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Applied and Environmental Microbiology, April 2006, p. 2471-2475, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2471-2475.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Male-Specific Coliphages as Indicators of Thermal Inactivation of Pathogens in Biosolids

Sharon P. Nappier, Michael D. Aitken,^* and Mark D. Sobsey

Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina 27599-7431

Received 5 October 2005/ Accepted 20 January 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
Male-specific (F⁺) coliphages have been proposed as a candidate indicator of fecal contamination and of virus reduction in waste treatment. However, in this and earlier work with a laboratory thermophilic anaerobic digester, a heat-resistant fraction of F⁺ coliphage populations indigenous to municipal wastewater and sludge was evident. We therefore isolated coliphages from municipal wastewater sludge and from biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific groups to thermal inactivation. Similar numbers of F⁺ DNA and F⁺ RNA coliphages were found in untreated sludge, but the majority of isolates in digested biosolids were group I F⁺ RNA phages. Separate experiments on individual isolates at 53°C confirmed the apparent heat resistance of group I F⁺ RNA coliphages as well as the susceptibility of group III F⁺ RNA coliphages. Although few F⁺ DNA coliphages were recovered from the treated biosolid samples, thermal inactivation experiments indicated heat resistance similar to that of group I F⁺ RNA phages. Hence, F⁺ DNA coliphage reductions during thermophilic anaerobic digestion are probably related to mechanisms other than thermal inactivation. Further studies should focus on the group III F⁺ RNA coliphages as potential indicators of reductions of heat-resistant pathogens in thermal processes for sludge treatment.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Of the total amount of land-applied biosolids derived from municipal wastewater treatment in the United States, nearly 85% is applied to agricultural lands used to raise crops for human or animal consumption (29). Current U.S. Environmental Protection Agency (EPA) regulations on land application require that class A biosolids contain no detectable pathogens in specified quantities of biosolids (23, 27). The class A criteria can be met with sludge treatment processes referred to as "processes to further reduce pathogens," which are assumed to reduce pathogenic bacteria, enteric viruses, and viable helminth ova to below the class A criterion levels (23, 27). There are other options by which a sludge treatment process can be considered to produce class A biosolids, including the use of specified combinations of time and temperatures of 50°C or higher (27). However, in these cases, it must be demonstrated that organisms in each pathogen category are reduced to nondetectable levels (27). Because many different taxonomic groups of viral pathogens have been detected at widely varying concentrations in human feces and domestic wastewater (12), direct monitoring and enumeration of viral pathogens associated with biosolids are impractical.

Indicator organisms are easier to measure than specific pathogens because they are often more abundant and consistently present in human and animal wastes (13, 26). Currently, fecal coliform bacteria are the standard microbial indicators in biosolids in the United States (27). However, with the exception of the spores of sulfite-reducing clostridia, indicator bacteria are less resistant than coliphages to thermal inactivation (22) and therefore may be less desirable as indicators of pathogen inactivation in thermal treatment processes. Enteric bacteriophages, specifically F⁺ RNA bacteriophages infecting Escherichia coli, have been proposed as more reliable indicators of human viral pathogens (13, 18) because they are similar to human enteric viruses in their physical structure, composition, and morphology, survivability in the environment, and persistence in treatment processes (10, 14, 18). F⁺ RNA coliphages can also be detected and quantified by simple, inexpensive, rapid, and reliable methods (13, 15). They are abundant in domestic wastewater, raw sewage sludge, and polluted waters (7, 17, 20) and originate almost exclusively from the feces of humans and other warm-blooded animals (14, 26). However, the thermal inactivation characteristics of coliphage taxonomic groups have not been well characterized at the temperatures used for thermal sludge treatment processes, and the extent to which coliphages are reliable indicators of pathogen reductions by these processes has not been evaluated.

In a previous study (2), we examined the inactivation of male-specific (F⁺) coliphages indigenous to municipal wastewater sludges in a lab-scale continuous-flow thermophilic anaerobic digester. A significant fraction of the F⁺ coliphages survived treatment at temperatures as high as 55°C, and in subsequent batch treatments of the effluent from the continuous digester, there was limited further reduction at any temperature studied (2). In the present study, the rates of inactivation of total F⁺ coliphages in biosolids were quantified at 51 and 53°C. F⁺ coliphages isolated from raw sludge and digested biosolids were characterized as either F⁺ DNA or F⁺ RNA, with the F⁺ RNA isolates further identified by serogroup. The isolates were also evaluated for susceptibility to inactivation in aqueous medium at 53°C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The construction and operation of the continuous and batch laboratory reactors are described elsewhere (2, 3). The concentrations of F⁺ coliphages in the untreated sludges ranged from about 3 log₁₀/g total solids (TS) to about 5 log₁₀/g TS (2). Concentrations in the treated biosolids were 0.4 to 3.1 log₁₀/g TS, depending on the source sludge and operating temperature (2).

Inactivation kinetics in biosolids.
Rates of F⁺ coliphage inactivation in biosolids were measured at 51 and 53°C, with duplicate experiments at each temperature, using procedures described elsewhere (1). The source of the coliphages was an ultrafiltered concentrate of membrane-filtered (0.45-µm pore size) raw wastewater from the municipal wastewater treatment plant in Beaufort, North Carolina. Total F⁺ coliphages were quantified by a modification of EPA method 1601 (28), in which the presence-absence test format for 1-liter volumes was replaced with a most-probable-number format (2).

Coliphage isolation and characterization.
Indigenous F⁺ coliphages in untreated sludge and those that survived thermophilic anaerobic digestion were isolated by picking representative lysis zones from plates of the Escherichia coli F_amp host (ATCC 700891; American Type Culture Collection, Manassas, VA) used to analyze the sludge or biosolid samples. The biosolid samples included effluent from the continuous digester, samples from further batch treatment of the continuous digester effluent for various time intervals, and samples from various time intervals of the batch thermal inactivation experiments described above. Isolates were picked from lysis zones by using a filter barrier pipette tip. After the pipette tip was used to touch the plaque and the pipette plunger was released, the contents were added to a sterile microcentrifuge tube containing 1.5 ml of Trypticase soy broth (TSB) to discharge the phages. These phages were enriched by adding 1 drop of log-phase E. coli F_amp host to the tube and incubating them overnight at 37°C.

Isolates were characterized as F⁺ RNA or F⁺ DNA by exposure to RNase (type 1-A; Sigma-Aldrich, St. Louis, MO) and scoring for suppression of infectivity, or they were genotyped by PCR or reverse transcription-PCR (RT-PCR) when RNase testing produced ambiguous results. Primers SL2 and SL3 were used to PCR amplify all F⁺ DNA viruses, and primers MJV82, JV81 (leviviruses), and JV41 (alloleviviruses) were used to RT-PCR amplify the F⁺ RNA viruses, using a One-Step RT-PCR kit (QIAGEN, Valencia, CA) as described by Vinjé et al. (30). Representative F⁺ DNA coliphages (n = 7) were genetically characterized by reverse line blot hybridization as described elsewhere (30). F⁺ RNA coliphages were serotyped by a spot infectivity neutralization test, using the following group antisera for prototype viruses: group I (MS2), group II (GA), group III (Qß), group IV (SP), and group IVa (FI) (19). In some cases, lysis zones contained more than one genotype, and these are referred to as "mixed" isolates. Isolates that were not identifiable using these methods (10 total) were not considered further in our data analysis.

Thermal inactivation experiments with coliphage isolates.
The following five categories of isolates were evaluated for inactivation kinetics: F⁺ DNA phages isolated from untreated sludge and from a biosolid sample, group I (MS2-like) and group III (Qß-like) F⁺ RNA phages isolated from untreated sludge, and group I F⁺ RNA phages isolated from biosolid samples. Positive control coliphages (MS2, ATCC 15597-B1; Qß, ATCC 23631-B1; Fd, ATCC 15669-B2; F1, ATCC 8074-B1; and M13, ATCC 15669-B1) were also tested under the same conditions.

For each isolate, an enrichment tube was defrosted and centrifuged at 16,000 x g for 5 minutes to separate the E. coli F_amp cells and cell debris. A 200-µl volume of the supernatant (containing approximately 10⁷ PFU/ml) was then distributed into four sterile microcentrifuge tubes. One tube was used to measure the initial coliphage concentration. Two tubes were placed into an automated, thermoregulated heat block (Isotemp 125D; Fisher Scientific, Pittsburgh, PA) at 53°C. The fourth tube was incubated at room temperature for the duration of the heat inactivation experiment. After 60 min of incubation at 53°C, one tube of each isolate was removed and immersed in ice for 15 seconds to quickly cool the phage suspension to 40°C. Surviving coliphages were assayed immediately after they were cooled, using the spot plaque assay described below.

Spot plaque assay.
Coliphage isolates were serially diluted 10-fold, and 10 µl of each dilution was spotted in duplicate on streptomycin-ampicillin-Trypticase soy agar plates containing a lawn of E. coli F_amp host cells. The spots were allowed to dry for 30 to 60 min, and the plates were then inverted and incubated overnight at 37°C. Phage were enumerated by counting the number of plaques in each 10-µl spot per dilution per sample. Between 1 and 40 plaques are countable and produce reproducible results comparable to those in the double-agar-layer plaque assay (unpublished results). Coliphage titers are expressed in PFU/ml.

Data analysis.
For the experiments in which the kinetics of total F⁺ coliphage inactivation in biosolids were measured, it was assumed that there was a heat-sensitive population and a heat-resistant population. The inactivation rate of the heat-resistant population was assumed to be negligible. If inactivation of the heat-sensitive fraction follows first-order kinetics, then the following equation can be used to estimate the rate coefficient: C = C_r + (C₀ – C_r)e^–kt (equation 1), where C is the total concentration, C₀ is the total concentration at time zero, C_r is the concentration of heat-resistant coliphages, and k is the inactivation rate coefficient for the heat-sensitive coliphages at a given temperature. Values of k and C_r were determined by nonlinear regression (ProStat; Poly Software International, Pearl River, NY) of the concentration versus time data.

For the inactivation experiments with the F⁺ DNA and F⁺ RNA coliphage isolates, the net inactivation at 53°C was determined by subtracting the reduction of coliphage infectivity at room temperature from the reduction observed at 53°C. Analysis of variance and Tukey-Kramer multicomparison posttests were performed, using INSTAT 3.0 (Graphpad Software, Inc., San Diego, CA), to evaluate the significance of differences among the types of coliphages. To perform statistical analysis for any sample that did not have a detectable concentration of coliphage, it was assumed that one phage was present in the total sample volume assayed. In such cases, the corresponding net reductions were assumed to be greater than the reported value.

Top Abstract Introduction Materials and Methods Results Discussion References

 
F⁺ coliphage inactivation kinetics in biosolids.
The rates of total F⁺ coliphage inactivation in biosolids were evaluated under batch conditions at 51 and 53°C (Fig. 1). In these tests, a concentrated stock of coliphages derived from raw wastewater was spiked into biosolids from a continuous-flow thermophilic anaerobic digester operated at the same temperature as the batch reactor. Therefore, the initial concentration of coliphages included those present in the spike and those already present in the biosolids. A brief temperature drop of 0.3 to 0.4°C caused by introducing the coliphage stock to the biosolids did not have a significant effect on the estimated inactivation rates (1).

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FIG. 1. Concentrations of male-specific coliphages as a function of time in inactivation rate experiments with digested biosolids at 51°C (a) and 53°C (b). Data from duplicate experiments at a given temperature are indicated with different symbols. Lines represent best fits to equation 1. Dashed lines correspond to the data represented by open symbols.

The results in Fig. 1 illustrate the apparent existence of at least one fraction of the F⁺ coliphage population that is heat sensitive and at least one fraction that is heat resistant. About a 2-log₁₀ reduction of the initial coliphage concentrations occurred within 2 hours at each temperature, but little further reduction in coliphage concentration was observed for the duration of the 24-hour experiment (data for 24-hour samples are not shown in Fig. 1). Figure 1 also contains fits to equation 1, with fitted parameter values summarized in Table 1. The inactivation rate coefficient k was not higher at 53°C than at 51°C and, in fact, was lower for one of the tests at 53°C. This difference was due largely to scatter in the data for that test, because the slope of the curve for the first three data points was similar to the initial slopes in the other inactivation tests. Differences in the apparent heat-resistant fraction (C_r; Table 1) between temperatures may have resulted in part from differences in the indigenous coliphage populations present in the biosolids after treatment in the continuous-flow digester.

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TABLE 1. Best-fit parameter values for inactivation of total male-specific coliphages in biosolidsa

F⁺ coliphage characterization.
Because the inactivation rate experiments suggested a difference in heat resistance between two or more populations of F⁺ coliphages, randomly picked isolates from untreated sludge and from digested biosolid samples were characterized to evaluate if the resistance was characteristic of the F⁺ RNA coliphages or the F⁺ DNA coliphages. F⁺ RNA coliphage isolates were further characterized by taxonomic group (Table 2). There were similar numbers of F⁺ DNA and F⁺ RNA coliphage isolates in the untreated sludge samples (16 F⁺ RNA and 20 F⁺ DNA isolates). After thermophilic anaerobic digestion, however, 73 of 74 isolates from the biosolid samples were confirmed to be F⁺ RNA coliphages. The only F⁺ DNA isolate obtained from these samples was found in a biosolid sample collected after 8 h of batch treatment at 51°C. Of the F⁺ RNA coliphages, only group I (MS2-like) and group III (Qß-like) isolates were found in the untreated sludge and biosolid samples (Table 2). Furthermore, there was a predominance of group I isolates in the biosolid samples. Group III F⁺ RNA coliphages were found only in biosolids treated in the batch reactor for 1 hour or less at 51°C and, in all cases, were associated with mixed plaques that contained both group I and group III F⁺ RNA coliphages.

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TABLE 2. F+ RNA serotypes (groups) found in sludge and biosolid samples

We also genotyped seven of the F⁺ DNA coliphages isolated from untreated sludge. Two were CH-like, two were Fd-like, two were CH-M13-like, and one was Fd-M13-like. With this limited sample size, it was not possible to determine if there were differences in thermal resistance among the different genotypes of F⁺ DNA coliphages. The one F⁺ DNA coliphage found in the digested biosolids was not genotyped.

Thermal inactivation of F⁺ coliphage isolates.
Further inactivation experiments with the F⁺ coliphage isolates were conducted by incubating them in TSB at 53°C for 60 min (Fig. 2). Prototype coliphages representing F⁺ DNA (M13, Fd, and F1), group I F⁺ RNA (MS2), and group III F⁺ RNA (Qß) were subjected to the same conditions. It is apparent from Fig. 2 that the group I F⁺ RNA and the F⁺ DNA coliphages are more resistant to thermal inactivation than the group III F⁺ RNA coliphages. Because 5 of the 10 group III F⁺ RNA isolates from biosolid samples were not detectable after 60 min of exposure at 53°C, the log₁₀ reductions for the group III isolates shown in Fig. 2 probably underestimate their actual heat sensitivities. Reductions of group III coliphages (both field isolates and the Qß prototype) were significantly higher than reductions of all the group I coliphages and the F⁺ DNA coliphages (P < 0.0001 by analysis of variance). The three categories of group I F⁺ RNA coliphages (isolates from sludge, isolates from digested biosolids, and the prototype) and the F⁺ DNA coliphages (isolates from sludge and the three different type strains) were not significantly different from each other (P > 0.05). Coliphages kept at room temperature were also susceptible to infectivity titer losses over the 60-minute incubation period, with reductions as high as 1.8 log₁₀ (data not shown), but these reductions were accounted for by subtracting them from the reductions at 53°C. In no case were reductions at room temperature significantly greater than those at 53°C.

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FIG. 2. Box-and-whisker plot of net reductions of F+ coliphages by group after 60 min of incubation at 53°C. Bars: A, F+ RNA group I sludge isolates (n = 9); B, F+ RNA group I biosolid isolates (n = 13); C, MS2 (n = 5); D, F+ RNA group III sludge isolates (n = 10); E, Qß (n = 8); F, F+ DNA isolates (n = 13 from untreated sludge and 1 from biosolids); G, F+ DNA from prototype strains Fd, M13, and F1 (n = 3).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inactivation rate measurements with F⁺ coliphages indigenous to domestic wastewater and sludge confirmed earlier observations (2) that some are appreciably inactivated at temperatures associated with thermophilic anaerobic digestion, but others are heat resistant. There was no difference in the rates of inactivation of the heat-sensitive F⁺ coliphages between temperatures of 51 and 53°C (Table 1). However, decimal reduction times for these F⁺ coliphages at 51°C were comparable to those for Ascaris suum and much higher than those for poliovirus in the same experiments (1). Thus, if the heat-sensitive fraction of F⁺ coliphages could be measured specifically, it might be useful as an indicator of thermal inactivation of pathogens in sludge treatment processes. The diversity of F⁺ coliphages and the possibility that relative proportions of more or less heat-sensitive groups can vary between sludges limit the use of total F⁺ coliphages as indicators of pathogen inactivation in thermal treatment processes.

Similar numbers of F⁺ DNA and F⁺ RNA coliphages were isolated from untreated sludge, which is consistent with their comparable abundances in waste sources (6). In the samples of biosolids after thermophilic anaerobic digestion, however, almost all of the isolates were F⁺ RNA coliphages. Of the F⁺ RNA coliphages isolated from untreated sludge, there were similar numbers of group I and group III isolates. In contrast, the F⁺ RNA isolates from digested biosolid samples were predominantly from group I. The group I F⁺ RNA coliphages therefore appear to be relatively heat resistant, while the group III F⁺ RNA coliphages are more susceptible to thermal inactivation.

The experiments on thermal inactivation of coliphage isolates and type strains in aqueous medium supported the observation from biosolid samples that group I F⁺ RNA coliphages are more resistant to thermal inactivation than the group III F⁺ RNA coliphages. Median reductions of group III (Qß-like) F⁺ RNA coliphage isolates were >2-log₁₀ greater than reductions of group I (MS2-like) isolates after 60 min of exposure to 53°C in TSB. These results are consistent with earlier work indicating that group I F⁺ RNA coliphages are more resistant to thermal inactivation than other groups of F⁺ RNA coliphages (9, 24).

The reasons for the differences in sensitivity to heat among the F⁺ RNA coliphage groups are unknown. Groups I and II belong to the Leviviridae family, and groups III and IV belong to the Alloleviviridae family. It is possible that differences in viral proteins between families (5) correspond to differences in the thermal stabilities of proteins associated with infectious virus particles. Based on inactivation kinetics measurements, protein denaturation has been suggested to be the primary mechanism of thermal inactivation for viruses (1, 4, 8).

It is uncertain why only one F⁺ DNA coliphage was isolated from the digested biosolid samples. F⁺ DNA coliphages were slightly more abundant than F⁺ RNA coliphages in untreated sludge, and the thermal inactivation experiments indicated that they are as heat resistant as the group I F⁺ RNA phages. F⁺ DNA coliphages have been found in the environment in summer months and are more stable than F⁺ RNA coliphages over different seasonal temperatures, suggesting that they may have some resistance to heat (21). It is possible that F⁺ DNA coliphages are more susceptible to matrix effects in biosolids, which would not have been manifested in the thermal inactivation experiments using laboratory medium. Of the typical biosolid matrix components, free ammonia is known to cause virus inactivation (4, 31). Group I coliphages have exhibited more resistance to ammonia than other F⁺ RNA coliphage groups (24), but the effect of ammonia on the F⁺ DNA coliphages is not known.

F⁺ RNA strains isolated from human feces are usually from groups II and III, while groups I and IV are usually found in animal feces (11, 14, 16, 19, 25). This source specificity has been considered useful for fecal source tracking (6, 25). However, Schaper et al. (24) suggested that using the distribution of genotypes of F⁺ RNA bacteriophages for understanding fecal origins in natural (environmental) samples requires previous knowledge of the comparative resistance of the four genotypes to inactivation by various factors. In this study, it was primarily the group I coliphages that survived thermophilic anaerobic digestion. If biosolids from thermophilic anaerobic digesters were to be applied to land, the presence of group I isolates in the field could reflect survival through the sludge treatment process rather than a high proportion or load of animal source inputs. Accordingly, the differential survival of F⁺ RNA coliphage groups may limit the usefulness of F⁺ RNA coliphage grouping for quantitative tracking of fecal sources.

In the inactivation experiments with the F⁺ coliphage isolates and the prototype phages (M13, Fd, and F1 for the F⁺ DNA phages and MS2 and Qß for groups I and III, respectively, of the F⁺ RNA phages), there were no statistically significant differences between the isolates and their prototype counterparts for any of the three groups evaluated. This finding suggests that further experiments to characterize and quantify the thermal inactivation properties of a particular F⁺ coliphage group could be conducted with prototype coliphages. Given their sensitivity to thermal inactivation and an apparent inactivation rate similar to that for Ascaris suum at 51°C, further efforts should be devoted to developing methods that selectively detect group III F⁺ RNA coliphages in sludge and biosolids. Of particular importance would be a comparison of inactivation rates for the group III coliphages to those for enteric viruses and helminths. A correspondence of these rates, particularly at temperatures near the low end of the acceptable temperature range for thermal sludge treatment processes (50°C), would provide further support for the use of these coliphages as indicators of thermal inactivation of pathogens.

 
This work was supported in part by the Board of Water Commissioners of the City of Columbus, Georgia.

We thank Billy Turner and Cliff Arnett of Columbus Water Works for their commitment to this work and Mina Shehee, Jan Vinjé, Tiina Pasanen, Nicole van Abel, Kimberly Blauth, Doug Wait, and Sjon Oudejans for their help and suggestions.

 
* Corresponding author. Mailing address: Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, NC 27599-7431. Phone: (919) 966-1481. Fax: (919) 966-7911. E-mail: mike_aitken{at}unc.edu.

Present address: Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Md.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, April 2006, p. 2471-2475, Vol. 72, No. 4
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.4.2471-2475.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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