Degradation and adsorption of synthetic DNA water tracers in environmental matrices.

Synthetic DNA tracers are gaining interest as tools for tracking contamination pathways and hydraulic connections in surface water and groundwater systems. However, few quantitative data exist that describe DNA tracer degradation and adsorption in environmental matrices. We undertook laboratory experiments to quantify the degradation of multiple double-stranded DNA tracers in stream water, groundwater, and domestic and dairy-shed effluent, and adsorption to stream sediments, soils, coastal sand aquifer media and alluvial sandy gravel aquifer media. Faster DNA tracer degradation seemed to be associated with high bacterial concentrations in the liquid phase. Overall, the degradation of the 352 base pair (bp) DNA tracers in the aqueous phase was significantly (P = 0.018) slower than that of the 302 bp DNA tracers. Although the tracers' internal amplicon lengths were similar, the longer non-amplified flanking regions of the 352 bp tracers may better protect them from environmental degradation. Thermodynamic analysis suggests that longer flanking regions contribute to greater tracer stability. This finding may explain our previous field observations that 352 bp tracer mass reductions were often lower than 302 bp tracer mass reductions. The 2 sets of DNA tracers did not differ significantly regarding their adsorption to stream sediment-stream water or aquifer media-groundwater mixtures (P > 0.067), but the 352 bp tracers showed significantly less adsorption to soil-effluent mixtures than the 302 bp tracers (P = 0.005). The DNA tracers' adsorption to soil-effluent mixtures was comparatively less than their adsorption to the aquifer media-groundwater and stream sediment-stream water mixtures, suggesting that DNA tracers may compete with like-charged organic matter for adsorption sites. These findings provide insights into the fate of DNA tracers in the environment. The DNA tracers' degradation rate constants determined in this study for a range of environmental conditions could assist the design of future field investigations.

Molecular risk assessment and epidemiological typing of Shiga toxin-producing Escherichia coli by using a novel PCR binary typing system.

Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes diarrheal disease in humans and is of public health concern because of its ability to cause outbreaks and severe disease such as hemorrhagic colitis or hemolytic-uremic syndrome. More than 400 serotypes of STEC have been implicated in outbreaks and sporadic human disease. The aim of this study was to develop a PCR binary typing (P-BIT) system that could be used to aid in risk assessment and epidemiological studies of STEC by using gene targets that would represent a broad range of STEC virulence genes. We investigated the distribution of 41 gene targets in 75 O157 and non-O157 STEC isolates and found that P-BIT provided 100% typeability for isolates, gave a diversity index of 97.33% (compared with 99.28% for XbaI pulsed-field gel electrophoresis [PFGE] typing), and produced 100% discrimination for non-O157 STEC isolates. We identified 24 gene targets that conferred the same level of discrimination and produced the same cluster dendrogram as the 41 gene targets initially examined. P-BIT clustering identified O157 from non-O157 isolates and identified seropathotypes associated with outbreaks and severe disease. Numerical analysis of the P-BIT data identified several genes associated with human or nonhuman sources as well as high-risk seropathotypes. We conclude that P-BIT is a useful approach for subtyping, offering the advantage of speed, low cost, and potential for strain risk assessment that can be used in tandem with current molecular typing schema for STEC.

Attenuation and transport of human enteric viruses and bacteriophage MS2 in alluvial sand and gravel aquifer media-laboratory studies.

Potable groundwater contamination by human enteric viruses poses serious health risks. Our understanding of virus subsurface transport has largely depended on studying bacteriophages as surrogates. Few studies have compared the transport behaviour of enteric viruses, especially norovirus, with phage surrogates. We conducted laboratory column experiments to investigate norovirus and bacteriophage MS2 (MS2) filtration in alluvial sand, and rotavirus, adenovirus and MS2 filtration in alluvial gravel aquifer media in 2 mM NaCl (pH 6.6-6.9) with pore velocities of 4.6-5.4 m/day. The data were analysed using colloid filtration theory and HYDRUS-1D 2-site attachment-detachment modelling. Norovirus removal was somewhat lower than MS2 removal in alluvial sand. The removal of rotavirus and adenovirus was markedly greater than MS2 removal in alluvial gravel. These findings concurred with the log10 reduction values, mass recoveries, attachment efficiencies and irreversible deposition rate constants. The modelling results suggested that the MS2 detachment rates were in the same order of magnitude as norovirus, but they were 1 order of magnitude faster than those of rotavirus and adenovirus. The attachment of viruses and MS2 was largely reversible with faster detachment than attachment rates, favouring free virus transport. These findings highlight the risk associated with continual virus transport through subsurface media if viruses are not inactivated and remobilising previously attached viruses could trigger contamination events. Thus, virus attachment reversibility should be considered in virus transport predictions in subsurface media. Further research is needed to compare surrogates with enteric viruses, especially norovirus, regarding their transport behaviours under different experimental conditions.

Potential to use ultraviolet-treated bacteriophages to control foodborne pathogens.

The use of replication-deficient UV-treated bacteriophages, or phages, presents an alternative to viable phages for food biocontrol applications. Nontransducing UV-treated phages, if used correctly, are unlikely to produce viable progeny phages, which might otherwise mediate undesirable horizontal gene transfer events. Phage T4 and Escherichia coli were used as a model system to examine this possibility. UV-treated phages were able to cause a reduction in the optical density of outer membrane-free cell suspensions and they also killed host cells under conditions not permitting their multiplication, that is, 24 degrees C for 2 h and 37 degrees C for 15 min. Host cell reductions were also demonstrated in broth and on meat at 5 degrees C when high concentrations of phages of 2.3 x 10(9) PFU mL(-1) and 1.8 x 10(8) PFU cm(-2), respectively, were used. At 24 degrees C and 37 degrees C, "lysis from without" was likely to be the mechanism responsible for the reduction in host cell concentrations, but at 5 degrees C this may not have been the case.

Water tracking in surface water, groundwater and soils using free and alginate-chitosan encapsulated synthetic DNA tracers.

Investigating contamination pathways and hydraulic connections in complex hydrological systems will benefit greatly from multi-tracer approaches. The use of non-toxic synthetic DNA tracers is promising, because unlimited numbers of tracers, each with a unique DNA identifier, could be used concurrently and detected at extremely low concentrations. This study aimed to develop multiple synthetic DNA tracers as free molecules and encapsulated within microparticles of biocompatible and biodegradable alginate and chitosan, and to validate their field utility in different systems. Experiments encompassing a wide range of conditions and flow rates (19 cm/day-39 km/day) were conducted in a stream, an alluvial gravel aquifer, a fine coastal sand aquifer, and in lysimeters containing undisturbed silt loam over gravels. The DNA tracers were identifiable in all field conditions investigated, and they were directly detectable in the stream at a distance of at least 1 km. The DNA tracers showed promise at tracking fast-flowing water in the stream, gravel aquifer and permeable soils, but were unsatisfactory at tracking slow-moving groundwater in the fine sand aquifer. In the surface water experiments, the microencapsulated DNA tracers' concentrations and mass recoveries were 1-3 orders of magnitude greater than those of the free DNA tracers, because encapsulation protected them from environmental stressors and they were more negatively charged. The opposite was observed in the gravel aquifer, probably due to microparticle filtration by the aquifer media. Although these new DNA tracers showed promise in proof-of-concept field validations, further work is needed before they can be used for large-scale investigations.

Genome Sequence of a Podovirus (AAPEc6) Isolated from Wastewater in New Zealand That Infects Escherichia coli O45:H10.

Bacteriophages are ideal candidates for pathogen biocontrol to mitigate outbreaks of prevalent foodborne pathogens, such as Escherichia coli We identified a bacteriophage (AAPEc6) from wastewater that infects E. coli O45:H10. The AAPEc6 genome sequence shares 93% identity (with 92% coverage) to enterobacterial phage K1E (Sp6likevirus) in the Autographivirinae subfamily (Podoviridae).

Growth of Yersinia enterocolitica and Y. pseudotuberculosis in Yersinia selective enrichment broth according to Ossmer.

The growth of Yersinia enterocolitica and Yersinia pseudotuberculosis in Yersinia enrichment broth according to Ossmer (YSEO) was investigated. Y. enterocolitica reached a higher concentration than Y. pseudotuberculosis but both always exceeded 10(6)CFU/ml. The medium may be useful for the detection of both species in foods.