Comparative evaluation of irrigation waters on microbiological safety of spinach in field.

AIMS: Effect of ground water (GW), secondary-treated wastewater (STWW) and roof-harvested rainwater (RHW) irrigation on microbiological quality of spinach in field was investigated. METHODS AND RESULTS: Spinach grown at the Fulton farm (Chambersburg, PA) was spray-irrigated with GW, STWW or RHW once a week for 2 weeks in summer and fall seasons. Four replicate spinach and soil samples collected from two plots for each group were analysed for indicator and pathogenic bacteria on 0, 1, 2 and 4 days postirrigation (dpi). While total coliforms remained unchanged on spinach regardless of the treatment waters and growing seasons, populations of faecal coliform significantly decreased on RHW- and STWW-irrigated spinach as compared to spinach irrigated with GW at 4 dpi of each week in fall. Irrigation with STWW that contained Escherichia coli population <1·0 log CFU per 100 ml resulted in the lowest E. coli recovery on spinach in fall. Bacterial pathogens were not detected in any sample. CONCLUSIONS: The transference of indicator micro-organisms from irrigation waters to plants was influenced by the type of water and growing season. SIGNIFICANCE AND IMPACT OF THE STUDY: Alternative water sources such as STWW and RHW containing low indicator bacterial populations may be suitable for spinach irrigation in the mid-Atlantic region. However, microbiological quality of these waters must be determined prior to their use for irrigation.

Microbiological quality of spinach irrigated with reclaimed wastewater and roof-harvest water.

AIMS: The effect of reclaimed wastewater (RCW) and roof-harvest rainwater (RHW) on the microbiological quality of irrigated spinach was investigated. METHODS AND RESULTS: Spinach grown in the controlled environment chamber was irrigated by RCW, RHW or creek water (CW; control water) for 4 weeks, and then six replicate spinach samples from each treatment were collected weekly at 0 h and 24 h postirrigation. Spinach samples were analysed for populations of faecal bacterial indicators and pathogens. Bacterial populations in alternative irrigation water samples were determined by the membrane filtration technique. The RCW samples contained the highest faecal bacterial indicator populations, followed by the CW and RHW throughout the entire study. Irrigation waters containing higher populations of total and faecal coliforms did not necessarily result in higher populations of these bacteria on the irrigated spinach. Higher numbers of E. coli-positive spinach samples were reported from RCW-irrigated spinach, especially with repeated irrigation. Pathogens were not detected from any water or spinach samples. CONCLUSIONS: Spinach irrigated with RHW did not significantly affect the populations of faecal indicator bacteria when compared with CW-irrigated spinach. Repeat irrigation with RCW is not recommended due to the increased contamination of E. coli on spinach leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: RHW may potentially be used as alternative irrigation water without deleteriously affecting the microbiological safety of the spinach.

Persistence of enterohaemorrhagic and nonpathogenic E. coli on spinach leaves and in rhizosphere soil.

AIMS: Survival of Escherichia coli O157:H7 and nonpathogenic E. coli on spinach leaves and in organic soil while growing spinach in a growth chamber was investigated. METHODS AND RESULTS: Spinach plants were maintained in the growth chamber at 20 degrees C (14 h) and 18 degrees C (10 h) settings at 60% relative humidity. Five separate inocula, each containing one strain of E. coli O157:H7 and one nonpathogenic E. coli isolate were applied to individual 4-week-old spinach plants (cultivar 'Whale') grown in sandy soil. Leaf and soil inocula consisted of 100 microl, in 5 microl droplets, on the upper side of leaves resulting in 6.5 log CFU plant(-1) and 1 ml in soil, resulting in 6.5 log CFU 200 g(-1) soil per plant. Four replicates of each plant shoot and soil sample per inoculum were analysed on day 1 and every 7 days for 28 days for E. coli O157:H7 and nonpathogenic E. coli (by MPN) and for heterotrophic plate counts (HPC). Escherichia coli O157:H7 was not detected on plant shoots after 7 days but did survive in soil for up to 28 days. Nonpathogenic E. coli survived up to 14 days on shoots and was detected at low concentrations for up to 28 days. In contrast, there were no significant differences in HPC from days 0 to 28 on plants, except one treatment on day 7. CONCLUSIONS: Escherichia coli O157:H7 persisted in soil for at least 28 days. Escherichia coli O157:H7 on spinach leaves survived for less than 14 days when co-inoculated with nonpathogenic E. coli. There was no correlation between HPC and E. coli O157:H7 or nonpathogenic E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: The persistence of nonpathogenic E. coli isolates makes them possible candidates as surrogates for E. coli O157:H7 on spinach leaves in field trials.

Effect of a reactive oxygen species-generating system for control of airborne microorganisms in a meat-processing environment.

The effectiveness of reactive oxygen species (ROS)-generating AirOcare equipment on the reduction of airborne bacteria in a meat-processing environment was determined. Serratia marcescens and lactic acid bacteria (Lactococcus lactis subsp. lactis and Lactobacillus plantarum) were used to artificially contaminate the air via a six-jet Collison nebulizer. Air in the meat-processing room was sampled immediately after aerosol generation and at various predetermined times at multiple locations by using a Staplex 6 stage air sampler. Approximately a 4-log reduction of the aerial S. marcescens population was observed within 2 h of treatment (P < 0.05) compared to a 1-log reduction in control samples. The S. marcescens populations reduced further by approximately 4.5 log after 24 h of exposure to ROS treatment. Approximately 3-log CFU/m3 reductions in lactic acid bacteria were observed following 2-h ROS exposure. Further ROS exposure reduced lactic acid bacteria in the air; however, the difference in their survival after 24 h of exposure was not significantly different from that observed with the control treatment. S. marcescens bacteria were more sensitive to ROS treatment than the lactic acid bacteria. These findings reveal that ROS treatment using the AirOcare unit significantly reduces airborne S. marcescens and lactic acid bacteria in meat-processing environments within 2 h.

Adenosylcobalamin inhibits ribosome binding to btuB RNA.

Expression of the btuB gene encoding the outer membrane cobalamin transporter in Escherichia coli is strongly reduced on growth with cobalamins. Previous studies have shown that this regulation occurs in response to adenosylcobalamin (Ado-Cbl) and operates primarily at the translational level. Changes in the level and stability of btuB RNA are consequences of the modulated translation initiation. To examine how Ado-Cbl affects translation, the binding of E. coli 30S ribosomal subunits to btuB RNA was investigated by using a primer extension inhibition assay. Ribosome binding to btuB RNA was much less efficient than to other RNAs and was preferentially lost when the ribosomes were subjected to a high-salt wash. Ribosome binding to btuB RNA was inhibited by Ado-Cbl but not by cyanocobalamin, with half-maximal inhibition around 0.3 microM Ado-Cbl. Ribosome-binding activity was increased or decreased by mutations in the btuB leader region, which affected two predicted RNA hairpins and altered expression of btuB-lacZ reporters. Finally, the presence of Ado-Cbl elicited formation of a single primer extension-inhibition product with the same specificity and Cbl-concentration dependence as the inhibition of ribosome binding. These results indicate that btuB expression is controlled by the specific binding of Ado-Cbl to btuB RNA, which then affects access to its ribosome-binding sequence.

Coupled changes in translation and transcription during cobalamin-dependent regulation of btuB expression in Escherichia coli.

The level of the vitamin B12 transport protein BtuB in the outer membrane of Escherichia coli is strongly reduced by growth in the presence of cobalamins. Previous analyses of regulatory mutants and of btuB-lacZ fusions indicated that the primary site of btuB gene regulation was at the translational level, and this required sequences throughout the 240-nucleotide (nt) leader region. Cobalamin-dependent regulation of transcriptional fusions was of a lesser magnitude but required, in addition to the leader, sequences within the first 100 nt of the coding sequence, termed the translated regulatory region (TRR). To analyze the process of transcription-level regulation of btuB in E. coli, the levels and metabolism of btuB RNA were analyzed by S1 nuclease protection assays, and mutations that alter the coupling of translational and transcriptional control were analyzed. Expression of transcriptional fusions was found to correlate with changes in the level of intact btuB RNA and was related to changes in the metabolic stability of the normally long-lived RNA. Mutational analysis showed that the btuB start codon and a hairpin structure that can sequester the Shine-Dalgarno sequence are necessary for cobalamin-dependent regulation and that translation of the TRR is necessary for extended RNA stability and for expression of the transcriptional fusion. The absence of regulation at the stage of transcription initiation was confirmed by the findings that several truncated btuB RNA fragments were expressed in a constitutive manner and that the normal regulatory response occurred even when the btuB promoter and upstream sequences were replaced by the heterologous bla and lac promoters. Transcription driven by phage T7 RNA polymerase was not regulated by cobalamins, although some regulation at the translational level was retained. Cobalamin-dependent changes in RNA structure were suggested from the RNase III-dependent production of a transcript fragment that is made only in the presence of cobalamin and is independent of the regulatory outcome. These results indicate that the primary control of btuB expression by cobalamin occurs at the level of translation initiation, which directly affects the level and stability of btuB RNA in a process that requires the presence of the intact translated regulatory region.

Differential binding of Lrp to two sets of pap DNA binding sites mediated by Pap I regulates Pap phase variation in Escherichia coli.

Pyelonephritis-associated pili (Pap) expression in Escherichia coli is subject to a phase variation control mechanism that is regulated by the leucine-responsive regulatory protein (Lrp), PapI, and deoxyadenosine methylase (Dam). In previous work, we found that the differential Dam methylation of two target sites in pap regulatory DNA, GATC-I and GATC II, is essential for the transition between active and inactive pap transcriptional states. Here, we identify six Lrp binding sites within the pap regulatory DNA, each separated by about three helical turns. Lrp binds with highest affinity to three sites (1, 2 and 3) proximal to the papBAp promoter. A mutational analysis indicates that the binding of Lrp to sites 2 and 3 inhibits pap transcription, which is consistent with the fact that Lrp binding site 3 is located between the --35 and --10 RNA polymerase binding region of papBAp. The addition of PapI decreases the affinity of Lrp for sites 1, 2 and 3 and increases its affinity for the distal Lrp binding sites 4 and 5. Mutations within Lrp binding sites 4 and 5 shut off pap transcription, indicating that the binding of Lrp to this pap region activates pap transcription. The pap GATC-I and GATC-II methylation sites are located within Lrp binding sites 5 and 2, respectively, providing a mechanism by which Dam controls Lrp binding and Pap phase variation.

Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli.

We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E. coli. Two Dam methyltransferase sites, GATC1028 and GATC1130, were shown previously to be differentially methylated in phase ON versus phase OFF cells. In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. Our results show that methylation of GATC1028 blocks formation of the ON state by inhibiting the binding of Lrp and PapI regulatory proteins to this site. Conversely, methylation of GATC1130 is required for the ON state. Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter. A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

Regulation of pyelonephritis-associated pili phase-variation in Escherichia coli: binding of the PapI and the Lrp regulatory proteins is controlled by DNA methylation.

Expression of pyelonephritis-associated pili (Pap) in Escherichia coli is under a phase-variation control mechanism in which individual cells alternate between pili+ (ON) and pili- (OFF) states through a process involving DNA methylation by deoxyadenosine methylase (Dam). Methylation of two GATC sites (GATC1028 and GATC1130) within the pap regulatory region is differentially inhibited in phase ON and phase OFF cells. The GATC1028 site of phase ON cells is non-methylated and the GATC1130 site is fully methylated. Conversely, in phase OFF cells the GATC1028 site is fully methylated whereas the GATC1130 site is non-methylated. Two transcriptional activators, PapI and Lrp (leucine-responsive regulatory protein), are required for this specific methylation inhibition. DNA footprint analysis using non-methylated pap DNAs indicates that Lrp binds to a region surrounding the GATC1130 site, whereas PapI does not appear to bind to pap regulatory DNA. However, addition of Lrp and PapI together results in an additional DNaseI footprint around the GATC1028 site. Moreover, Dam methylation inhibits binding of Lrp/PapI near the GATC1028 site and alters binding of Lrp at the GATC1130 site. Our results support a model in which Dam and Lrp/PapI compete for binding near the GATC1028 site, regulating the methylation state of this GATC site and, consequently, the pap transcription state.