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.     

Evaluation of thermophilic anaerobic digestion processes for full-scale Class A biosolids disinfection at Hyperion Treatment Plant

This paper describes 5 phases of full-scale testing at the City of Los Angeles Hyperion Treatment Plant (HTP) for producing Class A biosolids (U.S. EPA Part 503 Biosolids Rule) by thermophilic anaerobic digestion. Phases I and II were tests with a two-stage continuous-batch process in a thermophilic battery of six digesters and a designated post-digestion train that was isolated from mesophilic operations. These tests demonstrated that digester outflow biosolids met the Class A limits for fecal coliforms and Salmonella sp. However, fecal coliform densities sharply increased during post-digestion. The recurrence was possibly related to a combination of a large drop of the biosolids temperature after the dewatering centrifuges and contamination of thermophilically digested biosolids from mesophilic operations. Phase III was conducted after insulation and electrical heat-tracing of the post-digestion train to maintain a biosolids temperature throughout post-digestion at about the same level as in the digester outflow. Biosolids monitoring at the last points of plant control (silos at Truck Loading Facility and farm for land application) indicated that fecal coliform recurrence was prevented. After completing the conversion of HTP to thermophilic operation, certification tests of Phases IV and V demonstrated Class A compliance of a two-stage continuous-batch process under Alternatives 1 and 3 of the Part 503 Biosolids Rule, respectively. HTP received the permit for Class A (indeed exceptional quality) biosolids land application in Kern County, California, in December 2002 under Alternative 3. Since 2003, HTP has consistently complied with the federal and local standards for Class A biosolids, indicating that Class A limits can be met under conditions less stringent than defined by the Alternative 1 time-temperature requirement for batch treatment.     

Dynamics of carbon,nitrogen and phosphorus in soil amended with irradiated,pasteurized and limed biosolids

Sewage biosolids contain high concentrations of pathogens, which limits their use as soil amendment. This study investigated how application of lime (Ca(OH)2), irradiation, or pasteurization reduced pathogens in biosolids and how its application affected soil characteristics. A soil sampled outside the canopy of Mesquite trees (Prosopis laevigata) and from a pasture at Lerma (Mexico) was amended with treated or untreated biosolids, characterized and incubated aerobically while dynamics of carbon (C), nitrogen (N) and phosphorus (P) were monitored. Heavy metals concentrations in the biosolids were low, so it was of excellent quality (USEPA). The amount of pathogens in the biosolids made it a class "B" (USEPA) which can be used in forests. Only irradiation sufficiently reduced faecal coliforms to make it a class "A" biosolids without restrictions in application. C mineralization increased significantly when biosolids were added, but not concentrations of available P (P < 0.05). Ammonium (NH4+) concentrations in soil amended with biosolids were higher compared to unamended soil, but not the concentrations of nitrate (NO3-) except when biosolids treated with Ca(OH)2 was added to the Lerma soil.     

Occurrence and distribution of bacterial indicators and pathogens in canal communities along the Texas coast.

Increased construction of residential canal communities along the southern coastline of the United States has led to a concern about their impact on water quality. Pollution of such dead-end canals is potentially hazardous because of their heavy usage for recreational activities. Coliforms, fecal coliforms, and salmonellae in the surface water and bottom sediments of six selected residential coastal canals were monitored over a period of 17 months. No statistically significant relationship was observed between the organism concentrations and temperature, pH, turbidity, and suspended solids content of water. An inverse relationship between the concentration of indicator organism and salinity of water was found, however, to occur at a 99.9% level of significance. All of the microorganisms studied were found to be present in greater numbers in sediments than in the overlying water, often by a factor of several logs. Heavy rainfall resulted in large increases in the number of organisms in both water and sediment samples. Our results indicate that bottom sediments in the shallow canal systems can act as reservoirs of enteric bacteria, which may be resuspended in response to various environmental factors and recreational activities.     

Occurrence and distribution of bacterial indicators and pathogens in canal communities along the Texas coast.

Increased construction of residential canal communities along the southern coastline of the United States has led to a concern about their impact on water quality. Pollution of such dead-end canals is potentially hazardous because of their heavy usage for recreational activities. Coliforms, fecal coliforms, and salmonellae in the surface water and bottom sediments of six selected residential coastal canals were monitored over a period of 17 months. No statistically significant relationship was observed between the organism concentrations and temperature, pH, turbidity, and suspended solids content of water. An inverse relationship between the concentration of indicator organism and salinity of water was found, however, to occur at a 99.9% level of significance. All of the microorganisms studied were found to be present in greater numbers in sediments than in the overlying water, often by a factor of several logs. Heavy rainfall resulted in large increases in the number of organisms in both water and sediment samples. Our results indicate that bottom sediments in the shallow canal systems can act as reservoirs of enteric bacteria, which may be resuspended in response to various environmental factors and recreational activities.     

Comparative Survival of Indicator Bacteria and Enteric Pathogens in Well Water

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers. There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined. The comparative survival of the organisms tested follows: Aeromonas sp. > the shigellae (Shigella flexneri, S. sonnei, and S. dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser. paratyphi A and D, S. enteritidis ser. typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser. paratyphi B. S. bovis had a more rapid die-off than did S. equinus, but both had significantly shorter half-lives than the other streptococci. The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material.     

Removal of pathogen and indicator microorganisms from liquid swine manure in multi-step biological and chemical treatment

Concern has greatly increased about the potential for contamination of water, food, and air by pathogens present in manure. We evaluated pathogen reduction in liquid swine manure in a multi-stage treatment system where first the solids and liquid are separated with polymer, followed by biological nitrogen (N) removal using nitrification and denitrification, and then phosphorus (P) extraction through lime precipitation. Each step of the treatment system was analyzed for Salmonella and microbial indicators of fecal contamination (total coliforms, fecal coliforms, and enterococci). Before treatment, mean concentrations of Salmonella, total coliforms, fecal coliforms, and enterococci were 3.89, 6.79, 6.23 and 5.73 log(10) colony forming units (cfu)/ml, respectively. The flushed manure contained 10,590 mg/l TSS, 8270 mg/l COD, 688 mg/l TKN and 480 mg/l TP, which were reduced >98% by the treatment system. Results showed a consistent trend in reduction of pathogens and microbial indicators as a result of each step in the treatment system. Solid-liquid separation decreased their concentrations by 0.5-1 log(10). Additional biological N removal treatment with alternating anoxic and oxic conditions achieved a higher reduction with average removals of 2.4 log(10) for Salmonella and 4.1-4.5 log(10) for indicator microbes. Subsequent P treatment decreased concentration of Salmonella and pathogen indicators to undetectable level (<0.3 log(10) cfu/ml) due to elevated process pH (10.3). Our results indicate that nitrification/denitrification treatment after solids separation is very effective in reducing pathogens in liquid swine manure and that the phosphorus removal step via alkaline calcium precipitation produces a sanitized effluent which may be important for biosecurity reasons.     

Salmonellae in the Environment Around a Chicken Processing Plant

Studies have been conducted over a 2-year period to determine the extent to which a poultry processing plant served as a reservoir of salmonellae reaching the external environment, to examine the question of the importance of salmonellae in the environment, and to consider how best to control the spread of the organisms. The studies have been undertaken at a chicken processing plant handling between 75,000 and 80,000 birds per day. Populations of salmonellae and indicator bacteria were estimated in the raw wastes, through the waste treatment plant, and in the receiving stream waters. The results demonstrate that salmonellae are present in poultry processing wastes in a surprisingly constant relation to fecal coliforms (in excess of 1 Salmonella per 500 fecal coliforms), that serotypes in the environment are constantly changing, and that they may reflect unusual conditions in the processing plant, or a possible source of infection among human and animal residents of the environment. Disinfection of poultry processing wastes is recommended.     

Comparison of Methods to Identify Pathogens and Associated Virulence Functional Genes in Biosolids from Two Different Wastewater Treatment Facilities in Canada

The use of treated municipal wastewater residues (biosolids) as fertilizers is an attractive, inexpensive option for growers and farmers. Various regulatory bodies typically employ indicator organisms (fecal coliforms, E. coli and Salmonella) to assess the adequacy and efficiency of the wastewater treatment process in reducing pathogen loads in the final product. Molecular detection approaches can offer some advantages over culture-based methods as they can simultaneously detect a wider microbial species range, including non-cultivable microorganisms. However, they cannot directly assess the viability of the pathogens. Here, we used bacterial enumeration methods together with molecular methods including qPCR, 16S rRNA and cpn60 gene amplicon sequencing and shotgun metagenomic sequencing to compare pre- and post-treatment biosolids from two Canadian wastewater treatment plants (WWTPs). Our results show that an anaerobic digestion WWTP was unsuccessful at reducing the live indicator organism load (coliforms, generic E. coli and Salmonella) below acceptable regulatory criteria, while biosolids from a dewatering/pelletization WWTP met these criteria. DNA from other pathogens was detected by the molecular methods, but these species were considered less abundant. Clostridium DNA increased significantly following anaerobic digestion treatments. In addition to pathogen DNA, genes related to virulence and antibiotic resistance were identified in treated biosolids. Shotgun metagenomics revealed the widest range of pathogen DNA and, among the approaches used here, was the only approach that could access functional gene information in treated biosolids. Overall, our results highlight the potential usefulness of amplicon sequencing and shotgun metagenomics as complementary screening methods that could be used in parallel with culture-based methods, although more detailed comparisons across a wider range of sites would be needed.     

Bacteriology of Dehydrated Space Foods

The initial bacteriological requirement established in 1964 for space foods by the U.S. Army Natick Laboratories are: a total aerobic plate count ( 300,000), chocolate ice cream cubes (20,000), and each of four samples of chocolate candy (12,000 to 61,000); (ii) coliforms: two out of three vanilla milk drinks (16 and 127) and one beef hash bar (14); (iii) fecal coliforms: one sample of chicken soup and gravy base positive; (iv) fecal streptococci: two samples of peanut cubes (40 and 108), coconut cubes (75), chicken soup and gravy base (2,650), beef soup and gravy base (33), and five out of six flavored milk drinks (23 to 300); (v) salmonellae: one each of chicken and beef soup and gravy base were positive.     

Microbially mediated growth suppression and death of salmonella in composted sewage sludge

The role of compost microflora in the suppression of salmonella regrowth in composted sewage sludge was investigated. Microbial inhibition studies of salmonella growth were conducted on nutrient agar, in composts that had been subjected to different temperatures in compost piles, and in radiation sterilized composts inoculated with selected fractions of the compost microflora. Agar assays of inhibition indicated that bacteria and actinomycetes were not suppressive to salmonellae, but a few fungi were. However, compost inoculation assays showed consistently that fungi were not suppressive, but bacteria and actinomycetes were. In compost inoculation assays, microbial antagonists, when present, either killed salmonellae or reduced their growth rate. No suppression of salmonellae occurred in compost taken from 70°C compost-pile zones despite the presence and growth of many types of microbes. With greater numbers and kinds of microbes in 55°C compost, salmonella growth was suppressed 100–10,000-fold. Salmonellae died when inoculated into compost from unheated zones (25–40°C) of piles. Prior colonization of compost with only noncoliform gram-negative bacteria suppressed salmonellae growth 3,000-fold. Coliforms when inoculated prior to salmonellae accounted for 75% of salmonella die-off. Mesophilic curing to allow colonization of curing piles in their entirety by gram-negative bacteria, especially coliforms, should be an effective way to prevent repopulation by salmonellae.     

Survey of wastewater indicators and human pathogen genomes in biosolids produced by class a and class B stabilization treatments

Accurate modeling of the infectious aerosol risk associated with the land application of biosolids requires an in-depth knowledge of the magnitudes and changes in pathogen concentrations for a variety of class A and class B stabilization methods. The following survey used quantitative PCR (qPCR) and culture assays to detect environmentally resistant bacterial and viral pathogens and biosolid indicator organisms for 36 biosolid grab samples. Biosolids were collected from 14 U.S. states and included 16 class B mesophilic anaerobic digestion (MAD) samples and 20 class A biosolid samples from temperature-phased anaerobic digestion (TPAD), MAD plus composting (COM), and MAD plus heat pelletization processes. The indicator concentrations of fecal coliforms and male-specific coliphages as well as pathogen genome concentrations for human adenovirus species, Legionella pneumophila, Staphylococcus aureus, and Clostridium difficile were significantly lower in the class A samples, and a multivariate analysis of variance ranked the stabilization processes from the lowest pathogen/indicator load to the highest as (i) class A COM, (ii) class A TPAD, and (iii) class B MAD. Human adenovirus genomes were found in 88% of the class B samples and 70 to 100% of the class A samples. L. pneumophila, S. aureus, and C. difficile genomes were detected at the qPCR assay detection limits in 19 to 50% of the class B and class A anaerobic digestion samples, while L. pneumophila was detected in 50% of the class A compost samples. When considering all the stabilization methods, both the fecal coliform and the male-specific coliphage concentrations show a significant linear correlation with the pathogen genome concentrations. This survey provides the necessary pathogen concentrations to add to biosolid aerosol risk and pathogen exposure analyses and clarifies the effectiveness of class A stabilization methods with the pathogen and indicator loads in biosolids.     

Comparison of Media for Isolation of Salmonellae and Shigellae from Fecal Specimens

Five transport media, eight plating media, and three enrichment broth media for the isolation of salmonellae and shigellae were evaluated. Eight laboratories in widely separated regions of the United States participated in this evaluation by submitting 490 fecal specimens in the transport media provided. The results suggest that the newer transport media may not offer any advantage over the use of buffered glycerol-saline in the isolation of these enteric pathogens. Shigellae were best isolated by direct inoculation, whereas salmonellae were isolated in greater numbers after tetrathionate (without Brilliant Green) enrichment with subsequent culturing on the plating medium. The use of a variety of plating media is recommended for the recovery of a larger number of these enteric pathogens.     

Bacterial Contamination of Drinking Water Supplies in a Modern Rural Neighborhood

On six occasions during a 15-month period, the private well and spring water supplies in a modern rural neighborhood of 78 households were examined for total coliforms, fecal coliforms, Staphylococcus aureus, and standard plate count bacteria. More than one-third of the water supplies were unsatisfactory on at least one occasion as judged by standard plate counts over 10³/ml and the presence of coliforms, fecal coliforms, and/or S. aureus. Citrobacter freundii, Klebsiella pneumoniae, and Escherichia coli were the most frequently isolated total coliforms. At least 12 other genera of bacteria were identified from standard plate count agar. Coliform contamination was found to be higher after periods of rainfall, and high standard plate counts were more prevalent during warmer weather. These observations probably reflect leakage of surface water into improperly sealed wells or aquifer contamination during winter and the lack of chlorination to control microbial regrowth during the warm season. An inverse correlation was found between the presence of high standard plate counts and incidence of coliforms. Consumer education and at least a twice yearly monitoring of private water supplies (winter and summer) are suggested methods to signal that treatment may be necessary to reduce the risk of waterborne disease.     

Destruction by Anaerobic Mesophilic and Thermophilic Digestion of Viruses and Indicator Bacteria Indigenous to Domestic Sludges

In raw sludges and in mesophilically and thermophilically digested anaerobic sludges, large variations in numbers of viruses occurred over narrow ranges of numbers of fecal coliforms, total coliforms, and fecal streptococci, demonstrating that the bacteria were poor quantitative reflectors of the numbers of the viruses detected. Mesophilic and thermophilic digestion of anaerobic sludges destroyed all three indicator bacteria more rapidly than such digestion destroyed the viruses. The relative rates for the destruction of viruses, fecal coliforms, and fecal streptococci in the digested sludges were consistent over the 17-month study. Fecal coliforms were 7 to 8 times more sensitive than the viruses to mesophilic digestion and 9 to 10 times more sensitive to thermophilic digestion. Total coliforms were even more sensitive. The rates at which fecal streptococci were destroyed by mesophilic and thermophilic digestion of anaerobic sludges approached those at which the viruses were destroyed by those processes; this suggested that the rates at which fecal streptococci in sludges are destroyed by those processes may serve as useful indicators for the rates at which viruses in sludges are destroyed by those processes.     

Isolation of Fecal Coliform Bacteria from the Diamondback Terrapin (Malaclemys terrapin centrata)

Total and fecal coliform bacteria were isolated from the cloaca and feces of the estuarine diamondback terrapin. The majority of samples contained fecal coliforms. Escherichia coli was the predominant fecal coliform species isolated, and members of the genus Salmonella were isolated from 2 of 39 terrapins. Fecal coliform numbers are used to regulate shellfish harvests, and diamondback terrapins inhabit the brackish-water habitats where oyster beds are found; therefore, these findings have implications for the efficacy of current regulatory parameters in shellfishing waters.     

Field studies measuring the aerosolization of endotoxin during the land application of Class B biosolids

Endotoxins are a component of Gram-negative bacteria cell walls and are known to be present in biosolids. Endotoxins have been shown to be potent stimulators of the innate immune response causing airway irritation and shortness of breath. Class B biosolids are routinely applied to agricultural lands to enhance soil properties and can be used as an alternative to chemical fertilizers. This study investigated the aerosolized endotoxin dispersed during the land application of Class B biosolids on agricultural land and a concrete surface at two sites in Colorado, USA. Aerosolized endotoxin was captured using HiVol samplers fitted with glass fiber filters, polycarbonate filter cassettes (both open and closed) and BioSampler impinger air samplers. Endotoxins were also measured in the biosolids to allow for correlating bulk biosolids concentrations with aerosol emission rates. Endotoxin concentrations in biosolids, impinger solutions and filter extracts were determined using the kinetic Limulus amebocyte lysate assay. Aerosolized endotoxin concentration was detected from all sites with levels ranging from 0.5 to 642 EU/m³. The four types of sampling apparatus were compared, and the HiVol and open-faced cassette samplers produced higher time-weighted average (TWA) measurements (EU/m³) than the impinger and closed cassette samplers. Ambient wind speed was found to be the variable best describing the observed results with optimal wind speed for highest deposition estimated at 5 m s^?1. It is argued that HiVol air samplers are a particularly reliable approach and subsequent analyses relating TWA measurements to wind speed and biosolids characteristics were based on the measurements collected with those samplers.     

Comparison of four membrane filter methods for fecal coliform enumeration in tropical waters.

Four membrane filter methods for the enumeration of fecal coliforms were compared for accuracy, specificity, and recovery. Water samples were taken several times from 13 marine, 1 estuarine, and 4 freshwater sites around Puerto Rico, from pristine waters and waters receiving treated and untreated sewage and effluent from a tuna cannery and a rum distillery. Differences of 1 to 3 orders of magnitude in the levels of fecal coliforms were observed in some samples by different recovery techniques. Marine water samples gave poorer results, in terms of specificity, selectivity, and comparability, than freshwater samples for all four fecal coliform methods used. The method using Difco m-FC agar with a resuscitation step gave the best overall results; however, even this method gave higher false-positive error, higher undetected-target error, lower selectivity, and higher recovery of nontarget organisms than the method using MacConkey membrane broth, the worst method for temperate waters. All methods tested were unacceptable for the enumeration of fecal coliforms in tropical fresh and marine waters. Thus, considering the high densities of fecal coliforms observed at most sites in Puerto Rico by all these methods, it would seem that these density estimates are, in many cases, grossly overestimating the degree of recent fecal contamination. Since Escherichia coli appears to be a normal inhabitant of tropical waters, fecal contamination may be indicated when none is present. Using fecal coliforms as an indicator is grossly inadequate for the detection of recent human fecal contamination and associated pathogens in both marine and fresh tropical waters.     

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.     

Rainfall-induced release of fecal coliforms and other manure constituents: comparison and modeling

Modeling release of fecal coliforms is an important component of fate and transport simulations related to environmental water quality. Manure constituents other than fecal coliforms may serve as natural tracers of fecal contamination provided that their release from manure to runoff is similar to the fecal coliform release. The objectives of this work were to compare release of fecal coliforms (FC), chloride (Cl-), organic carbon (OC), and water-soluble phosphorus (P) from dissolving manure and to assess the performance of three models in describing the observed release. Bovine manure was applied on 0.5- by 0.3-m bare and vegetated subplots with 20% slope on sandy loam and clay loam soils. Concentrations of Cl-, FC, OC, and P were measured in runoff collected from troughs at the edges of the subplots at 5-min intervals during 1-h rainfall simulations. The one-parametric exponential model and two-parametric Vadas-Kleinman-Sharpley model and Bradford-Schijven model were fitted to the data. The Bradford-Schijven model had uncorrelated parameters, one of which was linearly related to the irrigation rate, and another parameter reflected the presence or the absence of vegetation. Kinetics of the FC release from manure was similar to the release kinetics of P and OC. The Bradford-Schijven model is recommended to simulate the release of manure constituents.