A national study on the residential impact of biological aerosols from the land application of biosolids.

AIMS: The purpose of this study was to evaluate the community risk of infection from bioaerosols to residents living near biosolids land application sites. METHODS AND RESULTS: Approximately 350 aerosol samples from 10 sites located throughout the USA were collected via the use of six SKC Biosamplers. Downwind aerosol samples from biosolids loading, unloading, land application and background operations were collected from all sites. All samples were analysed for the presence of HPC bacteria, total coliform bacteria, Escherichia coli, Clostridium perfringens, coliphage, enteroviruses, hepatitis A virus and norovirus. Total coliforms, E. coli, C. perfringens and coliphage were not detected with great frequency from any sites, however, biosolids loading operations resulted in the largest concentrations of these aerosolized microbial indicators. Microbial risk analyses were conducted on loading and land application operations and their subsequent residential exposures determined. CONCLUSIONS: The greatest annual risks of infection occurred during loading operations, and resulted in a 4 x 10(-4) chance of infection from inhalation of coxsackievirus A21. Land application of biosolids resulted in risks that were <2 x 10(-4) from inhalation of coxsackievirus A21. Overall bioaerosol exposure from biosolids operations poses little community risk based on this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluated the overall incidence of aerosolized micro-organisms from the land application of biosolids and subsequently determined that microbial risks of infection were low for residents close to biosolids application sites.

Bioaerosols from the land application of biosolids in the desert southwest USA.

This study evaluated bioaerosol emissions during land application of Class B biosolids in and around Tucson, Arizona, to aid in developing models of the fate and transport of bioaerosols generated from the land application of biosolids. Samples were collected for 20 min at distances between 2 m and 20 m downwind of point sources, using an SKC BioSampler impinger. A total of six samples were collected per sampling event, which consisted of a biosolid spray applicator applying liquid biosolids to a cotton field. Each application represented one exposure. Samples were collected in deionised water amended with peptone and antifoam agent. Ambient weather conditions were also monitored every 10 min following initiation of sampling. Concurrently with downwind samples, background (ambient) air samples were collected to compensate for any ambient airborne microorganisms. In addition, biosolids samples were collected for analysis of target indicator and pathogenic organisms. Soil samples were also collected and analysed. Significant numbers of heterotrophic plate count (HPC) bacteria were found in air samples collected during the biosolid application process. These could have arisen from soil particles being aerosolised during the land application process. Aerosolised soil may contribute significantly to the amount of aerosolised microorganisms. Soil particles may be able to more readily aerosolise, due to their low density, small particle size and low mass. Aerosolised HPC bacteria found during biosolids land application were similar to those found during normal tractor operation on non-biosolids applied fields. Coliforms and coliphages were not routinely detected even though they were found to be present in the biosolids at relatively high concentrations, 10(6) and 10(4)/g (dry weight) of biosolids respectively. This could be due to the die-off rate of aerosolised Gram-negative bacteria or sorption to the solid portion of the biosolids. Low numbers of aerosolised coliphages may likewise be due to sorption phenomena. We theorise that only organisms in the aqueous phase of the biosolids were available to desorb and be aerosolised. Animal viruses, which were not detected in the biosolids, were likewise not detected in the aerosol samples. Clostridium perfringens was detected in only a small percent of aerosol samples although it was detected during all weather conditions; other microorganisms were detected during more favourable environmental conditions (relative humidity >10%). Despite the fact that many of these organisms were present in the biosolids at significant concentrations, their presence in bioaerosols generated during the land application of biosolids was limited to only a small percentage of samples. Bacteria as well as viruses may sorb to biosolids, which contain a high percentage of organic matter, and desorption during land application of biosolids may not readily take place; therefore, these microorganisms may not be readily aerosolised.

Tracking the concentration of heterotrophic plate count bacteria from the source to the consumer's tap.

The goal of this project was to quantify the concentration of heterotrophic plate count (HPC) bacteria within water reaching consumer's taps, and from the sources used by a major utility serving the City of Tucson, AZ. With this information, the amounts and sources of HPC bacteria consumed at the tap could be determined. Samples of water were collected on a monthly basis from two well fields, the CAVSARP recovery well field and Southern Avra Valley well field which serves as one of the groundwater sources for Tucson, AZ, and the distribution system which serves the same homes from which tap water was also tested. The average concentration of HPC in source waters within Southern Avra Valley Wells was 56 CFU/ml (range 1-1995/ml). From the CAVSARP recovery well field, corresponding values were 38 CFU/ml (1 to 502 CFU/ml). Unblended groundwater in the chlorinated distribution system averaged 22 CFU/ml (range 1-794). Blended water at the chlorinated distribution site averaged 47 CFU/ml (range 10-158). There was a major shift in the percentage of gram negative to gram-positive bacteria from the wells to the distribution system, to the tap. In the surface CAP source water, 76% of the bacteria were gram-negative compared to 27% gram-negative in the CAVSARP recovery wells. In contrast, Avra Valley wells contained 17% gram-negative bacteria. In both the Tucson groundwater distribution sites and blended distribution sites, the corresponding number of gram negative bacteria was 12%. Finally at the tap, only 0.2% of the bacteria were gram-negative. The average number of bacteria in household taps averaged 3072 HPC/ml and was equal or greater than 500 ml 68% of the time. This study shows that the number of HPC bacteria increases dramatically from the distribution system to the consumers tap. Thus, the major source of bacteria ingested by the average consumer in Tucson originates from bacteria within the household distribution system or the household tap, rather than from source waters or the distribution system. It is also clear that consumers' regularly consume more than 500 HPC/ml from drinking water taken from the household tap.

Dual-bioaugmentation strategy to enhance remediation of cocontaminated soil.

Although metals are thought to inhibit the ability of microorganisms to degrade organic pollutants, several microbial mechanisms of resistance to metal are known to exist. This study examined the potential of cadmium-resistant microorganisms to reduce soluble cadmium levels to enhance degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under conditions of cocontamination. Four cadmium-resistant soil microorganisms were examined in this study. Resistant up to a cadmium concentration of 275 microg ml(-1), these isolates represented the common soil genera Arthrobacter, Bacillus, and Pseudomonas. Isolates Pseudomonas sp. strain H1 and Bacillus sp. strain H9 had a plasmid-dependent intracellular mechanism of cadmium detoxification, reducing soluble cadmium levels by 36%. Isolates Arthrobacter strain D9 and Pseudomonas strain I1a both produced an extracellular polymer layer that bound and reduced soluble cadmium levels by 22 and 11%, respectively. Although none of the cadmium-resistant isolates could degrade 2,4-D, results of dual-bioaugmentation studies conducted with both pure culture and laboratory soil microcosms showed that each of four cadmium-resistant isolates supported the degradation of 500-microg ml(-1) 2,4-D by the cadmium-sensitive 2,4-D degrader Ralstonia eutropha JMP134. Degradation occurred in the presence of up to 24 microg of cadmium ml(-1) in pure culture and up to 60 microg of cadmium g(-1) in amended soil microcosms. In a pilot field study conducted with 5-gallon soil bioreactors, the dual-bioaugmentation strategy was again evaluated. Here, the cadmium-resistant isolate Pseudomonas strain H1 enhanced degradation of 2,4-D in reactors inoculated with R. eutropha JMP134 in the presence of 60 microg of cadmium g(-1). Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.

Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture. Bioaugmentation effects on soil microorganisms.

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1,000 microg 3-chlorobenzoate g(-1) dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraders were enumerated on minimal salts media containing 3-chlorobenzoate. Culturable heterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1,000 microg 3-chlorobenzoate g(-1) dry soil inoculated microcosms, degraders increased from the initial inoculum and decreased following degradation of 3-CB. Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 x 10(7) colony-forming-units g(-1) dry soil in the 500 microg 3-chlorobenzoate g(-1) dry soil microcosms, but none were detected in the 1,000 microg 3-chlorobenzoate g(-1) dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species. In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1,000 microg 3-chlorobenzoate g(-1) dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.

Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria.

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.

An evaluation of ERIC PCR and AP PCR fingerprinting for discriminating Salmonella serotypes.

PCR fingerprints of 89 Salmonella isolates belonging to 22 serotypes were obtained using ERIC PCR (enterobacterial repetitive intergenic consensus PCR) and AP PCR (arbitrarily primed PCR) to evaluate the ability of different fingerprinting methods to differentiate or identify serotypes and subtypes. Fingerprints were scored and comparisons were made using a computer program. ERIC PCR produced a unique, complex fingerprint for almost every isolate, but these fingerprints did not identify serotypes. One AP PCR primer also produced complex fingerprints that discriminated among isolates, but again did not identify serotypes. A second AP PCR primer produced simple patterns, including one pattern shared by 35 isolates from 12 different serotypes. In general, the three sets of PCR fingerprints distinguished isolates, but were not correlated with serotypes. Matching fingerprints from different gels by computer was difficult, since similarities were based on both intense and faint bands. In addition, this study suggests that dendrograms created from PCR fingerprints should be viewed with caution.

Characterization and quantification of bacterial pathogens and indicator organisms in household kitchens with and without the use of a disinfectant cleaner.

This two year study evaluated the prevalence of indicator bacteria and specific pathogens in 10 'normal' kitchens in the United States. In Phase I, none of the kitchens was cleaned with an antimicrobial cleaner or disinfectant. Eight locations within the kitchens were monitored for: total heterotrophs, staphylococci, Pseudomonas, total coliforms and faecal coliforms. Almost all locations at all households exhibited contamination, with the sink and sponge samples exhibiting large bacterial concentrations. The faecal coliform concentrations in sink and sponge samples were very high, with 63 and 67% of all samples being positive, respectively. Escherichia coli was detected in 16.7% of all sink surfaces and 33.3% of all sponges. Salmonella was detected once and Campylobacter, on two occasions. In a second phase, households were provided with an antimicrobial disinfectant cleaner which families were encouraged to use but not forced to do so; in some cases, the product was used infrequently or not at all. This regimen did not demonstrate any consistent reduction in the incidence of bacterial contamination. By contrast, in the final phase of the study where disinfectant use was targeted for surfaces soon after contamination with foods or hands, the incidence of contamination decreased dramatically. These data show that normal kitchens can easily be contaminated with a variety of bacterial contaminants including faecal coliforms, E. coli, Salmonella and Campylobacter. Irregular use, or not using antimicrobial agents, is unlikely to reduce the risk of these infectious agents. By contrast, targeted use is likely to reduce the incidence of bacterial contaminants.

Frequency of horizontal gene transfer of a large catabolic plasmid (pJP4) in soil.

Limited work has been done to assess the bioremediation potential of transfer of plasmid-borne degradative genes from introduced to indigenous organisms in the environment. Here we demonstrate the transfer by conjugation of the catabolic plasmid pJP4, using a model system with donor and recipient organisms. The donor organism was Alcaligenes eutrophus JMP134 and the recipient organism was Variovorax paradoxus isolated from a toxic waste site. Plasmid pJP4 contains genes for mercury resistance and 2,4-dichlorophenoxyacetic (2,4-D) acid degradation. A transfer frequency of approximately 1/10(3) donor and recipient cells (parent cells) was observed on solid agar media, decreasing to 1/10(5) parent cells in sterile soil and finally 1/10(6) parent cells in 2,4-D-amended, nonsterile soil. Presumptive transconjugants were confirmed to be resistant to Hg, to be capable of degrading 2,4-D, and to contain a plasmid of size comparable to that of pJP4. In addition, we confirmed the transfer through PCR amplifications of the tfdB gene. Although transfer of pJP4 did occur at a high frequency in pure culture, the rate was significantly decreased by the introduction of abiotic (sterile soil) and biotic (nonsterile soil) stresses. An evaluation of the data from this model system implies that the reliance on plasmid transfer from a donor organism as a remediative strategy has limited potential.

Polymerase chain reaction detection of nonviable bacterial pathogens.

Polymerase chain reaction (PCR) methodologies for detection of pathogens in environmental samples are currently available. However, positive amplification products for any set of primers only signal that the appropriate target nucleic acid sequences were present in the sample. The presence of the amplification products does not imply that the target organisms were viable. Here we show that PCR will detect nonviable cells, as long as intact target nucleic acid sequences are available. In an environmental water sample, nucleic acids degraded quickly and were not detectable by PCR after 3 weeks even when stored at 4 degrees C. However, these data show that there is a window of opportunity for PCR analyses to result in false positives with respect to viable cells. We further show that care must be taken in the way samples are stored for future PCR amplifications and that filter sterilization of media is not acceptable for long-term preservation of samples for PCR.

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.

Rapid method for processing soil samples for polymerase chain reaction amplification of specific gene sequences.

Bacterial cells can be differentially separated from soil colloids on the basis of their buoyant densities. By using this principle, a modified sucrose gradient centrifugation protocol has been developed for separating bacterial cells from most of the soil colloids. Since the bacterial cell suspension still contained some colloidal soil particles, which inhibited polymerase chain reaction amplification, a new "double" polymerase chain reaction method of analysis was adopted for amplification of Tn5-specific gene sequences. This new protocol allowed rapid detection of small numbers (1 to 10 CFU/g) of bacterial cells present in soil samples.