Microorganisms Move a Short Distance into an Almond Orchard from an Adjacent Upwind Poultry Operation.

Over a 2-year period, drag swabs of orchard soil surface and air, soil, and almond leaf samples were collected in an almond orchard adjacent to (35 m from the first row of trees) and downwind from a poultry operation and in two almond orchards (controls) that were surrounded by other orchards. Samples were evaluated for aerobic plate count, generic Escherichia coli, other coliforms, the presence of Salmonella, bacterial community structure (analyzed through sequencing of the 16S rRNA gene), and amounts of dry solids (dust) on leaf surfaces on trees 0, 60, and 120 m into each orchard. E. coli was isolated from 41 of 206 (20%) and 1 of 207 (0.48%) air samples in the almond-poultry and control orchards, respectively. Salmonella was not isolated from any of the 529 samples evaluated. On average, the amount of dry solids on leaves collected from trees closest to the poultry operation was more than 2-fold greater than from trees 120 m into the orchard or from any of the trees in the control orchards. Members of the family Staphylococcaceae-often associated with poultry-were, on average, significantly (P < 0.001) more abundant in the phyllosphere of trees closest to the poultry operation (10% of relative abundance) than in trees 120 m into the orchard (1.7% relative abundance) or from any of the trees in control orchards (0.41% relative abundance). Poultry-associated microorganisms from a commercial operation transferred a short distance into an adjacent downwind almond orchard.IMPORTANCE The movement of microorganisms, including foodborne pathogens, from animal operations into adjacent plant crop-growing environments is not well characterized. This study provides evidence that dust and bioaerosols moved from a commercial poultry operation a short distance downwind into an almond orchard and altered the microbiome recovered from the leaves. These data provide growers with information they can use to assess food safety risks on their property.

A dry-inoculation method for nut kernels.

A dry-inoculation method for almonds and walnuts was developed to eliminate the need for the postinoculation drying required for wet-inoculation methods. The survival of Salmonella enterica Enteritidis PT 30 on wet- and dry-inoculated almond and walnut kernels stored under ambient conditions (average: 23 °C; 41 or 47% RH) was then compared over 14 weeks. For wet inoculation, an aqueous Salmonella preparation was added directly to almond or walnut kernels, which were then dried under ambient conditions (3 or 7 days, respectively) to initial nut moisture levels. For the dry inoculation, liquid inoculum was mixed with sterilized sand and dried for 24 h at 40 °C. The dried inoculated sand was mixed with kernels, and the sand was removed by shaking the mixture in a sterile sieve. Mixing procedures to optimize the bacterial transfer from sand to kernel were evaluated; in general, similar levels were achieved on walnuts (4.8-5.2 log CFU/g) and almonds (4.2-5.1 log CFU/g). The decline of Salmonella Enteritidis populations was similar during ambient storage (98 days) for both wet-and dry-inoculation methods for both almonds and walnuts. The dry-inoculation method mimics some of the suspected routes of contamination for tree nuts and may be appropriate for some postharvest challenge studies.

Impact of preinoculation culture conditions on the behavior of Escherichia coli O157:H7 inoculated onto romaine lettuce (Lactuca sativa) plants and cut leaf surfaces.

Inoculum preparation methods can impact growth or survival of organisms inoculated into foods, thus complicating direct comparison of results among studies. The objective of this study was to evaluate preinoculation culture preparation for impact on Escherichia coli O157:H7 inoculated onto leaves of romaine lettuce plants and cut leaf surfaces. E. coli O157:H7 was grown quiescently or shaken at 15, 25, or 37 degrees C to different growth phases in tryptic soy or M9 minimal salts broth or agar. Cells were harvested, washed, and suspended in 0.1% peptone, Milli Q water, or well water and refrigerated for 0 or 18 h. Prepared inoculum was spotted onto cut romaine lettuce (10 microl; 3 x 10(4) CFU/10 g) or onto romaine lettuce plants (20 microl; 3 x 10(6) CFU per leaf). Cut lettuce was sealed in 100-cm2 bags (made from a commercial polymer film) and incubated at 5 or 20 degrees C. Lettuce plants were held at 23 degrees C for 24 h. For all tested conditions, levels of E. coli O157:H7 increased at 20 degrees C on cut lettuce and decreased on cut lettuce stored at 5 degrees C or on leaves of lettuce plants. At 20 degrees C, preinoculation culture conditions had little impact on growth of E. coli O157:H7 on cut lettuce. However, survival at 5 degrees C was significantly better (P < 0.05) for cultures grown at 15 or 37 degrees C in minimal medium and to late stationary phase. Impact of preinoculation handling on survival on lettuce plants was less clear due to relatively high standard deviations observed among samples.

Survival of foodborne pathogens on inshell walnuts.

The survival of Salmonella enterica Enteritidis PT 30 or five-strain cocktails of S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes was evaluated on inshell walnuts during storage. Inshell walnuts were separately inoculated with an aqueous preparation of the pathogens at levels of 10 to 4 log CFU/nut, dried for 24 h, and then stored at either 4 °C or ambient conditions (23-25 °C, 25-35% relative humidity) for 3 weeks to more than 1 year. During the initial 24-h drying period, bacterial levels declined by 0.7 to 2.4 log CFU/nut. After the inoculum dried, further declines of approximately 0.1 log CFU/nut per month of Salmonella Enteritidis PT 30 levels were observed on inshell walnuts stored at 4 °C; at ambient conditions the rates of decline ranged from 0.55 to 2.5 log CFU/nut per month. Rates of decline were generally greater during the first few weeks of storage, particularly at lower inoculum levels. The survival of the five-strain cocktails inoculated at very low levels (under 400 CFU/nut) was determined during storage at ambient conditions. The pathogens could be recovered by either enumeration or enrichment from most samples throughout the 3-month storage period; reductions in bacterial levels from the beginning to end of storage were 0.7, 0.2, and 2.3 log CFU/nut for Salmonella, E. coli O157:H7, and L. monocytogenes, respectively. For 6% of all nut samples (14 of 234 samples), pathogens were isolated from the second but not first 24-h enrichment, suggesting that bacterial cells were viable but not easily culturable. Salmonella-inoculated walnuts were exposed for 2 min to water or a 3% solution of sodium hypochlorite (to mimic commercial brightening) either 24 h or 7 days after inoculation; treated nuts were dried for 24h and held at ambient conditions. Salmonella levels were reduced by less than 0.5 log or 2.4 to 2.6 log CFU/nut on water- or chlorine- treated walnuts, respectively, regardless of postinoculation treatment time. Additional reductions of 2.6 and 2.1 log CFU/nut were observed for water- and chlorine-treated walnuts, respectively, after storage for 2 weeks at ambient conditions. Bacterial foodborne pathogens are capable of long-term survival on the surface of inshell walnuts even when initial levels are low.