Survival and growth of Salmonella Enteritidis PT 30 in almond orchard soils.

AIMS: To evaluate factors potentially contributing to the long-term persistence of Salmonella enterica serovar Enteritidis phage type (PT) 30 in an almond orchard. METHODS AND RESULTS: Surface and subsurface soil temperatures, and air temperatures in a radiation shelter, were recorded during a 12-month period, and were used to identify relevant storage temperatures (20 or 35 degrees C) for microcosms of two different soil types (clay and sandy loams) with moisture levels near saturation or near field capacity. Salmonella Enteritidis PT 30 was inoculated into the microcosms at 6 log CFU g(-1) dry weight. Between 14 and 180 days of incubation, counts of S. Enteritidis PT 30 decreased rapidly at 35 degrees C and were significantly different (P < 0.05) from counts at 20 degrees C, regardless of the soil type or moisture level. Salmonella was detected by enrichment of 10-g samples from all microcosms after 180 days of incubation at 20 degrees C, but from none of the microcosms held at 35 degrees C. To measure the potential for the growth of S. Enteritidis PT 30 in clay loam soil, an aqueous extract of almond hulls (containing 1.6% mono and disaccharides) or equivalent volume of water was added 7 days after inoculation. Significant (P < 0.05) growth of S. Enteritidis PT 30 was observed within 8 or 24 h of adding hull extract, but not water, to soil. CONCLUSIONS: Opportunities may exist for S. Enteritidis PT 30 to survive for an extended time in almond orchard soils and to grow in these soils where hull nutrients are released. SIGNIFICANCE AND IMPACT OF THE STUDY: Temperature has a significant impact on the long-term survival of S. Enteritidis PT 30 in soil, and nutrients leached from almond hulls may result in Salmonella growth. These factors should be considered in the design of Good Agricultural Practices for almonds.

Effects of arbuscular mycorrhizas on ammonia oxidizing bacteria in an organic farm soil.

Arbuscular mycorrhizal fungi (AMF) are potentially important in nutrient cycling in agricultural soils and particularly in soils managed for organic production; little is known, however, about the interrelationships between AMF and other members of soil microbial communities. Ammonia oxidizing bacteria (AOB) are a trophic group of bacteria having an enormous impact on nitrogen availability in soils and are expected to be influenced by the presence of AMF. In a field study, we utilized a unique genetic system comprised of a mycorrhiza defective tomato mutant (named rmc) and its mycorrhiza wild-type progenitor (named 76RMYC+). We examined the effect of AMF by comparing AOB community composition and populations in soil containing roots of the two tomato genotypes in an organically managed soil. Responses of AOB to soil N and P amendments were also studied in the same experiment. Phylogenetic analysis of cloned AOB sequences, derived from excised denaturing gradient gel electrophoresis (DGGE) bands, revealed that the organic farm soil supported a diverse yet stable AOB community, which was neither influenced by mycorrhizal colonization of roots nor by N and P addition to the soil. Real-time TaqMan polymerase chain reaction (PCR) was used to quantify AOB population sizes and showed no difference between any of the treatments. An alternative real-time PCR protocol for quantification of AOB utilizing SYBR green yielded similar results as the TaqMan real-time PCR method, although with slightly lower resolution. This alternative method is advantageous in not requiring the detailed background information about AOB community composition required for adaptation of the TaqMan system for a new soil.

Detection and quantification of methyl tert-butyl ether-degrading strain PM1 by real-time TaqMan PCR.

The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribosomal DNA region, and specificity of the primers was confirmed with DNA from 15 related bacterial strains. A linear relationship was measured between the threshold fluorescence (C(T)) value and the quantity of PM1 DNA or PM1 cell density. The detection limit for PM1 TaqMan assay was 2 PM1 cells/ml in pure culture or 180 PM1 cells/ml in a mixture of PM1 with Escherichia coli cells. We could measure PM1 densities in solution culture, groundwater, and sediment samples spiked with PM1 as well as in groundwater collected from an MTBE bioaugmentation field study. In a microcosm biodegradation study, increases in the population density of PM1 corresponded to the rate of removal of MTBE.

Molecular ecological analysis of the succession and diversity of sulfate-reducing bacteria in the mouse gastrointestinal tract.

Intestinal sulfate-reducing bacteria (SRB) growth and resultant hydrogen sulfide production may damage the gastrointestinal epithelium and thereby contribute to chronic intestinal disorders. However, the ecology and phylogenetic diversity of intestinal dissimilatory SRB populations are poorly understood, and endogenous or exogenous sources of available sulfate are not well defined. The succession of intestinal SRB was therefore compared in inbred C57BL/6J mice using a PCR-based metabolic molecular ecology (MME) approach that targets a conserved region of subunit A of the adenosine-5'-phosphosulfate (APS) reductase gene. The APS reductase-based MME strategy revealed intestinal SRB in the stomach and small intestine of 1-, 4-, and 7-day-old mice and throughout the gastrointestinal tract of 14-, 21-, 30-, 60-, and 90-day-old mice. Phylogenetic analysis of APS reductase amplicons obtained from the stomach, middle small intestine, and cecum of neonatal mice revealed that Desulfotomaculum spp. may be a predominant SRB group in the neonatal mouse intestine. Dot blot hybridizations with SRB-specific 16S ribosomal DNA (rDNA) probes demonstrated SRB colonization of the cecum and colon pre- and postweaning and colonization of the stomach and small intestine of mature mice only. The 16S rDNA hybridization data further demonstrated that SRB populations were most numerous in intestinal regions harboring sulfomucin-containing goblet cells, regardless of age. Reverse transcriptase PCR analysis demonstrated APS reductase mRNA expression in all intestinal segments of 30-day-old mice, including the stomach. These results demonstrate for the first time widespread colonization of the mouse intestine by dissimilatory SRB and evidence of spatial-specific SRB populations and sulfomucin patterns along the gastrointestinal tract.

Desulfotomaculum genus- and subgenus-specific 16S rRNA hybridization probes for environmental studies.

Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum, a set of phylogenetically nested hybridization probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans), and five specific probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' using samples obtained from four different environments. Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized. The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3-2.1% of the total rRNA in the digesters, 2.6-6.6% in soil, 1.5-3.3% in human faeces and 2.5-6.2% in pig colon samples.