols (p-value<0.05). As the three methods provided similar results in clean water, the approach combining a SAL assay with the ISO bacterial host strain could be a useful option for coliphage analysis in this type of sample, as it does not require a concentration step.

Isolation and identification of specific bacteriophages against methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamases-producing Escherichia coli, extended-spectrum beta-lactamases-producing Klebsiella pneumoniae, and multidrug-resistant Acinetobacter baumanniiin vitro.

Drug resistance of methicillin-resistant Staphylococcus aureus, extended-spectrum beta-lactamases-producing Escherichia coli and Klebsiella pneumoniae and multidrug-resistant Acinetobacter baumannii are also cited as one of the most important causes of community and hospital acquired infections. Phage therapy can be used as a therapeutic method for the treatment of infections caused by these bacteria. The aim of this study was to isolate bacteriophages from municipal wastewater and assess their effects against drug resistant bacterial strains. The single agar layer technique was used to investigate the bacteriolytic effect of bacteriophages. Then, the double agar layer technique was used to observe phage plaques and the transmission electron microscopy was used to study the morphology of the bacteriophages. Transparent plaque formation in a double agar layer test of methicillin-resistant S. aureus and extended-spectrum beta-lactamases-producing E. coli and K. pneumoniae indicated the lysis of bacterial cells by isolated bacteriophages. No bacteriophage against A. baumannii was isolated from municipal wastewater. The morphology of these bacteriophages was also identified by electron microscopy. The results of this study showed that bacteriophages act specifically and due to the increasing level of antibiotic resistance, phage therapy as a new treatment can open a new horizon for the treatment of multidrug resistant bacteria.

Performance evaluation of a dead-end hollowfiber ultrafiltration method for enumeration of somatic and F+ coliphage from recreational waters

Dead-end hollow fiber ultrafiltration combined with a single agar layer assay (D-HFUF-SAL) has potential use in the assessment of sanitary quality of recreational waters through enumeration of coliphage counts as measures of fecal contamination. However, information on applicability across a broad range of sites and water types is limited. Here, we tested the performance of D-HFUF-SAL on 49 marine and freshwater samples. Effect of method used to titer the spiking suspension (SAL versus double agar layer [DAL]) on percent recovery was also evaluated. Average somatic coliphage recovery (72 % ± 27) was significantly higher (p < 0.0001) compared to F+ (53 % ± 19). This was more pronounced for marine (p ≤ 0.0001) compared to freshwaters (p = 0.0134). Neither method affected somatic coliphage, but DAL (28 % ± 12) significantly (p < 0.0001) underestimated F + coliphage recoveries compared to SAL (53 % ± 19). Overall, results indicate that, while D-HFUF-SAL performed well over a wide variety of water types, F + coliphage recoveries were significantly reduced for marine waters suggesting that some components unique to this habitat may interfere with the assay performance. More importantly, our findings indicate that choice of spike titer method merits careful consideration since it may under-estimate method percent recovery.

Virological Quality of Urban Rivers and Hospitals Wastewaters in Addis Ababa, Ethiopia

Background:Polluted rivers and hospital wastewater become a greater concern because of their public health and environmental hazards with high tendency to result in epidemics.Methods and Materials:The current study investigated 84 samples of Urban rivers and 30 samples of hospitals wastewaters in Addis Ababa, Ethiopia between February and April, 2017. The simultaneous detection of male-specific and somatic Coliphages from the samples was carried out usingEscherichia coliCB390 as the host according to the single agar layer plaque assay at public health microbiology laboratory of Ethiopian Public Health Institute.Results:Of the total 114 samples tested, coliphages were detected in 44 (52.4%) and 3 (10%) samples of urban rivers and hospital waste waters, respect ively. Total coliphages enumerations ranged from &lt;1pfu/100ml to 5.2×103pfu/100ml for urban rivers and &lt;1pfu/100ml to 4.92×103pfu/100ml for hospitals wastewaters.Conclusion:The detection of total coliphages in our study settings warrants the possibility that the pollution of urban rivers and hospital wastewaters may be a source for pathogenic viral infections. Unless coliphages, viral and fecal indicators are also examined in the waters by public health agencies, waterborne infections cause a major risk to public health.

Comparison of somatic and F+ coliphage enumeration methods with large volume surface water samples

Coliphages are alternative fecal indicators that may be suitable surrogates for viral pathogens, but majority of standard detection methods utilize insufficient volumes for routine detection in environmental waters. We compared three somatic and F+ coliphage methods based on a paired measurement from 1 L samples collected from the Great Lakes (n = 74). Methods include: 1) dead-end hollow fiber ultrafilter with single agar layer (D-HFUF-SAL); 2) modified SAL (M-SAL); and 3) direct membrane filtration (DMF) technique. Overall, D-HFUF-SAL outperformed other methods as it yielded the lowest frequency of non-detects [(ND); 10.8%] and the highest average concentrations of recovered coliphage for positive samples (2.51 ± 1.02 [standard deviation, SD] log10 plaque forming unit/liter (PFU/L) and 0.79 ± 0.71 (SD) log10 PFU/L for somatic and F+, respectively). M-SAL yielded 29.7% ND and average concentrations of 2.26 ± 1.15 (SD) log10 PFU/L (somatic) and 0.59 ± 0.82 (SD) log10 PFU/L (F+). DMF performance was inferior to D-HFUF-SAL and M-SAL methods (ND of 65.6%; average somatic coliphage concentration 1.52 ± 1.32 [SD] log10 PFU/L, no F+ detected), indicating this procedure is unsuitable for 1 L surface water sample volumes. This study represents an important step toward the use of a coliphage method for recreational water quality criteria purposes.

Use of coliphages to investigate norovirus contamination in a shellfish growing area in Republic of Korea.

A number of severe norovirus outbreaks due to the consumption of contaminated shellfish have been reported recently. In this study, we evaluated the distribution of coliphage densities to determine their efficacy as fecal indicators of enteric viruses, including noroviruses, in water samples collected from a shellfish growing area in Republic of Korea over a period of approximately 1year. Male-specific and somatic coliphages in water samples were analyzed using the single agar layer method, and norovirus genogroups I and II, which infect mainly humans, were analyzed using duplex reverse transcription quantitative PCR. Male-specific and somatic coliphages were detected widely throughout the study area. Several environmental parameters, including salinity, precipitation, temperature, and wind speed were significantly correlated with coliphage concentrations (P < 0.05). Moreover, the concentrations of male-specific coliphages were positively correlated with the presence of human noroviruses (r = 0.443; P < 0.01). The geospatial analysis with coliphage concentrations using a geographic information system revealed that densely populated residential areas were the major source of fecal contamination. Our results indicate that coliphage monitoring in water could be a useful approach to prevent norovirus contamination in shellfish.

Incidence of somatic and F+ coliphage in Great Lake Basin recreational waters

There is a growing interest for the use of coliphage as an alternative indicator to assess fecal pollution in recreational waters. Coliphage are a group of viruses that infect Escherichia coli and are considered as potential surrogates to infer the likely presence of enteric viral pathogens. We report the use of a dead-end hollow fiber ultrafiltration single agar layer method to enumerate F+ and somatic coliphage from surface waters collected from three Great Lake areas. At each location, three sites (two beaches; one river) were sampled five days a week over the 2015 beach season (n = 609 total samples). In addition, culturable E. coli and enterococci concentrations, as well as 16 water quality and recreational area parameters were assessed such as rainfall, turbidity, dissolved oxygen, pH, and ultra violet absorbance. Overall, somatic coliphage levels ranged from non-detectable to 4.39 log10 plaque forming units per liter and were consistently higher compared to F+ (non-detectable to 3.15 log10 PFU/L), regardless of sampling site. Coliphage concentrations weakly correlated with cultivated fecal indicator bacteria levels (E. coli and enterococci) at 75% of beach sites tested in study (r = 0.28 to 0.40). In addition, ultraviolet light absorption and water temperature were closely associated with coliphage concentrations, but not fecal indicator bacteria levels suggesting different persistence trends in Great Lake waters between indicator types (bacteria versus virus). Finally, implications for coliphage water quality management and future research directions are discussed.

Concentration and quantification of somatic and F+ coliphages from recreational waters

Somatic and F+ coliphages are promising alternative fecal indicators, but current detection methods are hindered by lower levels of coliphages in surface waters compared to traditional bacterial fecal indicators. We evaluated the ability of dead-end hollow fiber ultrafiltration (D- HFUF) and single agar layer (SAL) procedure to concentrate and enumerate coliphages from 1L and 10L volumes of ambient surface waters (lake, river, marine), river water with varying turbidities (3.74–118.7 NTU), and a simulated combined sewer overflow (CSO) event. Percentage recoveries for surface waters were 40–79% (somatic) and 35–94% (F+). The method performed equally well in all three matrices at 1L volumes, but percent recoveries were significantly higher in marine waters at 10L volumes when compared to freshwater. Percent recoveries at 1L and 10L were similar, except in river water where recoveries were significantly lower at higher volume. In highly turbid waters, D-HFUF-SAL had a recovery range of 25–77% (somatic) and 21–80% (F+). The method produced detectable levels of coliphages in diluted wastewater and in unspiked surface waters, emphasizing its applicability to CSO events and highlighting its utility in recovery of low coliphage densities from surface waters. Thus D-HFUF-SAL is a good candidate method for routine water quality monitoring of coliphages.

Comparison the effectiveness of using a magnetic field to control theColi phages isolated from rivulet water with water-treatment using magnetic field added iron filings

The present study aimed to use the magnetic field and nanotechnology in the field of water purification, which slots offering high efficiency to the possibility of removing biological contaminants such as viruses and bacteria rather than the use of chemical and physical transactions such as chlorine and bromine, and ultraviolet light and boiling and sedimentation and distillation, ozone and others that have a direct negative impact on human safety and the environment. Where they were investigating the presence in water samples under study Coli phages using Single agar layer method and then treated samples positive for phages to three types of magnetic field fixed as follows (North Pole - South Pole - Bipolar) and compare the results with samples of water treatment in the same conditions of the magnetic field with the addition of powder iron filings stated the results to the act synergistic to use a magnetic field with iron filling efficiency up to 100% better than the use of a magnetic field alone, where the disappearance of the emergence of spots Plaques treatment in the second. thus enhancing the possibility of making iron pipe nanoparticles to pump water in the treatment of water due to the high surface area (surface / volume). It is suggested that this could be used in the future in a wide range of water purification may be the best option for waste water treatment.

Comparison the effectiveness of using a magnetic field to control theColi phages isolated from rivulet water with water-treatment using magnetic field added iron filings

The present study aimed to use the magnetic field and nanotechnology in the field of water purification, which slots offering high efficiency to the possibility of removing biological contaminants such as viruses and bacteria rather than the use of chemical and physical transactions such as chlorine and bromine, and ultraviolet light and boiling and sedimentation and distillation, ozone and others that have a direct negative impact on human safety and the environment. Where they were investigating the presence in water samples under study Coli phages using Single agar layer method and then treated samples positive for phages to three types of magnetic field fixed as follows (North Pole - South Pole - Bipolar) and compare the results with samples of water treatment in the same conditions of the magnetic field with the addition of powder iron filings stated the results to the act synergistic to use a magnetic field with iron filling efficiency up to 100% better than the use of a magnetic field alone, where the disappearance of the emergence of spots Plaques treatment in the second. thus enhancing the possibility of making iron pipe nanoparticles to pump water in the treatment of water due to the high surface area (surface / volume). It is suggested that this could be used in the future in a wide range of water purification may be the best option for waste water treatment.

Surveillance of enteric viruses and coliphages in a tropical urban catchment

An assessment of the occurrence and concentration of enteric viruses and coliphages was carried out in highly urbanized catchment waters in the tropical city-state of Singapore. Target enteric viruses in this study were noroviruses, adenoviruses, astroviruses and rotaviruses. In total, 65 water samples were collected from canals and the reservoir of the Marina catchment on a monthly basis over a period of a year. Quantitative PCR (qPCR) and single agar layer plaque assay (SAL) were used to enumerate target enteric viruses and coliphages in water samples, respectively. The most prevalent pathogen were noroviruses, detected in 37 samples (57%), particularly norovirus genogroup II (48%), with a mean concentration of 3.7 × 102 gene copies per liter. Rotavirus was the second most prevalent virus (40%) with a mean concentration of 2.5 × 102 GC/L. The mean concentrations of somatic and male-specific coliphages were 2.2 × 102 and 1.1 × 102 PFU/100 ml, respectively. The occurrence and concentration of each target virus and the ratio of somatic to male-specific coliphages varied at different sampling sites in the catchment. For sampling sites with higher frequency of occurrence and concentration of viruses, the ratio of somatic to male-specific coliphages was generally much lower than other sampling sites with lower incidences of enteric viruses. Overall, higher statistical correlation was observed between target enteric viruses than between enteric viruses and coliphages. However, male-specific coliphages were positively correlated with norovirus concentrations. A multi-level integrated surveillance system, which comprises the monitoring of bacterial indicators, coliphages and selected enteric viruses, could help to meet recreational and surface water quality criteria in a complex urbanized catchment.

Comparison of methods for the detection of coliphages in recreational water at two California, United States beaches

Methods for detection of two fecal indicator viruses, F+ and somatic coliphages, were evaluated for application to recreational marine water. Marine water samples were collected during the summer of 2007 in Southern California, United States from transects along Avalon Beach (n=186 samples) and Doheny Beach (n=101 samples). Coliphage detection methods included EPA method 1601 - two-step enrichment (ENR), EPA method 1602 - single agar layer (SAL), and variations of ENR. Variations included comparison of two incubation times (overnight and 5-h incubation) and two final detection steps (lysis zone assay and a rapid latex agglutination assay). A greater number of samples were positive for somatic and F+ coliphages by ENR than by SAL (p<0.01). The standard ENR with overnight incubation and detection by lysis zone assay was the most sensitive method for the detection of F+ and somatic coliphages from marine water, although the method takes up to three days to obtain results. A rapid 5-h enrichment version of ENR also performed well, with more positive samples than SAL, and could be performed in roughly 24h. Latex agglutination-based detection methods require the least amount of time to perform, although the sensitivity was less than lysis zone-based detection methods. Rapid culture-based enrichment of coliphages in marine water may be possible by further optimizing culture-based methods for saline water conditions to generate higher viral titers than currently available, as well as increasing the sensitivity of latex agglutination detection methods.

F+ RNA coliphage-based microbial source tracking in water resources of South Korea

We previously demonstrated that genotyping followed by proper statistical analyses of F plus (F+)-specific RNA coliphages can effectively represent fecal origins of either humans or animals. Here, we performed microbial source tracking (MST) using F+ RNA coliphages as a target MST microorganism for identifying fecal sources contaminating ground and surface water in metropolitan Seoul and Gyeonggi Province in South Korea. In total, 71 groundwater and 5 surface water samples were collected and screened for the presence of F+ RNA coliphages. More than 124 F+ coliphages were isolated from six groundwater and five surface water samples by the single agar layer method. F+ RNA coliphages were predominant in both waters (100% and 91%, respectively). Genotyping of 118 F+ RNA coliphages revealed that most (51/60) of the groundwater F+ RNA coliphages belonged to group I, whereas both groups I (25/58) and IV (31/58) were predominantly observed in surface water. Further comparison of phage isolates from human and animal (pig, cow, goose, and chicken) fecal sources using nucleic acid sequencing and principal coordinate analysis showed that groundwater samples formed clusters associated with cow feces, whereas surface waters formed clusters related to chicken and human feces. These results indicate the potential of the F+ RNA coliphage-based MST for identifying fecal contamination sources, which may be further exploited and validated in different geographical regions of the world.

Use of Coliphages as an indicators of enteroviruses and faecal pollution in Water

In spite of increasing clinical cases which caused by enteroviruses transferred by water and no documents about entericviruses in the Iraqi water standards. The use of coliphages as an indicator of enteroviruses and fecal pollution were suggested two procedures were applied . The first is Two-Step Enrichment Method and the second is Single Agar Layer Method. Both methods gives good results in Identification of coliphages through testing fifty different water samples (Tap water, Surface water and Bottled water) the study shows the presence of coliphages in fourteen samples.

Use of Coliphages as an indicators of enteroviruses and faecal pollution in Water

In spite of increasing clinical cases which caused by enteroviruses transferred by water and no documents about entericviruses in the Iraqi water standards. The use of coliphages as an indicator of enteroviruses and fecal pollution were suggested two procedures were applied . The first is Two-Step Enrichment Method and the second is Single Agar Layer Method. Both methods gives good results in Identification of coliphages through testing fifty different water samples (Tap water, Surface water and Bottled water) the study shows the presence of coliphages in fourteen samples.

Microbial Fecal Indicator Concentrations in Water and Their Correlation to Environmental Parameters in Nine Geographically Diverse Estuaries

Fecal contamination of coastal recreational water is a public health concern, which coastal managers monitor using fecal indicator bacteria (FIB). In this study, 20 stations in nine United States (US) estuaries were monitored over three years for FIB (enterococci, fecal coliforms, Escherichia coli, and Clostridium perfringens) and fecal indicator virus (F+ coliphage and somatic coliphage) concentrations in water. Fecal indicator concentrations were significantly correlated with some environmental variables (rainfall, water temperature, salinity, and sample collection time) across all estuaries. Multilinear models utilizing environmental parameters predicted anywhere from 69% (C. perfringens) to 19% (F+ coliphages) of fecal indicator variability. Coliphage equivalence to US Environmental Protection Agency marine water enterococci criteria was estimated with 95% confidence intervals, though these findings should be interpreted with caution because enterococci densities were weakly positively correlated to coliphage densities (R=0.435 somatic coliphages, R=0.367 F+ coliphages). In a comparison of coliphage methods, enrichment (EPA Method 1601) outperformed single agar layer (EPA Method 1602) and membrane filtration. In summary, the relationships among coliphage methods, between fecal indicators, and their interactions with environmental variables represent findings from a geographically diverse set of US estuaries with different fecal waste sources and loads, which suggests these findings are highly generalizable.

지표미생물을 이용한 시화호 유입수의 수질평가

Lake Shiwha, an artificial lake located near metropolitan Seoul, offers a unique water environment and has been suspected to have high levels of chemical and microbiological contaminations. Lake Shiwha was originally connected to the sea but currently has four major surface water inputs from agricultural, municipal, industrial areas and in addition an occasional inflow from the sea. The objectives of this study are to investigate the relative contribution of microbial contaminants from each of the inflowing surface waters and to identify appropriate microbial indicator organisms in this unique water environment. We measured the levels of microbial contaminations in the four inflowing surface waters. A number of microbial indicator organisms including total coliform (TC), fecal coliform (FC), E. coli, Enterococci, somatic and male-specific coliphages were analyzed. Bacterial indicator microorganisms were detected and quantified by the <TEX>$Colilert^{(R)},\;Enterolert^{(R)}$</TEX> kit. Surface water (50 l) was sampled by <TEX>$ViroCap^{TM}\;5"$</TEX> cartridge filters and analyzed by the single agar layer method for detecting coliphages. The concentrations of TC, FC, E. coli, and Enterococci were 1543 CFU/100 ml<TEX>${\sim}1.99{\times}10^6$</TEX> CFU/100 ml, 0 CFU/100 ml<TEX>${\sim}202$</TEX> CFU/100ml, 0 CFU/100 ml<TEX>${\sim}1.80{\sim}10^5$</TEX> CFU/100ml, 74 CFU/100 ml<TEX>${\sim}3408$</TEX> CFU/100 ml, respectively. The male-specific and somatic coliphages were detected in three different inflowing surface waters. Isolated E. coli and Enterococci strains were further analyzed by 16s rDNA amplification and subsequent phylogenetic analysis from Jungwang-chun, Ansan-chun, Banwol-chun and penstock of inflowing surface water. Our results indicated that the concentrations of different fecal indicator microorganisms might not be highly correlated with each other. Multiple microbial indicator organisms should be used for monitoring microbial contamination and microbial source tracking methods.

F+RNA coliphage typing for microbial source tracking in surface waters

The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. Coliphages were isolated from 117 surface water samples by single agar layer (SAL) and enrichment presence/absence (EP/A) methods. Confirmed F+ RNA coliphages were typed for microbial source tracking using a library-independent approach. Concentrations of somatic coliphages using 37 and 44.5 degrees C incubation temperatures were found to be significantly different and the higher temperature may be more specific for faecal contamination. The EP/A technique detected coliphages infecting Escherichia coli Famp in 38 (66%) of the 58 surface water samples negative for F+ coliphages by the SAL method. However, coliphages isolated by EP/A were found to be less representative of coliphage diversity within a sample. Among the 2939 coliphage isolates tested from surface water and known source samples, 813 (28%) were found to be F+ RNA. The majority (94%) of surface water F+ RNA coliphage isolates typed as group I. Group II and/or III viruses were identified from 14 surface water stations, the majority of which were downstream of wastewater discharges. These sites were likely contaminated by human-source faecal pollution. The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. This study supports the premise that coliphage typing can provide useful, but not absolute, information to distinguish human from animal sources of faecal pollution. Furthermore, the comparison of coliphage isolation methods detailed in this study should provide valuable information to those wishing to incorporate coliphage detection into water quality assessments.

Development and evaluation of methods to detect coliphages in large volumes of water

New and improved methods have been developed to detect somatic and male-specific coliphages in large volumes of water by single agar layer (SAL), enrichment and membrane filter methods. Somatic coliphages were detected efficiently on E. coli hosts C and CN13, male-specific coliphages were detected more efficiently on E. coli Famp than on Salmonella typhimurium WG49 and both types of coliphages were detected simultaneously on E. coli C3000. For water volumes of up to 100 ml, the SAL method was efficient and reliable. For water volumes of <1 L and as many as 10 multiple 1 L volumes, the enrichment method was efficient in detecting very low numbers of coliphages. Membrane filter methods, in which coliphages were adsorbed to and eluted from filters, also were relatively efficient, but they were less efficient than SAL and enrichment methods and were considered to be more cumbersome. For filter adsorption-elution methods, coliphage recoveries were most efficient for cellulose ester filters, less efficient for electropositive 1 MDS filters and least efficient for a direct membrane filter method. Overall, the enrichment method was preferred because of its ability to easily and rapidly detect low levels of coliphages in large sample volumes by either presence-absence or most probable number quantification.

Optimisation of the ISO-method on enumeration of somatic coliphages (draft ISO 10705-2)

As part of the EU project "Bacteriophages in Bathing Waters" (January 1996-June 1999) research was carried out to optimise the method for detection and enumeration of somatic coliphages in water as described in ISO/CD 10705-2 of August 1995. It was concluded that this draft ISO standard needed to be amended in certain aspects. For determining the viable count of the host culture WG5 Escherichia coli, a membrane filtration technique should be used instead of spread plate technique as the latter gives lower and less reproducible results. A freshly prepared inoculum culture of host strain WG5 should be used instead of a frozen inoculum culture as freezing of the inoculum culture is found to negatively influence the phage counts. The double agar layer method (DAL) is preferred to the single agar layer method (SAL) for performing the phage analysis as the DAL method gives higher phage counts than the SAL method.