Sunlight inactivation of fecal bacteriophages and bacteria in sewage-polluted seawater.

Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k(D)s), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20 degrees C) than in winter (temperature range: 8 to 10 degrees C). Winter k(D)s were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k(S)), as a function of cumulative global solar radiation (insolation), were all higher than the k(D)s with a consistent k(S) ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k(S)s similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k(S) ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters.     

Comparison of methods of enumerating coliforms after UV disinfection.

In view of the differences that have been found between the most-probable-number and membrane filtration methods for the recovery of coliforms from chlorinated samples, the survival of total and fecal coliforms in UV-irradiated effluent samples, as tested by the most-probable-number and standard single-step membrane filtration methods, was compared. There were no significant differences in the survival of total and fecal coliforms, as tested by the two methods. In a separate set of experiments comparing total and fecal coliform survival, as tested by the most-probable-number method, only a very small but statistically significant difference of 0.1 log survival units was found. For UV-disinfected wastewater effluents, standard one-step membrane filtration procedures are comparable to standard most-probable-number procedures.     

Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis: use in predicting the source of fecal contamination in subtropical waters

The antibiotic resistance patterns of fecal streptococci and fecal coliforms isolated from domestic wastewater and animal feces were determined using a battery of antibiotics (amoxicillin, ampicillin, cephalothin, chlortetracycline, oxytetracycline, tetracycline, erythromycin, streptomycin, and vancomycin) at four concentrations each. The sources of animal feces included wild birds, cattle, chickens, dogs, pigs, and raccoons. Antibiotic resistance patterns of fecal streptococci and fecal coliforms from known sources were grouped into two separate databases, and discriminant analysis of these patterns was used to establish the relationship between the antibiotic resistance patterns and the bacterial source. The fecal streptococcus and fecal coliform databases classified isolates from known sources with similar accuracies. The average rate of correct classification for the fecal streptococcus database was 62.3%, and that for the fecal coliform database was 63.9%. The sources of fecal streptococci and fecal coliforms isolated from surface waters were identified by discriminant analysis of their antibiotic resistance patterns. Both databases identified the source of indicator bacteria isolated from surface waters directly impacted by septic tank discharges as human. At sample sites selected for relatively low anthropogenic impact, the dominant sources of indicator bacteria were identified as various animals. The antibiotic resistance analysis technique promises to be a useful tool in assessing sources of fecal contamination in subtropical waters, such as those in Florida.     

Sunlight Inactivation of Enterococci and Fecal Coliforms in Sewage Effluent Diluted in Seawater

Inactivation (loss of culturability) by sunlight of enterococci and fecal coliforms within sewage effluent diluted in seawater was investigated in field experiments. In most experiments, 500-ml flasks of pure silica were used to confine activated sludge effluent diluted to 2% (vol/vol) in seawater. Inactivation of bacteria in these flasks (diameter, 0.1 m) was faster than in either open chambers (depth, 0.25 m) or patches of dyed effluent (depth of order, 1 m), probably because of the longer light paths in the latter two types of experiment, which caused greater attenuation of sunlight. Inactivation of 90% of enterococci generally required 2.3 times the insolation required for 90% inactivation of fecal coliforms, because of both the presence of larger initial shoulders on survival curves and a lower final inactivation rate. Two parameters are required to model inactivation of enterococci, a shoulder constant as well as a rate coefficient. The depth dependence of inactivation rate for both fecal indicators matched the attenuation profile of UV-A radiation at about 360 nm. Inactivation by UV-B radiation (290 to 320 nm), which penetrates much less into seawater, is of minor importance compared with the UV-A and visible radiation in sunlight, contrary to expectations in consideration of published action spectra for bacterial inactivation.     

Persistence and distribution of pollution indicator bacteria on land used for disposal of piggery effluent.

Numbers of pollution indicator bacteria (fecal coliforms and fecal streptococci) were assessed on land to which effluent from intensively housed pigs had been applied. Topsoil (to a 30-mm depth) was found to provide a more favorable environment for fecal coliform persistence than was pasture or subsoil. Times required for a 90% reduction in number (T90) in topsoil (calculated by linear regression of log counts obtained in a 6-week period after effluent application) ranged from 7 to 20 days (mean T90, 11 days). T90 values for fecal coliforms fell within this range irrespective of the season of application and for a number of soil types and climatic conditions. The range in die-off times was encountered irrespective of the fecal coliform count in the applied effluent or the application regimen (125 to 1,000 kg of elemental nitrogen in the form of effluent per ha; return periods, 3 to 12 months). Autumn and winter conditions were conducive to the persistence of a survivor tail of these bacteria at 10(1) to 10(3) cells per g of topsoil. Fecal streptococci survived similarly on soil and pasture (T90, ca. 14 days) and appeared slightly more suited to survival in the environment than did fecal coliforms. Contamination of subsoils after effluent applications occurred at a rate well in excess of the infiltration capacity of the soil, presumably by percolation of the effluent through soil cracks. Contamination levels of subsoils in the experimental area generally remained low.     

Sunlight Inactivation of Fecal Bacteriophages and Bacteria in Sewage-Polluted Seawater

Sunlight inactivation rates of somatic coliphages, F-specific RNA bacteriophages (F-RNA phages), and fecal coliforms were compared in seven summer and three winter survival experiments. Experiments were conducted outdoors, using 300-liter 2% (vol/vol) sewage-seawater mixtures held in open-top chambers. Dark inactivation rates (k_Ds), measured from exponential survival curves in enclosed (control) chambers, were higher in summer (temperature range: 14 to 20°C) than in winter (temperature range: 8 to 10°C). Winter k_Ds were highest for fecal coliforms and lowest for F-RNA phages but were the same or similar for all three indicators in summer. Sunlight inactivation rates (k_S), as a function of cumulative global solar radiation (insolation), were all higher than the k_Ds with a consistent k_S ranking (from greatest to least) as follows: fecal coliforms, F-RNA phages, and somatic coliphages. Phage inactivation was exponential, but bacterial curves typically exhibited a shoulder. Phages from raw sewage exhibited k_Ss similar to those from waste stabilization pond effluent, but raw sewage fecal coliforms were inactivated faster than pond effluent fecal coliforms. In an experiment which included F-DNA phages and Bacteroides fragilis phages, the k_S ranking (from greatest to least) was as follows: fecal coliforms, F-RNA phages, B. fragilis phages, F-DNA phages, and somatic coliphages. In a 2-day experiment which included enterococci, the initial concentration ranking (from greatest to least: fecal coliforms, enterococci, F-RNA phages, and somatic coliphages) was reversed during sunlight exposure, with only the phages remaining detectable by the end of day 2. Inactivation rates under different optical filters decreased with the increase in spectral cutoff wavelength (50% light transmission) and indicated that F-RNA phages and fecal coliforms are more susceptible than somatic coliphages to longer solar wavelengths, which predominate in seawater. The consistently superior survival of somatic coliphages in our experiments suggests that they warrant further consideration as fecal, and possibly viral, indicators in marine waters.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

Sunlight inactivation of fecal indicator bacteria and bacteriophages from waste stabilization pond effluent in fresh and saline waters

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14C (mean river water temperature). Sunlight inactivation (k(S)) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k(D)) rates in enclosed (control) chambers. The overall k(S) ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms greater-than-or-equal E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

Antibiotic resistance patterns of fecal coliforms isolated from domestic sewage before and after treatment in an aerobic lagoon.

The resistance of 260 strains of fecal coliforms, isolated from raw domestic sewage and aerobic lagoon effluent, to ampicillin, aureomycin, chloromycetin, gentamicin, streptomycin, sulfadiazine, and tetracycline, was determined. Aerobic lagoon treatment produced a 20-fold reduction in the fecal coliform numbers. No statistically significant difference in antibiotic resistance was observed between the fecal coliforms found in raw sewage and in lagoon effluent despite a trend towards the loss of resistance in the latter. Antibiotic resistance, either single or multiple, did not contribute to, or detract from, bacterial survival in the aerobic lagoon. Of the isolates, 15% showed resistance to two or more antiobiotics.     

Sunlight Inactivation of Fecal Indicator Bacteria and Bacteriophages from Waste Stabilization Pond Effluent in Fresh and Saline Waters

Sunlight inactivation in fresh (river) water of fecal coliforms, enterococci, Escherichia coli, somatic coliphages, and F-RNA phages from waste stabilization pond (WSP) effluent was compared. Ten experiments were conducted outdoors in 300-liter chambers, held at 14°C (mean river water temperature). Sunlight inactivation (k_S) rates, as a function of cumulative global solar radiation (insolation), were all more than 10 times higher than the corresponding dark inactivation (k_D) rates in enclosed (control) chambers. The overall k_S ranking (from greatest to least inactivation) was as follows: enterococci > fecal coliforms ≥ E. coli > somatic coliphages > F-RNA phages. In winter, fecal coliform and enterococci inactivation rates were similar but, in summer, enterococci were inactivated far more rapidly. In four experiments that included freshwater-raw sewage mixtures, enterococci survived longer than fecal coliforms (a pattern opposite to that observed with the WSP effluent), but there was little difference in phage inactivation between effluents. In two experiments which included simulated estuarine water and seawater, sunlight inactivation of all of the indicators increased with increasing salinity. Inactivation rates in freshwater, as seen under different optical filters, decreased with the increase in the spectral cutoff (50% light transmission) wavelength. The enterococci and F-RNA phages were inactivated by a wide range of wavelengths, suggesting photooxidative damage. Inactivation of fecal coliforms and somatic coliphages was mainly by shorter (UV-B) wavelengths, a result consistent with photobiological damage. Fecal coliform repair mechanisms appear to be activated in WSPs, and the surviving cells exhibit greater sunlight resistance in natural waters than those from raw sewage. In contrast, enterococci appear to suffer photooxidative damage in WSPs, rendering them susceptible to further photooxidative damage after discharge. This suggests that they are unsuitable as indicators of WSP effluent discharges to natural waters. Although somatic coliphages are more sunlight resistant than the other indicators in seawater, F-RNA phages are the most resistant in freshwater, where they may thus better represent enteric virus survival.     

Evaluation of Standard and Modified M-FC, MacConkey, and Teepol Media for Membrane Filtration Counting of Fecal Coliforms in Water

MacConkey agar, standard M-FC agar, M-FC agar without rosolic acid, M-FC agar with a resuscitation top layer, Teepol agar, and pads saturated with Teepol broth, were evaluated as growth media for membrane filtration counting of fecal coliform bacteria in water. In comparative tests on 312 samples of water from a wide variety of sources, including chlorinated effluents, M-FC agar without rosolic acid proved the medium of choice because it generally yielded the highest counts, was readily obtainable, easy to prepare and handle, and yielded clearly recognizable fecal coliform colonies. Identification of 1,139 fecal coliform isolates showed that fecal coliform tests cannot be used to enumerate Escherichia coli because the incidence of E. coli among fecal coliforms varied from an average of 51% for river water to 93% for an activated sludge effluent after chlorination. The incidence of Klebsiella pneumoniae among fecal coliforms varied from an average of 4% for the activated sludge effluent after chlorination to 32% for the river water. The advantages of a standard membrane filtration procedure for routine counting of fecal coliforms in water using M-FC agar without rosolic acid as growth medium, in the absence of preincubation or resuscitation steps, are outlined.     

Incidence of coliphage in potable water supplies

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Incidence of coliphage in potable water supplies.

Samples of drinking water from different sources in greater Cairo, Egypt, and bottled drinking water were tested for total coliform, fecal coliform, and coliphage populations. Of the 147 samples tested, 4 samples were positive for both total coliforms and coliphage, 65 samples were negative for total coliforms, fecal coliforms, and coliphage, and 78 samples were positive for coliphage and negative for total coliforms and fecal coliforms. The incidence of coliphage in these potable water supplies reflects the probability of human pathogenic virus survival in these waters also.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Potential Regrowth and Recolonization of Salmonellae and Indicators in Biosolids and Biosolid-Amended Soil

This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.     

Potential regrowth and recolonization of salmonellae and indicators in biosolids and biosolid-amended soil

This study evaluated the potential for conversion of Class B to Class A biosolids with respect to salmonellae and fecal coliforms during solar drying in concrete lined drying beds. Anaerobically (8% solids) and aerobically (2% solids) digested Class B biosolids were pumped into field-scale drying beds, and microbial populations and environmental conditions were monitored. Numbers of fecal coliforms and salmonellae decreased as temperature and rate of desiccation increased. After 3 to 4 weeks, Class A requirements were achieved in both biosolids for the pathogens and the indicators. However, following rainfall events, significant increase in numbers was observed for both fecal coliforms and salmonellae. In laboratory studies, regrowth of fecal coliforms was observed in both biosolids and biosolid-amended soil, but the regrowth of salmonellae observed in the concrete-lined drying beds did not occur. These laboratory studies demonstrated that pathogens decreased in numbers when soil was amended with biosolids. Based on serotyping, the increased numbers of salmonellae seen in the concrete lined drying beds following rainfall events was most likely due to recolonization due to contamination from fecal matter introduced by animals and not from regrowth of salmonellae indigenous to biosolids. Overall, we conclude that the use of concrete-lined beds created a situation in which moisture added as rainfall accumulated in the beds, promoting the growth of fecal coliforms and salmonellae added from external sources.     

Rapid, single-step most-probable-number method for enumerating fecal coliforms in effluents from sewage treatment plants.

A single-step most-probable-number method for enumerating fecal coliforms in sewage treatment plant effluents is described. The method requires the use of only one lactose-based medium and a single incubation temperature of 44.5 degrees C, and it can be completed in 18 h or less, as compared with up to 72 h for the standard most-probable-number method. The appearance of growth is the sole criterion used for designating positives, which can be determined either by increases in the electrical impedance ratio of inoculated medium, as compared to an uninoculated control using a Bactometer model 32, or by visual examination of inoculated medium for turbidity. In trials with 53 samples of unchlorinated sewage treatment plant effluent, fecal coliform counts by the single-step most-probable-number method, throughout a range of less than 10 to almost 10(7) fecal coliforms per 100 ml of effluent, were in excellent agreement with counts abtained by the standard most-probable-number procedure. Similar agreement was obtained in comparative trials with 31 chlorinated effluent samples from two sewage treatment plants. Overall, 87% of 452 positives were confirmed as containing fecal coliforms. The applicability of the single-step most-probable-number method to automated sewage treatment plant operations is discussed.     

Survival of Candida albicans in tropical marine and fresh waters.

A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.     

Survival of Candida albicans in tropical marine and fresh waters

A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.