Bacterial populations within copper mine tailings: long-term effects of amendment with Class A biosolids.

AIM: This study evaluates the effect of surface application of dried Class A biosolids on microbial populations within copper mine tailings. METHODS AND RESULTS: Mine tailing sites were established at ASARCO Mission Mine close to Sahuarita Arizona. Site 1 (December 1998) was amended with 248 tons ha(-1) of Class A biosolids. Sites 2 (December 2000) and 3 (April 2006) were amended with 371 and 270 tons ha(-1), respectively. Site D, a neighbouring native desert soil, acted as a control for the evaluation of soil microbial characteristics. Surface amendment of Class A biosolids showed a 4 log(10) increase in heterotrophic plate counts (HPCs) compared to unamended tailings, with the increase being maintained for 10-year period. Microbial activities such as nitrification, sulphur oxidation and dehydrogenase activity were also sustained throughout the study period. 16S rRNA clone libraries obtained from community DNA suggest that mine tailings amended with biosolids achieve diversity and bacterial populations similar to native soil bacterial phyla, 10 years postapplication. CONCLUSION: Addition of Class A biosolids to copper mine tailings in the desert south-west increased soil microbial numbers, activity and diversity relative to unamended mine tailings. SIGNIFICANCE AND IMPACT OF THE STUDY: The amended tailings resulted in a functional soil with respect to microbial characteristics, which were sustainable over a 10-year period enabling the development of appropriate vegetation.

Influence of long-term land application of Class B biosolids on soil bacterial diversity.

AIM: To evaluate the effect of long-term annual land applications of Class B biosolids on soil bacterial diversity at University of Arizona Marana Agricultural Field Center, Tucson, Arizona. METHODS AND RESULTS: Following the final of 20 consecutive years of application of Class B biosolids in March 2005, followed by cotton growth from April to November 2005 surface soil samples (0-30 cm) were collected from control (unamended) and biosolid-amended plots. Total bacterial community DNA was extracted, amplified using 16S rRNA primers, cloned, and sequenced. All 16S rRNA sequences were identified by 16S rRNA sequence analysis and comparison to known sequences in GenBank (NCBI BlastN and Ribosomal Database Project II, RDP). Results showed that the number of known genera (identifiable > 96%) increased in the high rate biosolid plots compared to control plots. Biosolids-amended soils had a broad phylogenetic diversity comprising more than four major phyla: Proteobacteria (32%), Acidobacteria (21%), Actinobacteria (16%), Firmicutes (7%), and Bacteroidetes (6%) which were typical to bacterial diversity found in the unamended arid southwestern soils. CONCLUSION: Bacterial diversity was either enhanced or was not negatively impacted following 20 years of land application of Class B biosolids. SIGNIFICANCE AND IMPACT OF THE STUDY: This study illustrates that long-term land application of biosolids to arid southwestern desert soils has no deleterious effect on soil microbial diversity.

Diversity of aerosolized bacteria during land application of biosolids.

AIMS: The purpose of this study was to determine the diversity of bacterial communities associated with bioaerosols generated during land application of biosolids using 16S ribosomal RNA (16S rRNA) PCR. METHODS AND RESULTS: Anaerobically digested Class B biosolids were land applied to an agricultural site located in South Central Arizona. Aerosol samples were collected downwind of the biosolids operations and were collected via the use of SKC Biosamplers and subsequently extracted for the presence of bacterial community DNA. All DNA was amplified using 16S rRNA primers, cloned and sequenced. All sequences were aligned and phylogenetic trees were developed to generate community profiles. The majority of aerosolized bacterial clone sequences belonged to the Actinobacteria and alpha- and beta-proteobacterial taxa. Aerosol samples collected downwind of soil aerosolization produced similar profiles. These profiles differed from upwind and background samples. CONCLUSIONS: No one clone sequence isolated from the aerosol samples could be solely attributed to biosolids; on the contrary, the majority appeared to have arisen from soil. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that in dry, arid climates the majority of aerosols associated with biosolids land application appear to be associated with the onsite soil.

Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids.

The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities.

Expression of gentisate 1,2-dioxygenase (gdoA) genes involved in aromatic degradation in two haloarchaeal genera.

Gentisate-1,2-dioxygenase genes (gdoA), with homology to a number of bacterial dioxygenases, and genes encoding a putative coenzyme A (CoA)-synthetase subunit (acdB) and a CoA-thioesterase (tieA) were identified in two haloarchaeal isolates. In Haloarcula sp. D1, gdoA was expressed during growth on 4-hydroxybenzoate but not benzoate, and acdB and tieA were not expressed during growth on any of the aromatic substrates tested. In contrast, gdoA was expressed in Haloferax sp. D1227 during growth on benzoate, 3-hydroxybenzoate, cinnamate and phenylpropionate, and both acdB and tieA were expressed during growth on benzoate, cinnamate and phenylpropionate, but not on 3-hydroxybenzoate. This pattern of induction is consistent with these genes encoding steps in a CoA-mediated benzoate pathway in this strain.

The measurement of aerosolized endotoxin from land application of Class B biosolids in Southeast Arizona.

The purpose of this study was to determine aerosolized endotoxin concentrations downwind of a biosolids land application site. Aerosol samples were collected from biosolids land application sites, tractor operation, and an aeration basin located within an open-air wastewater treatment plant. Aerosolized endotoxin above background concentrations was detected from all sites, at levels ranging from below detection up to 1800 EU m-3 of air. Biosolids loading operations resulted in the greatest concentrations of endotoxin (mean 344 EU m-3). As downwind (perpendicular to wind vector) distance increased from sources (2-200 m), levels of endotoxin decreased to near background (without biosolids application) concentrations. Overall, the detected levels of aerosolized endotoxin were within past proposed aerosolized endotoxin limits (250-2000 EU m-3) by other occupational exposure studies. Occasionally, peak concentrations were found to be above these limits. Sites in which soil was being aerosolized resulted in greater concentrations of endotoxin with or without biosolids, which suggested that the majority of endotoxin may in fact be of soil origin. This study evaluated the presence of aerosolized endotoxin from the land application of biosolids and showed that these levels were within ranges for concern suggested by other studies and that this area of research needs further investigation.

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.

Estimation of bioaerosol risk of infection to residents adjacent to a land applied biosolids site using an empirically derived transport model.

AIM: The purpose of this study was to develop an empirically derived transport model, which could be used to predict downwind concentrations of viruses and bacteria during land application of liquid biosolids and subsequently assess microbial risk associated with this practice. METHODS AND RESULTS: To develop the model, coliphage MS-2 and Escherichia coli were aerosolized after addition to water within a biosolids spray application truck, and bioaerosols were collected at discrete downwind distances ranging from 2 to 70 m. Although coliphage were routinely detected, E. coli did not frequently survive aerosolization. Data on aerosolized coliphage was then used to generate a virus transport model. Risks of infection were calculated for various ranges of human virus concentrations that could be found in biosolids. CONCLUSIONS: A conservative estimate at 30.5 m (assumed to be nearest adjacent residences) downwind, resulted in risks of infection of 1 : 100,000, to the more realistic 1 : 10,000,000 per exposure. Conservative annual risks were calculated to be no more than 7 : 100,000 where as a more realistic risk was no greater than 7 : 10,000,000. Overall, the viral risk to residences adjacent to land application sites appears to be low, both for one time and annual probabilities of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a simple approach towards modelling viral pathogens aerosolized from land applied liquid biosolids, and offers insight into the associated viral risk.

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.

Chlorobenzoate-degrading bacteria in similar pristine soils exhibit different community structures and population dynamics in response to anthropogenic 2-, 3-, and 4-chlorobenzoate levels.

A study was conducted to determine the diversity of 2-, 3-, and 4-chlorobenzoate (CB) degraders in two pristine soils with similar physical and chemical characteristics. Surface soils were collected from forested sites and amended with 500 microg of 2-, 3-, or 4-CB g(-1) soil. The CB levels and degrader numbers were monitored throughout the study. Degraders were isolated, grouped by DNA fingerprints, identified via 16S rDNA sequences, and screened for plasmids. The CB genes in selected degraders were isolated and/or sequenced. In the Madera soil, 2-CB and 4-CB degraded within 11 and 42 d, respectively, but 3-CB did not degrade. In contrast, 3-CB and 4-CB degraded in the Oversite soil within 14 and 28 d, respectively, while 2-CB did not degrade. Approximately 10(7) CFU g(-1) of degraders were detected in the Madera soil with 2-CB, and the Oversite soil with 3- and 4-CB. No degraders were detected in the Madera soil with 4-CB even though the 4-CB degraded. Nearly all of the 2-CB degraders isolated from the Madera soil were identified as a Burkholderia sp. containing chromosomally encoded degradative genes. In contrast, several different 3- and 4-CB degraders were isolated from the Oversite soil, and their populations changed as CB degradation progressed. Most of these 3-CB degraders were identified as Burkholderia spp. while the majority of 4-CB degraders were identified as Bradyrhizobium spp. Several of the 3-CB degraders contained the degradative genes on large plasmids, and there was variation between the plasmids in different isolates. When a fresh sample of Madera soil was amended with 50, 100, or 200 microg 3-CB g(-1), 3-CB degradation occurred, suggesting that 500 microg 3-CB g(-1) was toxic to the degraders. Also, different 3-CB degraders were isolated from the Madera soil at each of the three lower levels of 3-CB. No 2-CB degradation was detected in the Oversite soil even at lower 2-CB levels. These results indicate that the development of 2-, 3-, and 4-CB degrader populations is site-specific and that 2-, 3-, and 4-CB are degraded by different bacterial populations in pristine soils. These results also imply that the microbial ecology of two soils that develop under similar biotic and abiotic environments can be quite different.

A risk assessment of emerging pathogens of concern in the land application of biosolids.

Since the development of the United States Environmental Protection Agency's 503 biosolids Rule, which includes treatment requirements to reduce the threat of pathogen transmission, many new pathogens have been recognized which could be transmitted by biosolids. A risk analysis was performed assess which emerging pathogens would be most likely to survive treatments required for Class B biosolids before land application. The literature was reviewed on the resistance of emerging pathogens to temperature and other environmental factors to assess their probability of surviving various biosolids treatment processes. In addition existing information on occurrence in biosolids and dose response models for each pathogen was reviewed. It was concluded that adenoviruses and hepatitis A virus are the most thermally resistant viruses and can survive for prolonged periods in the environment. The protozoan parasites microsporidia and Cyclospora were unlikely to survive the temperatures achieved in anaerobic digestion and do not survive well under low moisture conditions. A risk model was used to assess the risk of infection and illness from enteric viruses after application of class B biosolids.

Dispersal of plasmid pJP4 in unsaturated and saturated 2,4-dichlorophenoxyacetic acid contaminated soil.

Little is known regarding plasmid fate within contaminated soils. Column studies were used to evaluate dissemination of plasmid pJP4 under unsaturated or saturated flow conditions in a 2,4-dichlorophenoxyacetic acid (2,4-D) contaminated soil. Columns were destructively sampled following 1 week of percolation to assess the vertical distribution of donors, transconjugants, and 2,4-D concentrations within the soil. In unsaturated soil, pJP4 was detected in both culturable donor and transconjugant cells within soil 10.5 cm from the inoculated end of the column. In saturated soil, no transconjugants were detected; however, donors were found throughout the entire length of the column (30.5 cm). These results suggest that donor transport, particularly in conjunction with plasmid transfer to indigenous recipients, allows for significant dispersal of introduced genes through contaminated soil.

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.

Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors.

A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.

Use of integrated cell culture-PCR to evaluate the effectiveness of poliovirus inactivation by chlorine.

Current standards, based on cell culture assay, indicate that poliovirus is inactivated by 0.5 mg of free chlorine per liter after 2 min; however, integrated cell culture-PCR detected viruses for up to 8 min of exposure to the same chlorine concentration, requiring 10 min for complete inactivation. Thus, the contact time for chlorine disinfection of poliovirus is up to five times greater than previously thought.

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.

Evaluation of methodologies including immunofluorescent assay (IFA) and the polymerase chain reaction (PCR) for detection of human pathogenic microsporidia in water.

Microsporidia is a term used to describe a group of emerging protozoan pathogens whose environmental occurrence has only recently been documented due to lack of detection methodologies. This study evaluates and describes current methods for detection of microsporidia in water. Standard methods, for the collection and processing of large volumes of water to detect protozoa, showed only a 4.8% recovery, of microsporidia spores, from 100 l volumes of tap. Immunofluorescent assay (IFA) analysis was assessed using two different antibodies specific for human pathogenic microsporidia. Results indicated that the use of IFA for routine screening of water for microsporidia was not an acceptable approach. The antibodies tested for the IFA resulted in false positives and false negatives and did not react with Enterocytozoon bieneusi, which is an important human pathogenic microsporidia. Finally, the small sizes of the human pathogenic microsporidia prevent confirmation and species determination by light microscopic methods. Two methods for isolating microsporidia DNA from water for use in polymerase chain reaction (PCR) amplification of microsporidia target sequences were assessed. Both of these DNA isolation methods when combined with the PCR showed the ability to detect less than ten spores in purified water concentrates. Thus, this study represents the first documentation and evaluation of current methods for the detection of human pathogenic microsporidia in water.

Incidence of enteroviruses in Mamala Bay, Hawaii using cell culture and direct polymerase chain reaction methodologies.

The consequence of point and nonpoint pollution sources, discharged into marine waters, on public recreational beaches in Mamala Bay, Hawaii was evaluated using virus cell culture and direct reverse transcriptase-polymerase chain reaction (RT-PCR). Twelve sites, nine marine, two freshwater (one stream and one canal), and one sewage, were assessed either quarterly or monthly for 1 year to detect the presence of human enteric viruses. Water samples were concentrated from initial volumes of 400 L to final volumes of 30 mL using Filterite electronegative cartridge filters and a modified beef extract elution procedure. Cell culture was applied using the Buffalo Green Monkey kidney cell line to analyze samples for enteroviruses. Positive samples were also evaluated by RT-PCR, using enterovirus-specific primers. Levels of RT-PCR inhibition varied with each concentrated sample. Resin column purification increased PCR detection sensitivity by at least one order of magnitude in a variety of sewage outfall and recreational marine water samples but not in the freshwater canal samples. Using cell culture, viable enteroviruses were found in 50 and 17% of all outfall and canal samples, respectively. Samples were positive at beaches 8% of the time. These data illustrate the potential public health hazard associated with recreational waters. Using direct PCR, viruses were detected at the outfall but were not found in any beach or canal samples, in part, owing to substances that inhibit PCR. Therefore, conventional cell culture is the most effective means of detecting low levels of infectious enteroviruses in environmental waters, whereas direct RT-PCR is rendered less effective by inhibitory compounds and low equivalent reaction volumes.