Methodology for enumeration of coliphages in foods

The effects of eluent composition, pH, and chaotropic agents on the recovery of T2, MS2, and indigenous coliphages from various foods were investigated. Additionally, methods of sample suspension and clarification were evaluated for coliphage recovery and application to various foods. Clarified sample suspensions were assayed for coliphages with a modified agar layer technique and appropriate Escherichia coli hosts. Centrifugation and polypropylene mesh filtration were more rapid and effective than glass wool filtration for clarification of sample suspensions and subsequent recovery of coliphages. Blending, stomaching, and shaking procedures were generally comparable for sample liquefaction and release of coliphages from foods. Complex basal eluents, EC medium and 1% casein, were generally more effective than a less complex eluent, phosphate buffer, for elution of coliphages from foods. For most foods, incorporation of sodium chloride or chaotropic agents, i.e., sodium trichloroacetate, urea, Tween 80, Triton X-100, and sodium nitrate, into basal eluents did not enhance recovery of coliphages. Indigenous coliphage recovery was not affected by sample suspension pH over a range of 6.0 to 9.0. With an optimal procedure, i.e., EC medium eluent, blending, and centrifugation, the recovery of T2 and MS2 ranged from 48 to 81% and from 58 to 100%, respectively, depending on the food type.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water.

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

Comparison of selective media for assay of coliphages in sewage effluent and lake water

Selective media, including EC medium, gram-negative broth, nutrient broth (with 0.05% sodium deoxycholate), and lactose broth (with 0.05% sodium deoxycholate), as well as nonselective nutrient and lactose broths, were compared for the enumeration of coliphages by the agar layer method from activated-sludge effluent and eutrophic-lake water from a lake receiving treated sewage effluent. Samples were plated directly or after chloroform treatment with Escherichia coli B, E. coli C, or a mixed host of both E. coli B and C. With the exception of gram-negative broth, direct assays of all samples with the selective media generally resulted in significantly higher (P less than 0.05) recoveries of coliphages than did assays of chloroform-treated samples with nutrient broth medium regardless of the host used. In addition, chloroform pretreatment resulted in decreased recovery of coliphages with each selective medium in most analyses. The highest recoveries of coliphages from all samples with each host, except lake water with E. coli C, were obtained by direct assay on EC medium. The selectivity of the EC and gram-negative media resulted in suppression of bacterial interference on direct assay plates comparable to that observed in nutrient agar medium with chloroform-treated samples. The use of certain selective media for the direct assay of environmental materials for coliphage may enhance the recovery of coliphages and obviate bacterial decontamination procedures.     

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

AIMS: The utility of coliphages to detect and track faecal pollution was evaluated using South Carolina surface waters that exceeded State faecal coliform standards. METHODS AND RESULTS: 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. CONCLUSIONS: The results suggest that faecal contamination in surface waters can be detected and source identifications aided by coliphage analyses. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.     

Pretreatment to reduce somatic salmonella phage interference with FRNA coliphage assays: successful use in a one-year survey of vulnerable groundwaters

Somatic salmonella (SS) phages were commonly found in higher numbers than F-specific RNA (FRNA) coliphages in a multi-site survey of contamination-vulnerable groundwaters. The relative abundance of SS phages required that a pretreatment procedure be implemented to reduce the SS phage content of samples before FRNA coliphage assay with Salmonella typhimurium WG49. Pretreatment involved selective SS phage removal by Salm. typhimurium WG45 cells. This pretreatment proved effective in producing interference-free samples throughout the one-year survey period and in seeded evaluation, was shown not to affect the detection of representative FRNA coliphage MS2. During the survey, 30 groundwater sites located in the continental United States, Puerto Rico and the Virgin Islands were examined for FRNA coliphages and SS phages at monthly intervals. FRNA coliphages were detected at six of the 30 sites and in 33 of 329 monthly samples. SS phages were also detected at six sites and in 28 of 329 monthly samples. Five of the phage-positive sites were positive for both phage groups. At those five sites, 58 monthly samples were collected during the survey period. Those 58 samples yielded an average FRNA coliphage concentration of 140 pfu per 100 1 of groundwater as compared to an average SS phage concentration of 565 pfu per 100 1 of groundwater. Twenty of the 58 samples were positive for both FRNA coliphages and SS phages. In those samples, FRNA coliphages were more abundant in five samples; SS phages were more abundant in 15 samples. Because these results demonstrate that SS phage levels may often exceed FRNA coliphage levels in environmental waters, it is clear that SS phage removal procedures will greatly enhance the effectiveness of the WG49-based FRNA coliphage assay.     

Evaluation of F+ RNA and DNA coliphages as source-specific indicators of fecal contamination in surface waters

Male-specific (F+) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F+ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F+ DNA and group II and III F+ RNA coliphages. Bovine wastewaters also contained a high proportion of F+ DNA coliphages, but group I and IV F+ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F+ DNA and RNA coliphages, and group I and III F+ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F+ RNA coliphages of groups I and IV. No F+ coliphages were isolated from the feces of the other species examined. F+ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F+ DNA and RNA coliphages. Group I F+ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F+ RNA coliphages detected was greatest at background sites, and the percentage of group II F+ RNA coliphages was highest at human-impacted sites. Monitoring of F+ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.     

Direct serum injection in micellar liquid chromatography : Recovery of serum proteins and assay of hydrophilic drugs

The recovery of serum proteins from reversed-phase and internal-surface reversed-phase (ISRP) silica supports following direct serum injection was investigated using an eluent containing a micellar solution of sodium dodecyl sulphate (SDS). The results indicated that the recoveries of serum proteins were 98–103% for both supports. On the basis of the above findings, the separation and recovery of hydrophilic drugs (cephalosporins and salicylic acid) from human serum were investigated using acidic eluents including micellar solutions of SDS. They were completely separated from the components of serum, and the recoveries were 94–98% despite protein binding. Although the recommended eluent pH range is 6.0–7.5 for the ISRP support, eluents of pH 2–8 can be used with the micellar chromatographic system.     

应用TFF微孔过滤澄清白喉、破伤风毒素培养液及超滤浓缩的试验

用ＴＦＦ微孔过滤系统对白喉、破伤风毒素培养液分别进行了两次澄清过滤试验,澄清过滤后的毒素上清液立即用ＴＦＦ超滤系统浓缩至原培养液体积的１／１０－１／２０。白喉毒素总回收率分别为８６％、７６．６％,破伤风毒素总回收率为９４．１％、９２％。澄清过滤中白喉毒素平均Ｆ１ｕｘ分别为２３．４、１４．３Ｌ／ｍ２／ｈ,破伤风毒素平均Ｆｌｕｘ为２４及２２．９Ｌ／ｍ２／ｈ。结果表明破伤风两次试验有很好的一致性,白喉毒素回收率差别不甚明显,而两次试验Ｆｌｕｘ相差较大,ＴＦＦ微孔过滤系统用于澄清白喉培养液,滤膜使用后的清洗程序仍需改良,以提高膜滤过功能的恢复。     

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.     

Effects of pyrrol-carboxylic acid derivatives on the growth of coliphages.

Effects of 14 pyrrol-carboxylic acid derivatives and analogues (PY-compounds) on the growth of coliphage MS2 using E. coli E102 (Hfr) as the host were measured by the agar double-layer method. Enlargements of plaque size were observed with 7 PY-compounds but increase in plaque numbers was not induced. These enlargements of plaque size were specific to RNA coliphages MS2, GA and qbeta and not found with DNA coliphages delta AC and T4. Furthermore, the interaction between PY-compound PY-10 and the coliphage MS2 was dependent on the host bacterium (indicator strain). When E102 (Hfr) was used, the enlargement was marked, in the case of substrain W1895 (Hfr) it was less, while in the case of substrain W6 (F+) it was undetectable. The one-step growth of the phage MS2 and the production of intracellular phage MS2 were little affected by the PY-compound PY-10. However, the rate of one-step growth was increased in the early stage after infection. Accordingly, the enlargements of plaque size by the PY-compounds might be correlated with an increase in rate of release of phage particles.     

Ecology of coliphages in southern California coastal waters

Aims: This study aims to investigate the ecology of coliphages, an important microbial pollution indicator. Specifically, our experiments address (i) the ability of environmental Escherichia coli (E. coli) to serve as hosts for coliphage replication, and (ii) the temporal and spatial distribution of coliphages in coastal waters. Methods and Results: Water samples from three locations in California’s Newport Bay watershed were tested for the presence of coliphages every 2 weeks for an entire year. A total of nine E. coli strains isolated from various sources served as hosts for coliphage detection. Coliphage occurrence was significantly different between freshwater, estuarine and coastal locations and correlated with water temperature, salinity and rainfall in the watershed. The coliphages isolated on the environmental hosts had a broad host‐range relative to the coliphages isolated on an E. coli strain from sewage and a US EPA recommended strain for coliphage detection. Conclusions: Coliphage occurrence was related to the temperature, rainfall and salinity within the bay. The adaptation to a broad host‐range may enable the proliferation of coliphages in the aquatic environment. Significance and Impact of the Study: Understanding the seasonal variation of phages is useful for establishing a background level of coliphage presence in coastal waters. The broad host‐range of coliphages isolated on the environmental E. coli host calls for investigation of coliphage replication in the aquatic environment.     

Evaluation of F+ RNA and DNA Coliphages as Source-Specific Indicators of Fecal Contamination in Surface Waters

Male-specific (F+) coliphages have been investigated as viral indicators of fecal contamination that may provide source-specific information for impacted environmental waters. This study examined the presence and proportions of the different subgroups of F+ coliphages in a variety of fecal wastes and surface waters with well-defined potential waste impacts. Municipal wastewater samples had high proportions of F+ DNA and group II and III F+ RNA coliphages. Bovine wastewaters also contained a high proportion of F+ DNA coliphages, but group I and IV F+ RNA coliphages predominated. Swine wastewaters contained approximately equal proportions of F+ DNA and RNA coliphages, and group I and III F+ RNA coliphages were most common. Waterfowl (gull and goose) feces contained almost exclusively F+ RNA coliphages of groups I and IV. No F+ coliphages were isolated from the feces of the other species examined. F+ coliphage recovery from surface waters was influenced by precipitation events and animal or human land use. There were no significant differences in coliphage density among land use categories. Significant seasonal variation was observed in the proportions of F+ DNA and RNA coliphages. Group I F+ RNA coliphages were the vast majority (90%) of those recovered from surface waters. The percentage of group I F+ RNA coliphages detected was greatest at background sites, and the percentage of group II F+ RNA coliphages was highest at human-impacted sites. Monitoring of F+ coliphage groups can indicate the presence and major sources of microbial inputs to surface waters, but environmental effects on the relative occurrence of different groups need to be considered.     

Method for recovery of enteric viruses from estuarine sediments with chaotropic agents.

An evaluation was made of the ability of chaotropes, low-molecular-weight ionic compounds which enhance the solubilization of hydrophobic compounds in water, to improve the recovery of enteric viruses from highly organic estuarine sediments. Chaotropic agents alone were poor eluents of polioviruses from sediment but were effective when combined with 3% beef extract. Chaotropes of lower potency, NaNO3, NaCl, and KCl, were more efficient eluents than the stronger chaotropes, guanidium hydrochloride or sodium trichloroacetate. The most effective eluent was 2 M NaNO3 in 3% beef extract at pH 5.5, which eluted 71% of sediment-associated polioviruses. Efficient concentration of the sodium nitrate-beef extract eluate by organic flocculation required the addition of the antichaotrope (NH4)2SO4 to a 2 M concentration and Cat-Floc T (Calgon, Pittsburgh, Pa.) a cationic polyelectrolyte, to a 0.01% concentration. Dialysis of the final concentrate was necessary to reduce salts to nontoxic levels before assay in cell cultures. Trials with highly organic estuarine sediment seeded with high or low numbers of poliovirus 1, echovirus 1, or rotavirus SA-11 demonstrated the superiority of this method over two other methods currently in use.     

Comparison of microporous filters for concentration of viruses from wastewater.

The 1-MDS Virosorb filter and the 50S and 30S Zeta-plus filters, all with a net positive charge, were compared with the negatively charged Filterite filter for concentration of naturally occurring coliphages and animal viruses from sewage effluent. When Filterite filters were used, the effluent was adjusted to pH 3.5 and AlCl3 was added before filtration to facilitate virus adsorption. No adjustment was required with the positively charged filters. Sets of each filter type were eluted with 3% beef extract (pH 9.5) or eluted with 0.05 M glycine (pH 11.5). A maximum volume of 19 liters could be passed through 142-mm diameter Filterite filters before clogging, whereas only 11, 11, and 15 liters could be passed through the 1-MDS, 50S, and 30S filters, respectively. For equal volumes passed through the filters, coliphage recoveries were 14, 15, 18, and 37% in primary effluent and 40, 97, 50, and 46% in secondary effluent for the Filterite , 1-MDS, 50S, and 30S filters, respectively. No statistically significant difference was observed in the recovery of animal viruses among the filters from secondary effluent, whereas in the Filterite and 50S filters, higher numbers of viruses from primary effluent were recovered than in the 1-MDS and 30S filters in two of three collections. Glycine was found to be a less-efficient eluent than beef extract in the recovery of naturally occurring viruses.     

Comparison of microporous filters for concentration of viruses from wastewater

The 1-MDS Virosorb filter and the 50S and 30S Zeta-plus filters, all with a net positive charge, were compared with the negatively charged Filterite filter for concentration of naturally occurring coliphages and animal viruses from sewage effluent. When Filterite filters were used, the effluent was adjusted to pH 3.5 and AlCl3 was added before filtration to facilitate virus adsorption. No adjustment was required with the positively charged filters. Sets of each filter type were eluted with 3% beef extract (pH 9.5) or eluted with 0.05 M glycine (pH 11.5). A maximum volume of 19 liters could be passed through 142-mm diameter Filterite filters before clogging, whereas only 11, 11, and 15 liters could be passed through the 1-MDS, 50S, and 30S filters, respectively. For equal volumes passed through the filters, coliphage recoveries were 14, 15, 18, and 37% in primary effluent and 40, 97, 50, and 46% in secondary effluent for the Filterite , 1-MDS, 50S, and 30S filters, respectively. No statistically significant difference was observed in the recovery of animal viruses among the filters from secondary effluent, whereas in the Filterite and 50S filters, higher numbers of viruses from primary effluent were recovered than in the 1-MDS and 30S filters in two of three collections. Glycine was found to be a less-efficient eluent than beef extract in the recovery of naturally occurring viruses.     

A simplified method for coliphage detection in natural waters

The ARCAT (A Rapid Coliphage Analysis Technique) method for detecting coliphages in water has been modified. Modifications to the original method include media optimization, the use of frozen host cultures, the use of a single agar coliphage assay and optimized plaque resolution with 2, 3, 4-triphenyltetrazolium chloride. Detection of 5 coliphages per 100 ml of water sample is accomplished in 6 hours for rapid estimation of water quality.     

Inactivation of hepatitis A virus and indicator organisms in water by free chlorine residuals

Hepatitis A virus (HAV) and selected indicator organisms were mixed together in chlorine-demand-free buffers at pH 6, 8, or 10 and exposed to free chlorine residuals, and the survival kinetics of individual organisms were compared. HAV was enumerated by a most-probable-number dilution assay, using PLC/PRF/5 liver cells for propagation of the virus and radioimmunoassay for its detection. At all pH levels, HAV was more sensitive than Mycobacterium fortuitum, coliphage V1 (representing a type of phage common in some sewage-polluted waters), and poliovirus type 2. Under certain conditions, HAV was more resistant than Escherichia coli, Streptococcus faecalis, coliphage MS2, and reovirus type 3. It was always more resistant than SA-11 rotavirus. Evidence is presented that conditions generally specified for the chlorine disinfection of drinking-water supplies will also successfully inactivate HAV and that HAV inactivation by free chlorine residuals can reliably be monitored by practical indicator systems consisting of appropriate combinations of suitable indicators such as coliform and acid-fast bacteria, coliphages, the standard plate count, and fecal streptococci.     

The diversity of coliphages and coliforms in horse feces reveals a complex pattern of ecological interactions

The diversity of coliphages and indigenous coliform strains (ICSs) simultaneously present in horse feces was investigated by culture-based and molecular methods. The richness of coliforms (as estimated by the Chao1 method) is about 1,000 individual ICSs distinguishable by genomic fingerprinting present in a single sample of feces. This unexpectedly high value indicates that some factor limits the competition of coliform bacteria in the horse gut microbial system. In contrast, the diversity of phages active against any selected ICS is generally limited to one to three viral genotypes present in the sample. The sensitivities of different ICSs to simultaneously present coliphages overlap only slightly; the phages isolated from the same sample on different ICSs are usually unrelated. As a result, the titers of phages in fecal extract as determined for different Escherichia coli strains and ICSs may differ by several orders of magnitude. Summarizing all the data, we propose that coliphage infection may provide a selection pressure that maintains the high level of coliform diversity, restricting the possibility of a few best competitors outgrowing other ICSs. We also observed high-magnitude temporal variations of coliphage titers as determined using an E. coli C600 test culture in the same animal during a 16-day period of monitoring. No correlation with total coliform count was observed. These results are in good agreement with our hypothesis.     

Evaluation of positively charged alumina nanofibre cartridge filters for the primary concentration of noroviruses,adenoviruses and male‐specific coliphages from seawater

Aim: To evaluate the electropositive, alumina nanofibre (NanoCeram) cartridge filter as a primary concentration method for recovering adenovirus, norovirus and male‐specific coliphages from natural seawater. Methods and Results: Viruses were concentrated from 40 l of natural seawater using a NanoCeram cartridge filter and eluted from the filter either by soaking the filter in eluent or by recirculating the eluent continuously through the filter using a peristaltic pump. The elution solution consisted of 3% beef extract and 0·1 mol l^?1 of glycine. The method using a peristaltic pump was more effective in removing the viruses from the filter. High recoveries of norovirus and male‐specific coliphages (>96%) but not adenovirus (<3%) were observed from seawater. High adsorption to the filter was observed for adenovirus and male‐specific coliphages (>98%). The adsorption and recovery of adenovirus and male‐specific coliphages were also determined for fresh finished water and source water. Conclusion: The NanoCeram cartridge filter was an effective primary concentration method for the concentration of norovirus and male‐specific coliphages from natural seawater, but not for adenovirus, in spite of the high adsorption of adenovirus to the filter. Significance and Impact of the Study: This study demonstrates that NanoCeram cartridge filter is an effective primary method for concentrating noroviruses and male‐specific coliphages from seawater, thereby simplifying collection and processing of water samples for virus recovery.