On-farm and postharvest processing sources of bacterial contamination to melon rinds.

Multistate and international foodborne illness outbreaks, particularly involving cantaloupe and often involving rare Salmonella spp., have increased dramatically over the past 13 years. This study assessed the sources and extent of melon rind contamination in production fields and at processing and packing facilities. In the spring of 1999, cantaloupe (Cucumis melo L. [reticulatus group] cv. Cruiser) sampled from two sites in the Rio Grande River Valley showed that postharvest-processed melon rinds often had greater plate counts of bacterial contaminants than field-fresh melons. Cantaloupe in the field had 2.5 to 3.5 log CFU g(-1) rind total coliforms by aerobic plate counts, whereas washed melons had 4.0 to 5.0 log CFU g(-1). In the fall of 1999, coliforms on honeydew melons (C. melo [inodorous group] cv. Honey Brew) ranged from 2.6 to 3.7 log CFU g(-1) after processing, and total and fecal coliforms and enterococci never fell below 2.5 log CFU g(-1). A hydrocooler at another site contaminated cantaloupe rinds with up to 3.4 log CFU g(-1) total and fecal enterococci; a secondary rinse with chlorinated water incompletely removed these bacteria. Sources of coliforms and enterococci were at high levels in melon production soils, especially in furrows that were flood irrigated, in standing water at one field, and in irrigation water at both sites. At one processing facility, wash water pumped from the Rio Grande River may not have been sufficiently disinfected prior to use. Because soil, irrigation water, and process water were potential sources of bacterial contamination, monitoring and management on-farm and at processing and packing facilities should focus on water quality as an important control point for growers and packers to reduce bacterial contamination on melon rinds.

Influence of the zinc hyperaccumulator Thlaspi caerulescens J. & C. Presl. and the nonmetal accumulator Trifolium pratense L. on soil microbial populations.

Metal hyperaccumulator plants like Thlaspi caerulescens J. & C. Presl. are used for phytoremediation of contaminated soils. Since little is known about the rhizosphere of hyperaccumulators, the influence of T. caerulescens was compared with the effects of Trifolium pratense L. on soil microbes. High- and low-metal soils were collected near a zinc smelter in Palmerton, Penn. Soil pH was adjusted to 5.8 and 6.8 by the addition of Ca(OH)2. Liming increased bacterial populations and decreased metal toxicity to levels allowing growth of both plants. The effects of the plants on total (culturable) bacteria, total fungi, as well as cadmium- and zinc-resistant populations were assessed in nonrhizosphere and rhizosphere soil. Both plants increased microbial populations in rhizosphere soil compared with nonrhizosphere soil. Microbial populations were higher in soils planted with T. pratense, but higher ratios of metal-resistant bacteria were found in the presence of T. caerulescens. We hypothesize that T. caerutescens acidifies its rhizosphere. Soil acidification in the rhizosphere of T. caerulescens would affect metal uptake by increasing available metals around the roots and consequently, increase the selection for metal-resistant bacteria. Soil acidification may be part of the hyperaccumulation process enhancing metal uptake from soil.

Intact soil-core microcosms compared with multi-site field releases for pre-release testing of microbes in diverse soils and climates.

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

Leaching of Escherichia coli O157:H7 in diverse soils under various agricultural management practices.

Application of animal manures to soil as crop fertilizers is an important means for recycling the nitrogen and phosphorus which the manures contain. Animal manures also contain bacteria, including many types of pathogens. Manure pathogen levels depend on the source animal, the animal's state of health, and how the manure was stored or treated before use. Rainfall may result in pathogen spread into soil by runoff from stored or unincorporated manure or by leaching through the soil profile. Steady rainfall consisting of 16.5 mm h(-1) was applied to 100-mm disturbed soil cores that were treated with manure and inoculated with Escherichia coli O157:H7 strain B6914. The level of B6914 in leachate was near the inoculum level each hour for 8 h, as was the level of B6914 at several soil depths after 24 h, indicating that there was a high rate of growth. Bacterial movement through three different types of soil was then compared by using disturbed (tilled) and intact (no-till) soil cores and less intense rainfall consisting of 25.4 mm on 4 consecutive days and then four more times over a 17-day period. Total B6914 levels exceeded the inoculum levels for all treatments except intact clay loam cores. B6914 levels in daily leachate samples decreased sharply with time, although the levels were more constant when intact sandy loam cores were used. The presence of manure often increased total B6914 leachate and soil levels in intact cores but had the opposite effect on disturbed soil cores. Ammonia and nitrate levels correlated with B6914 and total coliform levels in leachate. We concluded that tillage practice, soil type, and method of pathogen delivery affect but do not prevent vertical E. coli O157:H7 and coliform transport in soil and that soluble nitrogen may enhance transport.

Validation of Microcosms for Examining the Survival of Pseudomonas aureofaciens (lacZY) in Soil.

Evaluating the safety and efficacy of a recombinant bacterium prior to its release into the terrestrial environment requires that risk assessment data be collected in the laboratory. Much of this information is obtained with the use of microcosms. The design of the microcosm significantly affects the ability of the recombinant microorganism to survive in soil and, thus, complicates the risk assessment process. To standardize microcosms for future use, we evaluated the survival of Pseudomonas aureofaciens 3732 RN-L11 (lacZY Rif(supr) Nal(supr)) in intact soil cores (5.0 by 15 cm; polyvinyl chloride core) and disturbed soil microcosms (50 g of fresh, sieved soil). Survival data were compared with those obtained during a field release. The intact soil core microcosm was shown to closely simulate results obtained in the field. The intact soil core microcosm closely predicts survival in bulk soil and in the rhizosphere of wheat. Data obtained with the microcosm were also similar when evaluated in separate studies in two different years. In 1993, P. aureofaciens survived for approximately 63 days in bulk soil and 96 days in the rhizosphere. The disturbed soil microcosm exhibited a much more rapid decline in population size (34 days to zero) than the intact core microcosm. We speculate that drying and sieving of soil for the disturbed soil microcosm affected the ability of the soil to support the survival of P. aureofaciens. These results demonstrate that a small, inexpensive, and simple intact soil core microcosm may be appropriate for risk assessment.

Pseudomonas aureofaciens in soil: survival and recovery efficiency.

Efficient methods for the recovery of genetically engineered organisms (GEM) added to soil are critical if the safety of potential releases is to be evaluated and the actual release is to be monitored. Pseudomonas aureofaciens strain 3732 RN-L11 (lacZY) was added to 10 g sieved soil microcosms and incubated for 5 and 28 days. Various diluents, shaking methods, and settling of soil were examined to determine the optimum method for recovery of the GEM from the soil. Of the diluents examined, 0.1% agar gave significantly lower numbers than distilled water, 1.0% sodium metaphosphate, 1% peptone, and phosphate-buffered water. After 5 days of incubation, shaking for 10 min with glass beads and shaking for 30 min without glass beads resulted in the highest recovery of the GEM from soil, while sonification resulted in the lowest recovery. After 28 days of incubation, sonification produced significantly lower numbers than any of the other treatments. The addition of 1% CaCl2 to enhance settling significantly increased recovery efficiency. Although the use of CaCl2 in distilled water and shaking for 10 min was an effective method for recovering P. aureofaciens from a Maryland soil, when the same extraction procedure was compared with a standard technique (dd H2O, shaking for 10 min) for eight divergent soils, neither extraction method was consistently better than the other. Statistical analysis of the data showed the need for log transformation of the raw data. Four microcosm and two plate replicates for each dilution provided the greatest ability to detect differences between treatment means while maximizing experimental efficiency.

Naloxone reversal of mild neurobehavioral depression in normal newborn infants after routine obstetric analgesia.

To investigate the presence of subtle narcotic depression following maternal narcotic analgesia, we have evaluated the effects of naloxone versus placebo in a double-blind parallel group study in 43 normal term newborn infants whose mothers had received routine narcotic analgesia within six hours prior to delivery. Infants were given either an intramuscular injection of 20 microgram/kg naloxone or 0.20 ml/kg placebo after determination of the one-minute Apgar score, and the following measurements were compared: Apgar scores at one and five minutes, capillary blood gas values at one, 60, 120, and 240 minutes, and neurobehavioral assessments at one, 4, and 24 hours. No adverse effects from naloxone were observed. Neither Apgar scores nor capillary blood gas determinations differed significantly between the two groups. Response to sound was significantly higher in the naloxone group at 24 hours. The alertness score was significantly higher for the naloxone group at one and four hours; the general assessment score for the naloxone group was significantly higher at four and 24 hours. Average scores of naloxone and placebo groups were also different at four and 24 hours of age. These data demonstrate that maternal narcotic analgesia may produce subtle changes in alertness and general behavior not reflected by Apgar scores or respiratory status, potentially reversible by administration of naloxone shortly following delivery.