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. 2007 Feb 16;73(7):2191–2198. doi: 10.1128/AEM.02213-06

Extractable Organic Components and Nutrients in Wastewater from Dairy Lagoons Influence the Growth and Survival of Escherichia coli O157:H7▿

Subbarao V Ravva 1,*, Anna Korn 1
PMCID: PMC1855652  PMID: 17308194

Abstract

The influence of nutrients in wastewater from dairy lagoons on the survival of Escherichia coli O157:H7 was monitored. Initially, the survival of E. coli O157:H7 in wastewater from which the competing native organisms had been removed by filter sterilization or autoclaving was compared with that in wastewater from which competing organisms had not been removed. Numbers of E. coli O157:H7 or E. coli ONT (O-nontypeable):H32 cells declined rapidly in filter-sterilized water and exhibited a slower decline in nonsterile water, while the organisms proliferated in autoclaved water. Subsequently, the growth of E. coli O157:H7 strains was monitored in 300 μl of Luria-Bertani (LB) broth supplemented with incremental proportions of filter-sterilized wastewater. E. coli O157:H7 and E. coli ONT:H32 strains failed to grow in filter-sterilized wastewater, and their growth was reduced incrementally with wastewater supplementation of LB broth. Consequently, the influence of organic extracts of wastewater on the growth of E. coli O157:H7 and E. coli ONT:H32 in reduced-strength LB was monitored, followed by scale-up tests in wastewater. Acidic and basic extracts inhibited growth of both strains, while the neutral aqueous extract improved growth. However, a scale-up with a threefold increase in the acidic components supplementing the wastewater did not result in any additional decline in numbers of E. coli O157:H7 cells. When protected inside a 300-kDa dialysis tube and exposed to diffusible components, E. coli O157:H7 survived longer, with a decimal reduction time of 18.1 days, compared to 3.5 days when inoculated directly into wastewater. Although wastewater can potentially provide nutrients to naturally occurring human pathogens, the chemical components, protozoa, and coliphages in wastewater can inhibit the growth of freshly introduced pathogens from manure.

Produce was the most common single-food vehicle linked to food-borne illness outbreaks during 1990 to 2004 (9). Consumption of contaminated produce and produce dishes resulted in 639 outbreaks involving 31,496 cases of infection during that period. On-farm contamination of fruit and vegetable produce, meat, and dairy products is often suspected as the cause of these illness outbreaks (4, 29, 30). Designing effective intervention strategies to minimize the risk of on-farm contamination requires an understanding of pathogen survival and proliferation strategies.

Escherichia coli O157:H7, a causal agent of hemolytic uremic syndrome, is to blame for 48 produce-related and 144 beef-related outbreaks in the United States (9). The pathogen is known to be shed in manure by cattle and survives in animal waste and wastewater (2, 11, 14, 22, 25). Owing to its low prevalence (13, 14, 25), the pathogen could not be detected in manure and wastewater from dairies in the central valley of California (27, 31). It is likely that the pathogen could escape detection at low cell numbers or as nonculturable cells (32). Thus, the elusive pathogen was not found in locations that had been sampled for 5 years (38a) prior to the September 2006 multistate outbreak linked to spinach that sickened 204 individuals and killed 3. The deadly Walkerton (Canada) outbreak of E. coli O157:H7 was most likely a result of contamination of drinking water with low levels of pathogen that grew in biofilms (19). The recurrence of outbreaks (9) related to produce suggests that contamination with the pathogen may occur through wastewater or manure applied to the crops. In fact, the pathogenic strain of E. coli O157:H7 from the September 2006 outbreak was traced to cattle feces from ranches implicated in the outbreak (6). The same strain was also detected in samples from a stream and from wild pigs that have been in the spinach fields and the ranches. Once contamination occurs, regrowth in nutrient-rich root zones may further enhance the risk of transfer to the above-ground portions of plants (23, 34).

Although E. coli O157:H7 can be isolated even after 21 months (24) by selective enrichments from a manure pile from inoculated sheep, the pathogen failed to grow and decline rapidly in manure, manure slurries, and wastewater from dairy lagoons (2, 17, 31). In addition, feeding cattle with a straw-rich diet (12), silage made with bacteriocin-producing bacteria (3), and competitive exclusion products using lactic acid bacteria (7), colicinogenic E. coli (8, 33) and O157-specific bacteriophages (35) has the potential to inhibit pathogen replication inside the animals. Nevertheless, releasing even a few cells into environments that favor pathogen regrowth (10) may pose an epidemiological risk. Other studies have not provided any insights into the failure of pathogenic E. coli to proliferate in manure or wastewater that are rich in nutrients. In this study, we determined the influence of dairy wastewater components on the survival and proliferation of pathogenic strains of E. coli O157:H7. The growth and survival of organisms was monitored in microcosms of 300 μl and scaled up to larger volumes of wastewater supplemented with or without nutrients or organic extracts of wastewater. To do this, wastewater was extracted and fractionated into acidic, basic, and neutral organic components and tested for their influence on pathogen growth. In addition, the influence of diffusible components of wastewater was evaluated by comparing the growth of E. coli O157:H7 inside dialysis tubes to growth in wastewater. These comparisons also indirectly highlight the influence of other biotic factors in controlling pathogenic E. coli in dairy wastewater.

MATERIALS AND METHODS

Survival of E. coli O157:H7 in sterile and nonsterile wastewater.

Wastewater used in this study was collected from an aerated manure lagoon from a medium-sized (ca. 800 milking head) dairy in central California (Oakdale, CA) and acclimated overnight at room temperature prior to inoculations. Survival of three strains of E. coli O157:H7 (MM100, MM149, and MM151) and one of E. coli ONT:H32 (MM158), selected for rifampin and nalidixic acid resistance (Table 1), in wastewater was compared with their fate in 0.2-μm-filter-sterilized and autoclaved wastewaters. The inoculum was an overnight growth of organisms in LB-RN broth (Luria-Bertani broth supplemented with 100 μg/ml rifampin and 50 μg/ml nalidixic acid) centrifuged and resuspended in 0.01 M phosphate-buffered saline (PBS; pH 7.4). The tests were conducted in 250-ml Erlenmeyer flasks containing 50-ml water samples inoculated to a final concentration of 1 × 105 to 10 × 105 CFU/ml. The flasks were incubated at 25°C on a gyratory shaker operated at 100 rpm, and samples were taken at various intervals for monitoring pathogen survival.

TABLE 1.

E. coli O157:H7 strains

Straina Sourceb
MM100 Odwalla apple juice outbreak; wt, FDA strain SEA13B88
MM149 Dairy manure, isolated 8/93, northwestern Oregon; wt, RM2543
MM151 Dairy manure, isolated 8/94, western Washington; wt, RM2608
MM158 E. coli ONT:H32c isolated from 4-wk-old calf, California veal farm; wt, VMTRC 8051-B
MM147 Manure; wt, RM1673
MM148 Manure; wt, RM1695
MM150 Manure, western Washington; wt, RM2563
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a

Strains selected for rifampin (110 μg/ml) and nalidixic acid (50 μg/ml) resistance. All strains, except MM158, were O157:H7.

b

Odwalla outbreak strain and wild types (wt) were from Robert Mandrell, the Produce Safety and Microbiology Unit (RM strains), USDA, ARS, Albany, CA. VMTRC 8051-B was from the Veterinary Medical Teaching and Research Center, Tulare, CA. Dates are given in the form month/year.

c

Strain ONT:H32 was serotyped and characterized by PCR for virulence factors at the Gastroenteric Disease Center, Pennsylvania State University, State College, PA. It tested negative for virulence factors STa, stx1, stx2, eae, cnf1, cnf2, K99, CS31A, and F1845.

Pathogen populations were monitored, as described earlier (31), by plating 100-μl portions of serial dilutions of wastewater in PBS on LB agar supplemented with 100 μg/ml of rifampin and 50 μg/ml each of nalidixic acid and cycloheximide (LB-RNC agar). Petrifilm count plates were used to enumerate aerobic bacteria and total coliforms (31) from source wastewater, and chemical analysis of filter-sterilized and nonsterile wastewaters was performed (A & L Western Agricultural Labs. Inc., Modesto, CA) (Table 2).

TABLE 2.

Chemical properties of filtered and unfiltered wastewater

Propertya Level in water type
Filtered (0.2 μm) Unfiltered
Concn (ppm) of:
    N 222 484
    P 8 67
    K 351 620
    S 68 72
    Mg 77 125
    Ca 104 270
    Na 174 330
    Fe 1 8
    Al 1 3
    Mn 0.1 1
    Cu 0.1 1
    Zn 0.1 2
    B 0.6 13
    OM 303 3,395
    BOD 158 714
    COD 400 4,600
    TSS 90 940
EC (mS/cm) 3.8 7.2
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a

EC, electrical conductivity; OM, organic matter; BOD, biological oxygen demand; COD, chemical oxygen demand; TSS, total suspended solids.

Growth kinetics of E. coli O157:H7 and native organisms in LB broth and wastewater.

Using a Bioscreen C microbial incubation system (Growth Curves USA, Piscataway, NJ), the growth kinetics of six strains of E. coli O157:H7 and one strain of E. coli ONT:H32 (Table 1) was monitored in LB broth supplemented with incremental proportions of 0.2-μm-filter-sterilized wastewater. The treatments contained LB broth supplemented with 0%, 50%, 90%, 95%, 99%, or 100% filter-sterilized wastewater. Wells of Bioscreen plates contained 290 μl of LB broth-wastewater inoculated with 10 μl of overnight growth of E. coli O157:H7 or ONT:H32 resuspended in PBS, while the optical density of all resuspensions was adjusted to 0.3 at 600 nm prior to inoculations. The inoculated plates were constantly shaken at medium speed during incubations at 25°C, and growth was monitored at 10-min intervals by an onboard spectrophotometer equipped with a wide-band filter (420 to 580 nm). A separate study was conducted to monitor growth of native bacteria in wastewater. The above experiment was repeated by adding incremental amounts of nonsterile, coarsely (glass wool) filtered wastewater to LB. As all treatments contained organisms from wastewater, only the wells containing 100% LB broth were inoculated with 10 μl of wastewater. Two replicate treatments were used for all growth kinetics studies.

Extraction of organic components from wastewater.

Five hundred milliliters of wastewater was acidified to pH 2 with 6 N HCl and extracted twice with 100-ml portions of ethyl acetate. The aqueous portion was made alkaline to pH 10 with 5 N NaOH and extracted twice with 100 ml ethyl acetate, and the resultant aqueous portion was adjusted to pH 7 and extracted again with ethyl acetate. The ethyl acetate extracts were concentrated to near dryness in a rotary flash evaporator and reconstituted in 1 to 5 ml of ethanol. A 100-ml portion of the extracted wastewater was concentrated to less than 10 ml and reconstituted back to 10 ml with distilled water to produce a neutral aqueous extract. Weighed portions of each extract were evaporated to dryness, and the dry weight of solids was determined.

Influence of extracts of wastewater on growth of E. coli O157:H7 and ONT:H32 strains.

The growth of MM149 and MM158 was evaluated in Bioscreen wells containing 265 μl of Murashige and Skoog basal salts (Fisher Scientific, Fair Lawn, NJ) medium with either 5 or 50% LB broth (LB-MS) at pH 7.0, 15 μl of wastewater extract, and 20 μl of inoculum consisting of 5 × 106 CFU. The growth was monitored for 2 days at 25°C. Since the quantity of solids in each extract was different, the amount of solids in 15 μl extract and the corresponding amount of wastewater extracted to obtain the solids are shown in Table 3. The pH of the growth medium was also monitored at the termination of the experiment.

TABLE 3.

Details of extracts of wastewater used in Bioscreen treatments

Extract Amt (μg)a/300 μl medium Concn (μg/ml) in medium Wastewater equivalents (ml)
Acidic organic 95 315 3.4
Basic organic 27 90 6.8
Neutral organic 35 115 0.8
Neutral aqueous 1,100 5,500 4.7
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a

Dry weight of solids in 15 μl of the extract.

Influence of acidic extract on survival of E. coli ONT:H32 in wastewater.

As cell death cannot be monitored by the optical density-based Bioscreen system, the influence of acidic organic components of wastewater on the growth of MM158 was monitored in 50 ml of nonsterile wastewater supplemented with acidic extract from 137.5 ml of wastewater. The extract applied was an exact scale-up from the Bioscreen test volume, but this treatment contained wastewater instead of LB-MS. Since the acidic extract was added as a 1.65-ml solution in ethanol, a treatment with the same amount of ethanol was included for growth comparisons. A parallel treatment of wastewater plus acidic extract without ethanol was also included. For this treatment, ethanol from acidic extract was removed by flash evaporation and the residue was reconstituted in 50 ml of nonsterile wastewater. The wastewaters in all the flasks were inoculated with MM158 at a concentration of 1 × 107 CFU/ml and incubated at 25°C for 10 days on a shaker operating at 100 rpm. Survival of the organism was monitored at various intervals by enumeration on LB-RNC agar plates as described earlier.

Influence of diffusible components of wastewater on the survival of E. coli O157:H7.

The survival of MM149 was monitored in 1 liter of wastewater and in dialysis tubes exposed to the same amount of wastewater. Freshly collected wastewater acclimated overnight at room temperature was used as the medium in monitoring the fate of inoculated pathogens incubated in magnetically stirred tissue culture vessels (Nalge Nunc International, Rochester, NY). For the indirect exposure of wastewater components, 6 ml of 0.2-μm-filtered wastewater in dialysis tubes was inoculated with the same amount of bacteria and exposed to 1 liter of unfiltered wastewater in a 2-liter beaker. The wastewater was mixed at a low speed on a magnetic stir plate during incubations. To differentiate the influence of high-molecular-weight components of wastewater from that of smaller molecules, survival of the pathogen incubated in 1,000- and 300,000-molecular-weight-cutoff dialysis tubes (Float-A-Lyzer; Spectrum Labs, Rancho Dominguez, CA) was monitored. The treatments of wastewater with direct inoculations were replicated three times, whereas two replicates were used to determine the influence of dialyzable components. Both treatments received ∼2 × 109 cells of overnight growth of MM149 in LB-RN broth centrifuged and resuspended in PBS as the inoculum. The microcosms were incubated at 23 ± 2°C for 27 days or less if the organism could no longer be detected.

Pathogen populations and native aerobic bacteria were monitored at various intervals as described above. The volume of water in the dialysis tubes was measured, and the volume in excess of 6 ml was removed at every sampling interval to minimize the chance of bursting due to diffusion of water into the tubes. The decline of organisms inside the dialysis tube was calculated based on 1 liter of total volume, since the organisms were exposed to the components from a liter of wastewater.

In a separate experiment, the influence of nutrient depletion on the survival of pathogens was monitored by exposing the organisms inside the dialysis tubes to dilutions of wastewater. MM149 incubated in 1,000- and 300,000-molecular-weight-cutoff dialysis tubes was exposed to 10%, 50% and 100% wastewater. Distilled water was used for making the dilutions.

Statistical analysis.

Repeated-measures two-way analysis of variance (ANOVA) (Prism 4.0; GraphPad Software, Inc., San Diego, CA) was used to compare differences in growth of E. coli O157:H7 or native organisms in LB broth supplemented with incremental amounts of filtered or unfiltered wastewater. Growth measurements of two replicate treatments at hourly intervals were statistically compared, and extremely significant (P < 0.0001) differences between growth curves of different treatments were observed. The Bonferroni posttest was used to determine the earliest interval at which the growth of E. coli O157:H7 significantly (P < 0.05) differed at various concentrations of wastewater or wastewater components used to amend growth media in Bioscreen wells. The same statistical tests were applied to determine if pathogen growth inside the dialysis tubes was significantly influenced by the diffusible components of wastewater.

RESULTS

Survival of E. coli O157:H7 in sterile and nonsterile wastewater.

Since E. coli O157:H7 disappeared rapidly from wastewater in a previous study (31), an attempt was made to determine the reasons for failure of this pathogen in wastewater by eliminating competing organisms by autoclaving or filter sterilization. While autoclaving removes all competing organisms including parasites and predators, filter-sterilized water would contain phages. In addition, filter sterilization also removed most suspended solids along with nutrient-rich organic matter compared to the nonsterile water (Table 2). As a result, two strains of E. coli O157:H7 (MM100 and MM151) and a strain of E. coli ONT:H32 (MM158) completely disappeared from wastewater 4 to 5 days after inoculation, and all four strains disappeared even more rapidly from filter-sterilized wastewater (Fig. 1). These strains survived longer in steam-sterilized wastewater, and two of them (MM149 and MM158) grew during the first 2 days. However, none of them could be recovered from steam-sterilized or other wastewaters after 15 days of incubation (data not shown). The source wastewater used in this study contained (1.5 ± 0.5) × 106 CFU/ml of aerobic bacteria and (9.4 ± 1.5) × 103 CFU/ml of coliforms. Similar results were obtained a month later in a repeat study with fresh wastewater.

FIG. 1.

FIG. 1.

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Fate of E. coli O157:H7 strains in filter-sterilized (○), autoclaved (▵), and nonsterile (□) wastewater from a dairy lagoon.

Growth kinetics of E. coli O157:H7 and native organisms in LB broth supplemented with wastewater.

The ability of wastewater to supply nutrients and the growth responses of six strains of E. coli O157:H7 and a strain of E. coli ONT:H32 (Table 1) to chemical components of wastewater were evaluated using the Bioscreen system. Increasing the proportion of filter-sterilized or nonsterile wastewaters in growth medium to >50% significantly (P < 0.01, repeated-measures two-way ANOVA) inhibited the growth of all seven strains (Table 1) and native organisms in wastewater (Fig. 2). A further increase in proportion of wastewater to >90% significantly (P < 0.001) inhibited their growth. Furthermore, all seven strains and native organisms failed to grow in unsupplemented wastewater. In general, growth patterns shown in Fig. 2A for strain MM100 were similar to those for other strains (data not shown), but the lag time for significant growth to occur at incremental concentrations of wastewater varied among strains. For example, a shortest lag of 10 h was observed for MM100 in LB broth with 50% wastewater, whereas a 32-h lag was observed with MM147. Although growth was not evident after 7 days in wells with 100% wastewater, viable pathogens were detected by enumeration on LB-RNC agar plates. An inoculum of (2 ± 1) × 106 CFU per well was used in comparing growth of different strains.

FIG. 2.

FIG. 2.

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Growth of E. coli O157:H7 strain MM100 and native organisms from wastewater in LB broth supplemented with incremental amounts of 0.2-μm-filter-sterilized (A) or nonsterile, glass wool-filtered (B) wastewaters. LB broth was supplemented with 0% (curve 1), 50% (curve 2), 90% (curve 3), 95% (curve 4), 99% (curve 5), and 100% (curve 6) filter-sterilized or nonsterile wastewaters. Data points are averages of two replicates. Growth of native organisms in LB with 99% wastewater was not measured, and the optical densities in nonsterile wastewater treatments (B) were not corrected for time zero values.

In contrast to the observance of prolonged lag times with E. coli O157:H7 or ONT:H32, significant (P < 0.001) growth of native organisms was observed within 3 h in LB broth supplemented with or without 50% wastewater (Fig. 2B). This treatment contained 2.3 × 107 CFU of aerobic bacteria at the beginning of incubations. In addition, a wider range of initial bacterial counts ([1.5 ± 0.5] × 105 CFU in 100% LB broth plus 10 μl wastewater as the inoculum to 4.5 × 107 CFU in 100% wastewater) did not make any difference in lag times to significant growth.

Influence of organic extracts of wastewater on growth of E. coli O157:H7 and E. coli ONT:H32.

As all seven strains failed to grow in wastewater, attempts were made to determine if any organic component of wastewater was inhibitory. Growth of two E. coli strains (one each of O157:H7 and ONT:H32) was monitored in reduced-strength LB-MS broth supplemented with organic extracts of neutral, acidic, or alkaline fractions of wastewater (Fig. 3). Since an enhanced effect of organic extracts in nutrient-limited media was expected, we chose 5 and 50% LB-MS as basal media for these comparisons. The acidic extract completely inhibited the growth of both strains in media containing 5 and 50% LB broth. The basic extract, on the other hand, inhibited the growth of both strains in 5% LB-MS but not in 50% LB-MS. The neutral aqueous extracts encouraged the growth of organisms in both media, while the neutral organic fraction slightly inhibited the growth. On a weight basis, neutral aqueous extract added to Bioscreen wells contained the most solids (Table 3). At termination of the experiment, the pH of all growth media plus extracts was 6.8 ± 0.2.

FIG. 3.

FIG. 3.

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Growth of E. coli ONT:H32 (MM158) and O157:H7 (MM149) in 5% and 50% LB-MS broth supplemented with organic extracts of wastewater. LB-MS broth was supplemented with acidic (⋄), basic (□), and neutral (○) organic extracts and neutral aqueous concentrate (▵). The growth in LB-MS supplemented with wastewater extracts was compared with growth in unsupplemented LB broth (solid line).

Survival of E. coli ONT:H32 in wastewater supplemented with acidic wastewater extract.

Since the Bioscreen system monitors optical density and cannot monitor the decline of organisms, the influence of acidic components of wastewater on survival of E. coli ONT:H32 in wastewater was monitored by plate counts on LB-RNC agar (Fig. 4). In this scale-up experiment, MM158 declined from 107 to 103 CFU/ml during a 10-day incubation in wastewater supplemented with or without an additional 2.75 equivalents of acidic organic fraction from wastewater (Fig. 4). However, the organisms grew in treatments containing ethanol as a carrier for the acidic organic fraction and in wastewater treated with only ethanol. The quantity of ethanol added was 3.3% (vol/vol) of total volume of wastewater and was a proportional scale-up from the Bioscreen studies (Fig. 3).

FIG. 4.

FIG. 4.

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Survival of E. coli ONT:H32 (MM158) in wastewater supplemented with acidic organic extract of wastewater. Growth of MM158 was monitored in wastewater (▵), wastewater amended with 2.75 equivalents of acidic organic extracts without ethanol carrier (▴), wastewater amended with 2.75 equivalents of acidic organic extract in ethanol (•), and wastewater amended with only ethanol (○). The treatments were not replicated.

Influence of diffusible components of wastewater on the survival of E. coli O157:H7.

To determine the reasons for unexpected decline of E. coli ONT:H32 or E. coli O157:H7 strains in filter-sterilized wastewater (Fig. 1), the fate of E. coli O157:H7 (MM149) in dialysis tubes with filter-sterilized wastewater and exposed to the components of wastewater was monitored (Fig. 5). As coliphages cannot pass through either 1-kDa or 300-kDa dialysis tubes, this test would distinguish nutrient deficiency from native coliphages (if present) in reducing E. coli O157:H7 numbers inside the dialysis tubes. A >3-log decline was observed during a 5-day period with MM149 inoculated directly in wastewater compared to a fractional decline from the dialysis tubes exposed to the same amount of wastewater (Fig. 5). E. coli O157:H7 survived better in the presence of diffusible components of <300 kDa than when exposed to components of <1 kDa. However, the components of <300 kDa significantly enhanced the survival of MM149 only after 20 days (P < 0.01, repeated-measures two-way ANOVA). In addition, the decline rate of pathogenic E. coli in 300-kDa dialysis tubes was comparable to that of native aerobic bacteria. The rates were 0.055 and 0.044 log CFU ml−1 day−1, respectively, for E. coli O157:H7 and native bacteria, based on plate counts for days 13 to 15. Meanwhile, the pathogen directly inoculated into wastewater declined at 0.283 log CFU ml−1 day−1. In other words, the directly inoculated pathogen, pathogen inside the 300-kDa dialysis tube, and native aerobic bacteria declined with decimal reduction times (time for 1-log decline) of 3.5, 18.1, and 22.5 days, respectively.

FIG. 5.

FIG. 5.

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Influence of diffusible components of wastewater on the survival of E. coli O157:H7. The fate of strain MM149 directly inoculated in wastewater (○) was compared with its growth inside 1,000 (▵)- and 300,000 (□)-molecular-weight-cutoff dialysis tubes. The same amount of inoculum was used for all three treatments. Aerobic bacterial counts (⋄) are from wastewater treated directly with the pathogen.

In a separate study, the capacity of wastewater to maintain MM149 in dialysis microcosms was determined indirectly by exposing the pathogen to dilutions of wastewater (Fig. 6). MM149 declined rapidly in 10% wastewater, while the population remained at a constant level even when the nutrients were diluted by half.

FIG. 6.

FIG. 6.

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Survival of E. coli O157:H7 strain MM149 inside 300,000-molecular-weight-cutoff dialysis tube exposed to dilutions of wastewater. Dialysis tubes were incubated in 1-liter solutions of 100% (○), 50% (▵), and 10% (□) wastewater dilutions (vol/vol) in distilled water. Similar results were obtained for the survival of MM149 inside the 1-kDa dialysis tube (not shown).

DISCUSSION

Manure and wastewater from dairy lagoons are an excellent source of nutrients for agricultural crops, but they are also known to be a potential source of contamination with human pathogens like E. coli O157:H7 (30). Although E. coli O157:H7 occurs at low prevalence (13, 14, 25), conditions that favor regrowth (10) may increase the risk of contamination of crops fertilized with manure or wastewater. Furthermore, E. coli O157:H7 persists longer in manure and wastewater at low temperatures (24, 31), in subsurface soils (1), and in dairy wastewater wetlands (22). However, the pathogen fails to establish in manure, manure slurries, and wastewater from dairy holding lagoons (17, 18, 21, 31). We found that the pathogen fails to establish during repeat inoculations that simulate continuous fecal input into lagoons through lane flushing (31). In this study, E. coli O157:H7 populations also declined rapidly in wastewater, while they proliferated in autoclaved wastewater. Pathogen regrowth in autoclaved wastewater may be a result of nutrient release from organic matter (16) and elimination of competing organisms (26, 39) during steam sterilization. It is surprising, however, that pathogens survived slightly longer in nonsterile wastewater than in filter-sterilized water, which did not contain any competing organisms. A higher level of inorganic nutrients in unfiltered water (Table 2) and supplemental release of nutrients from organic matter by native commensals may be responsible for the improved survival in unfiltered nonsterile wastewater. Nonetheless, pathogens declined to undetectable levels after 2 weeks in either sterile or nonsterile wastewater. In addition to low nutrient levels, coliphages (35) that pass through 0.2-μm filters were also suspected of being responsible for the enhanced decline of E. coli O157:H7 or E. coli ONT:H32 strains in filter-sterilized wastewater. An earlier observation (31) of wastewater sustaining native aerobic bacteria but not native coliforms or inoculated E. coli O157:H7 also implies that pathogen survival in dairy lagoons could be regulated by factors other than nutrient availability.

Even though the failure of pathogens to establish in wastewater as a result of competition from native organisms is expected, dialysis tube incubations resulted in unpredicted results. Although pathogen numbers declined rapidly in directly inoculated wastewater, they persisted at high levels inside both the dialysis tubes. Surprisingly, the prolonged survival of pathogens in dialysis tubes was comparable to maintenance of native aerobic bacteria in wastewater outside the tube. Therefore, native organisms appear to have a limited influence, if any, on the nutrient supply to pathogens inside the dialysis tube. This assumption is further confirmed by the maintenance of pathogens inside the dialysis tube even at 50% reduction of nutrients in wastewater. Since there were sufficient nutrients to prolong growth, pathogen decline in wastewater is suspected to be a result of antagonistic interactions other than competition for nutrients by native organisms or nutrient deficiency in wastewater. As bacteriocins and antibiotics can pass through the high-molecular-weight cutoff dialysis tube, protection from predation and lytic phages appears to be the most plausible explanation for prolonged survival of the pathogens inside dialysis tubes. Thus, directly inoculated pathogens in wastewater declined rapidly, with a decimal reduction time of 3.5 days, while they persisted inside 300-kDa dialysis tubes (decimal reduction time of 18.5 days). Yet the significant decline in pathogen numbers in 1-kDa tubes compared to that in the high-molecular-weight-cutoff tube is unexpected, although the pathogen may also be protected from colicins (8) and antibiotics produced from native organisms in wastewater. Exclusion of nutrients and growth factors of >1 kDa is hypothesized for the pathogen decline inside the low-molecular-weight-cutoff dialysis tube.

Although the dialysis microcosms suggest that the nutrient-rich wastewater can potentially sustain growth, all E. coli O157:H7 strains failed to grow in Bioscreen wells, and also their populations in both filter-sterilized and nonsterile wastewater declined rapidly in scale-up experiments. The results from Bioscreen studies also show that incremental addition of wastewater to LB broth enhanced the inhibition of pathogens. As anticipated, later experiments showed growth inhibition by extractable components from wastewater. Most inhibition of E. coli O157:H7 was observed with the ethyl acetate-extractable fraction of acidified wastewater. An earlier report of rapid killing of Shiga-toxigenic E. coli in acidified piggery effluent was linked with the antibacterial properties of volatile fatty acids (15) at pH levels below their pKa. However, these organisms were not killed when the pH of the effluent was raised to 6.8 (15). Acidification with organic acids is also an established practice for pathogen reduction in foods (5, 36-38). In contrast, our studies suggest that components of wastewater are inhibitory to the growth of E. coli O157:H7 at neutral pH. The basic organic fraction also inhibited pathogen growth, but to a lesser extent than the acidic components. This inhibition was observed only in media containing 5% LB broth, not at 50% LB broth. Higher nutrient levels appear to offset the inhibition by basic organic components. Similarly, the pathogen grew in wastewater supplemented with ethanol as a carrier for the acidic extract in a scale-up experiment using nonsterile wastewater. Since the pathogen dies rapidly in wastewater that already contains acidic inhibitors, a threefold supplemental addition of acidic components without ethanol did not result in a further decline. Ethanol acts as an energy source at the concentration applied (3.3%) and is not known to be inhibitory to E. coli O157:H7 or other Shiga-toxigenic strains at concentrations of <6% (20, 28). In addition, ethanol appears to have countered the inhibitory effects of acidic components of wastewater. The enhanced growth of organisms with neutral aqueous extract added to either 5 or 50% LB broth is remarkable, as this extract delivered the highest amount of soluble solids (5.5 mg/ml) to the assay medium. Growth stimulation by neutral aqueous extract may be a result of sequential removal of inhibitory acidic and basic fractions during extractions. Coincidentally, an earlier study (12) showed that E. coli O157:H7 declines rapidly in manure derived from dairy cattle fed with pure straw compared to manure derived from a highly digestible grass or maize silage diet. Although the chemistry of these manures is not known, it may be possible to modify the bovine diet to increase the levels of acidic or basic components in manure that would enhance the pathogen decline. In addition, pathogen decline in wastewater may be increased by limiting nutrients through removal of manure solids. Thus, the practice of separating manure solids to recycle wastewater may have the added benefit of on-farm pathogen control. However, daily washing of the lanes in dairy barns replenishes the lagoon with nutrients and possibly pathogens through fresh manure. Although nutrients are available, the failure of freshly introduced pathogens to survive in this study and others (18, 21, 31) suggests that the pathogens have difficulty competing for the available nutrients with the more acclimated native organisms.

Overall, these results demonstrate the possibility of controlling pathogenic E. coli by altering the chemistry and nutrients in wastewaters, and such modifications of chemistry should be explored in combination with other promising techniques that minimize pathogen replication and release from cattle (3, 7, 8, 12, 33, 35). An equally significant finding of prolonged survival of E. coli O157:H7 when protected in dialysis tubes suggests that coliphages (35) and protozoan predators may be responsible for the rapid decline of pathogenic E. coli in wastewaters from dairy lagoons. Nonetheless, the environmental significance of coliphages and protozoa in controlling pathogenic E. coli requires further scrutiny.

Acknowledgments

We thank Jeri Barak, J. Mark Carter, and Amarnath Ravva for helpful discussions, critical review, and advice and Chester Sarreal for technical assistance.

The work was funded by the U.S. Department of Agriculture, Agricultural Research Service, under CRIS project 5325-32000-005.

Footnotes

▿

Published ahead of print on 16 February 2007.

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