Long-amplicon propidium monoazide-PCR enumeration assay to detect viable Campylobacter and Salmonella.

AIMS: The effect of amplicon length on the ability of propidium monoazide-PCR (PMA-PCR) to reliably quantify viable cells without interference from dead cells was tested on heat- and ultraviolet (UV)-killed Salmonella enterica and Campylobacter jejuni, two important enteric pathogens of concern in environmental, food and clinical samples. METHODS AND RESULTS: PMA treatment followed by quantitative PCR (qPCR) amplification of short DNA fragments (<200 bp) resulted in incomplete signal inhibition of heat-treated Salm. enterica (3 log reduction) and Camp. jejuni (1 log reduction), whereas PCR amplification of a long DNA fragment (1·5 and 1·6 kb) completely suppressed the dead cell signal. PMA pretreatment of UV-irradiated cells did not affect PCR amplification, but long-amplicon PCR was shown to detect only viable cells for these samples, even without the addition of PMA. CONCLUSIONS: The long-amplicon PMA-PCR method was effective in targeting viable cells following heat and UV treatment and was applicable to enteric pathogens including Salmonella and Campylobacter that are difficult to enumerate using culture-based procedures. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR amplicon length is important for effective removal of the dead cell signal in PMA pretreatment methods that target membrane-damaged cells, and also for inactivation mechanisms that cause direct DNA damage.

The occurrence of Campylobacter in river water and waterfowl within a watershed in southern Ontario, Canada.

AIMS: Quantitative PCR and a culture method were used to investigate Campylobacter occurrence over 3 years in a watershed located in southern Ontario, Canada that is used as a source of drinking water. METHODS AND RESULTS: Direct DNA extraction from river water followed by quantitative PCR analysis detected thermophilic campylobacters at low concentrations (<130 cells 100 ml(-1) ) in 57-79% of samples taken from five locations. By comparison, a culture-based method detected Campylobacter in 0-23% of samples. Water quality parameters such as total Escherichia coli were not highly correlated with Campylobacter levels, although higher pathogen concentrations were observed at colder water temperatures (<10°C). Strains isolated from river water were primarily nalidixic acid-susceptible Campylobacter lari, and selected isolates were identified as Campylobacter lari ssp. concheus. Campylobacter from wild birds (seagulls, ducks and geese) were detected at a similar rate using PCR (32%) and culture-based (29%) methods, and although Campylobacter jejuni was isolated most frequently, C. lari ssp. concheus was also detected. CONCLUSIONS: Campylobacter were frequently detected at low concentrations in the watershed. Higher prevalence rates using quantitative PCR was likely because of the formation of viable but nonculturable cells and low recovery of the culture method. In addition to animal and human waste, waterfowl can be an important contributor of Campylobacter in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study show that Campylobacter in surface water can be an important vector for human disease transmission and that method selection is important in determining pathogen occurrence in a water environment.

Molecular biological detection and characterization of Clostridium populations in municipal landfill sites.

Primer sets specific for 16S rRNA genes were designed for four phylogenetic groups of clostridia known to contain mesophilic cellulolytic species. Specific amplification of these groups from landfill leachate DNA extracts demonstrated the widespread occurrence of clostridia from the Clostridium thermocellum and C. leptum groups. In contrast, the C. botulinum group was never detected, and the C. coccoides-C. lentocellum group was only occasionally detected. Amplification products were analyzed by temporal thermal gel electrophoresis to generate profiles of the clostridial groups and to identify dominant bands. Sequence analysis of 17 landfill clones confirmed that the primers were specific for the clostridial subgroups and that the cloned sequences had a close relationship with known cellulose-degrading clostridia. The primers have therefore been authenticated for use in the rapid identification of clostridia in anaerobic environments.

Survival of luxAB-marked Alcaligenes eutrophus H850 in PCB-contaminated soil and sediment.

A rifampicin-resistant PCB-degrading Alcaligenes eutrophus H850 strain was marked with luxAB reporter genes and designated H850Lr. This strain was enumerated in soil by viable plating and counting of light-emitting colonies. The marked strain was also inoculated into soil and sediment microcosms contaminated with PCBs and treated with rhamnolipid biosurfactants produced by Pseudomonas aeruginosa UG2Lr or inoculated with the P. aeruginosa UG2Lr strain. A. eutrophus H850Lr exhibited similar survival in sandy loam soil in the absence or presence of PCBs over 56 days. Survival of A. eutrophus H850Lr in PCB-contaminated sediment was less than in sandy soil under the same incubation conditions. Addition of P. aeruginosa UG2 rhamnolipids to soil increased the culturable indigenous heterotrophic population, and numbers of A. eutrophus H850Lr cells. P. aeruginosa UG2Lr cells did not affect survival of A. eutrophus H850, as cell enumerations after 2 months were the same as in microcosms containing only A. eutrophus H850 inoculum. P. aeruginosa UG2Lr survived in soils as demonstrated by the slight decrease in CFU from 1 x 10(8) to 2 x 10(6) CFU cm-3 after 2 months. Direct extraction of DNA from soil and purification for use in PCR amplification using primers specific for the bphC gene detected 8 x 10(2) A. eutrophus H850Lr CFU g-1 soil in PCB-contaminated soils. Colony lifts of bacteria isolated from microcosms containing PCB-contaminated soil did not hybridize with LB400 bphC probe. However, enrichment of PCB-contaminated soil with biphenyl, followed by DNA extraction and probing with bphC gene probe detected indigenous PCB-degrading bacteria containing a similar gene sequence in PCB-contaminated sediment. This study demonstrates the usefulness of using the lux reporter system in monitoring bacterial survival in PCB-contaminated soils and sediments.

Pseudomonas aeruginosa UG2 rhamnolipid biosurfactants: structural characterization and their use in removing hydrophobic compounds from soil.

The structure of two rhamnolipid biosurfactants produced by Pseudomonas aeruginosa UG2 was studied. Analyses by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed these two rhamnolipids to be alpha-L-rhamnopyranosyl-beta-hydroxydecanyol-beta-hydroxydecanoate and 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxydecan oyl-beta- hydroxydecanoate. The ability of UG2 rhamnolipid biosurfactants to enhance removal of naphthalene, anthracene, phenanthrene, fluorene, 2,2',5,5'-tetrachlorobiphenyl, and 3,3',4,4',5,5'-hexachlorobiphenyl into the aqueous phase was affected by soil type, hydrocarbon equilibration time, and biosurfactant adsorption to soil. Partially purified UG2 biosurfactants at a concentration of 5 g/L removed approximately 10% more hydrocarbon from a sandy loam soil than slit loam soil. High levels of UG2 rhamnolipids adsorbed to soil. In 18% (w/v) soil slurries 74, 49, 38, and 20% of 0.5, 1, 2, and 5 g UG2 rhamnolipids/L, respectively, were bound to the soil phase. Sodium dodecyl sulphate recovered lower levels and Witconol SN70 higher levels of phenanthrene and 2,2',5,5'-tetrachlorobiphenyl than UG2 biosurfactants.

Germanium and silver resistance, accumulation, and toxicity in microorganisms.

Germanium is an inert metal with no known biological function in prokaryotic or eukaryotic organisms. Its toxicity is low compared to that of silver. Germanium is accumulated in certain bacterial strains by either energy-independent passive binding or an energy-dependent mechanism. Little is known about the molecular aspects of silver resistance, toxicity, and accumulation in bacterial strains. This is surprising because silver has been used as an antimicrobial agent in the medical field for centuries. It is likely that silver ions are excluded (resulting in decreased silver accumulation) from certain bacterial strains or immobilized intracellularly to prevent toxic effects from being exerted. These mechanisms of silver resistance have not been fully elucidated. This review examines the toxicity and accumulation of germanium and silver in selected microbial species. In addition, resistance mechanisms to these biologically nonessential metals is discussed, with more emphasis placed on silver-resistant bacteria due to the knowledge available.

Applications of microbial surfactants.

Biosurfactants are amphiphilic compounds produced by microorganisms that are capable of decreasing surface and interfacial tensions. They are useful in remediation of insoluble organic pollutants in soil and marine environments. There are also a large number of industrial uses for biosurfactants. This paper reviews recent research on applications of microbially-produced surfactants.

Microbial interactions with germanium.

Germanium is a semi-precious, widely-dispersed, and biologically non-essential metal with considerable potential for application in the fields of electronics, computer engineering and medicine. Research on the physiology and genetics of germanium toxicity and accumulation in microorganisms has received scant attention. An understanding of these aspects is important from both fundamental and applied points of view. This review will examine the diverse range of interactions between germanium and microbial species.