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°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.

We identify key environmental and geochemical factors that shape the arid soil microbiome along aridity and vegetation gradients spanning over 300 km of the Atacama Desert, Chile. Decreasing average soil relative humidity and increasing temperature explain significant reductions in the diversity and connectivity of these desert soil microbial communities and lead to significant reductions in the abundance of key taxa typically associated with fertile soils. This finding is important because it suggests that predicted climate change-driven increases in aridity may compromise the capacity of the arid-soil microbiome to sustain necessary nutrient cycling and carbon sequestration functions as well as vegetative cover in desert ecosystems, which comprise one-third of the terrestrial biomes on Earth.

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 BiosamplersÒ. 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 · 10 )4 chance of infection from inhalation of coxsackievirus A21. Land application of biosolids resulted in risks that were <2 · 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 microorganisms from the land application of biosolids and subsequently determined that microbial risks of infection were low for residents close to biosolids application sites.

This two year study evaluated the prevalence of indicator bacteria and specific pathogens in10 ‘normal’ kitchens in the United States. In Phase I, none of the kitchens wascleaned with an antimicrobial cleaner or disinfectant. Eight locations within the kitchens weremonitored for: total heterotrophs, staphylococci, Pseudomonas, total coliforms andfaecal coliforms. Almost all locations at all households exhibited contamination, with the sink andsponge samples exhibiting large bacterial concentrations. The faecal coliform concentrations insink 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 and33·3% of all sponges. Salmonella was detected once and Campylobacter, on two occasions. In a second phase, households were provided with an antimicrobialdisinfectant 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 consistentreduction in the incidence of bacterial contamination. By contrast, in the final phase of the studywhere 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 caneasily be contaminated with a variety of bacterial contaminants including faecal coliforms, E.coli, Salmonella and Campylobacter. Irregular use, or not using antimicrobialagents, is unlikely to reduce the risk of these infectious agents. By contrast, targeted use is likelyto reduce the incidence of bacterial contaminants.

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 g 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-g ml Cocontaminated soils, soils contaminated with both metals and organics, are considered difficult to remediate because of the mixed nature of the contaminants. A treatment alternative to expensive excavation and incineration (9) of metal-contaminated soils is bioaugmentation with metal-detoxifying and/or organic-degrading microorganisms (1,3,4,6,18). Many microorganisms are known to degrade a variety of organics, and likewise, a number of metal-resistant microorganisms are known to detoxify metals, such as selenium, mercury, and cadmium (23,27). In cocontaminated sites, metal toxicity inhibits the activity of organic-degrading microorganisms (24). Consequently, bioremediation efforts focus on reducing metal toxicity in sites with mixed contaminants. Until recently, bioaugmentation studies focused on the introduction of a microorganism that was both metal resistant and capable of organic degradation. Under field conditions, such an approach is often unsuccessful. One reason may be that the energy requirements to maintain concurrent metal resistance and organic degradation are too high, and the introduced organism cannot perform both activities under environmental conditions. The issue of cocontamination is a serious one, since approximately 37% of all contaminated sites in the United States alone contain both metal and organic contaminants (20; W. W. Kovalich, Jr., keynote lecture, 4th World Congr. Chem. Eng., p. 281-295, 1991).The approach used in this study was to coinoculate with a metal-detoxifying population and an organic-degrading population that cooperatively functioned to remediate both metal and organic pollutants in a cocontaminated system. We hypothesized that the metal-resistant population could protect the metal-sensitive organic-degrading population from metal toxicity. Stephen et al. (27) used metal-resis...

No abstract

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.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect SalmoneUla species. phoP primers specific to the phoPlphoQ loci of coliform pathogenic bacteria such as SalnoneUla, ShigeUla, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-li primers, which targeted a 236-bp region of hinIH2 and a 173-bp region of the H-li flagellin gene, respectively, were used. Both Hin and H-li primers are specific to motile SalmoneUla species and are not present in ShigeUa, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonela species including Salmonella typhi, SalmoneUla typhimurium, Salmonella paratyphi A, and SalmoneUa enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of SalmoneUla species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 102 CFU after 25 cycles of PCR and 1 (100) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as SalmoneUla species by the use of conventional cultural techniques. In addition, positive amplifications resulted from SalmoneUla species in environmental samples including soil and water.