Stuart J. Chirtel

Inactivation of indicator microorganisms in estuarine waters

Abstract In the United States, shellfish growing areas are classified, in part, using standards based on the densities of either the total or fecal coliform groups in surface waters. However, the standards currently employed may not reliably index the presence of certain enteric pathogens, particularly enteric viruses responsible for human illnesses, even though both the pathogens and indicators derive from the same fecal contamination. To some extent, this may be due to differences in the survival of these pathogens in the environment relative to that of the bacterial indicators. This investigation was conducted to assess the effects of temperature, salinity, dissolved oxygen, geographic location, season, and solar radiation on the survival of selected indicator microorganisms in estuarine waters. The indicators examined included fecal coliforms, Escherichia coli, Clostridium perfringens, and male-specific bacteriophage (MSB), a potential indicator of enteric viruses. In situ experiments were performed in estuarine waters of Alabama and Rhode Island. Among the parameters examined, sunlight and/or temperature most significantly affected indicator decay rates. In general, the effects from exposure to sunlight accounted for up to 83, 84, and 99% of the density reductions of MSB, C. perfringens and fecal coliforms, respectively. Thus, the effects from sunlight were greatest on fecal coliforms and much less pronounced on MSB and C. perfringens. For fecal coliforms, the effect of sunlight was more pronounced during the winter than the summer. In the absence of sunlight, the rate of MSB decline was strongly negatively correlated with estuarine water temperatures and dissolved oxygen. Overall, fecal coliform decay rates were dissimilar to those found for MSB. From this, it would appear that fecal coliforms may not be reliable indicators of viruses in estuarine waters.

Potential for internalization, growth, and survival of Salmonella and Escherichia coli O157:H7 in oranges

Internalization potential, survival, and growth of human pathogens within oranges were investigated in a series of laboratory experiments. Submerging oranges into dye solutions at various temperature differentials was used to assess internalization potential. Conditions in which dye internalization was observed were further studied by applying Escherichia coli O157:H7 or Salmonella onto the stem scar, subjecting the oranges to a temperature differential, juicing, and measuring numbers of pathogens in the resulting juice. Pathogens for growth and survival studies were applied to or injected into simulated peel punctures. Oranges with small peel holes of selected sizes were also placed into solutions containing these pathogens. Bacterial survival was also evaluated in orange juice at 4 and 24 degrees C. Oranges internalized pathogens at a frequency of 2.5 to 3.0%, which mirrored dye internalization frequency (3.3%). Pathogens were internalized at an uptake level of 0.1 to 0.01% of the challenge applied. Bacteria grew within oranges at 24 degrees C, but not at 4 degrees C. Thirty-one percent of oranges with 0.91-mm surface holes showed pathogen uptake, whereas 2% of oranges with 0.68-mm holes showed pathogen uptake. Pathogens added to fresh orange juice and incubated at 24 degrees C declined 1 log CFU/ml within 3 days. These results suggest that internalization, survival, and growth of human bacterial pathogens can occur within oranges intended for producing unpasteurized juice.

Salmonella and the sanitary quality of aquacultured shrimp.

In this study, we examined the prevalence of Salmonella and coliform bacteria on shrimp aquaculture farms to develop guidelines or preventative measures for reducing Salmonella and fecal contamination on products harvested from these farms. The U.S. Food and Drug Administration, in conjunction with foreign government regulatory agencies, the aquaculture industry, and academia affiliates, analyzed 1,234 samples from 103 shrimp aquaculture farms representing six countries between July 2001 and June 2003 for fecal coliforms, Escherichia coli, and Salmonella. A significant relationship was found (P = 0.0342) between the log number of fecal bacteria and the probability that any given sample would contain Salmonella. The likelihood of any given sample containing Salmonella was increased by 1.2 times with each 10-fold increase in either fecal coliform or E. coli concentration. The statistical relationship between Salmonella concentration and that of both fecal coliforms and E. coli was highest in grow-out pond water (P = 0.0042 for fecal coliforms and P = 0.0021 for E. coli). The likelihood of finding Salmonella in grow-out pond water increased 2.7 times with each log unit increase in fecal coliform concentration and 3.0 times with each log unit increase in E. coli concentration. Salmonella is not part of the natural flora of the shrimp culture environment nor is it inherently present in shrimp grow-out ponds. The occurrence of Salmonella bacteria in shrimp from aquaculture operations is related to the concentration of fecal bacteria in the source and grow-out pond water.

Rat liver clone‐9 cells in culture as a model for screening hepatotoxic potential of food‐related products: hepatotoxicity of deoxynivalenol

Deoxynivalenol (DON) is a mycotoxin food contaminant found in several cereal grains. The literature on the liver toxicity of DON in vivo is conflicting and does not clearly characterize its hepatotoxic effects. Cultured rat liver clone‐9 cells were used as a model to assess the hepatotoxic potential of DON. The cell cultures, seeded onto 96‐well plates, were treated at confluence with varying concentrations of DON (0–100 µg ml−1) for 48 h at 37 °C in 5% CO2. After the treatment period, the cells were assayed for a number of hepatotoxic endpoints that included cytotoxicity, double‐stranded DNA (ds‐DNA) content, oxidative stress and mitochondrial function. The concentration‐dependent toxicity of DON, as measured by cytotoxicity and ds‐DNA content, was observed over the entire concentration range studied beginning at 0.5 µg ml−1. DON also induced a significant concentration‐dependent increase in oxidative stress at DON concentrations starting at 10 µg ml−1. The mitochondrial function of the treated cells decreased with the increasing concentration of DON exposure, but it was not statistically different from that of the control value. Liver histopathology observed at 3, 24 and 72 h following a single intraperitoneal administration dose of DON (10 mg kg−1 BW) to adult male rats is consistent with early mild hepatotoxicity. The overall results of this study suggest that acute DON exposure has early mild cytotoxic effects on hepatocytes in vivo that are expressed as severe effects in rat liver clone‐9 cells in vitro. Published in 2008 by John Wiley & Sons, Ltd.

Effect of growth on the thermal resistance and survival of Salmonella Tennessee and Oranienburg in peanut butter, measured by a new thin-layer thermal death time device.

In published data the thermal destruction of Salmonella species in peanut butter deviates from pseudo-first-order kinetics. The reasons for such deviation are unknown. This study examined both the method used to measure the thermal destruction rate and the method of growth of the microorganisms to explain variations in destruction kinetics. Growth on a solid matrix results in a different physiological state that may provide greater resistance to adverse environments. In this study, Salmonella Tennessee and Oranienburg were grown for 24 h at 37°C under aerobic conditions in broth and agar media to represent planktonic and sessile cell growth, respectively. Peanut butter was held at 25°C and tested for Salmonella levels immediately after inoculation and at various time intervals up to 2 weeks. Thermal resistance was measured at 85°C by use of a newly developed thin-layer metal sample holder. Although thermal heat transfer through the metal device resulted in longer tau values than those obtained with plastic bags (32.5 ± 0.9 versus 12.4 ± 1.9 s), the bags have a relative variability of about 15 % compared with about 3 % in the plates, allowing improved uniformity of sample treatment. The two serovars tested in the thin-layer device showed similar overall thermal resistance levels in peanut butter regardless of growth in sessile or planktonic states. However, thermal destruction curves from sessile cultures exhibited greater linearity than those obtained from planktonic cells (P = 0.0198 and 0.0047 for Salmonella Oranienburg and Salmonella Tennessee, respectively). In addition, both Salmonella serovars showed significantly higher survival in peanut butter at 25°C when originally grown on solid media (P = 0.001) with a <1-log loss over 2 weeks as opposed to a 1- to 2-log loss when grown in liquid culture. Consequently, the use of cells grown on solid media may more accurately assess the survival of Salmonella at different temperatures in a low-water-activity environment such as peanut butter.

Factors Influencing the Growth of Salmonella during Sprouting of Naturally Contaminated Alfalfa Seeds

In this study, the factors that affect Salmonella growth during sprouting of naturally contaminated alfalfa seeds associated with two previous outbreaks of salmonellosis were examined. A minidrum sprouter equipped with automatic irrigation and rotation systems was built to allow sprouting to be conducted under conditions similar to those used commercially. The growth of Salmonella during sprouting in the minidrum was compared with that observed in sprouts grown in glass jars under conditions commonly used at home. The level of Salmonella increased by as much as 4 log units after 48 h of sprouting in jars but remained constant during the entire sprouting period in the minidrum. The effect of temperature and irrigation frequency on Salmonella growth was examined. Increasing the sprouting temperature from 20 to 30 degrees C increased the Salmonella counts by as much as 2 log units on sprouts grown both in the minidrum and in the glass jars. Decreasing the irrigation frequency from every 20 min to every 2 h during sprouting in the minidrum or from every 4 h to every 24 h during sprouting in the glass jars resulted in an approximately 2-log increase in Salmonella counts. The levels of total aerobic mesophilic bacteria, coliforms, and Salmonella in spent irrigation water closely reflected those found in sprouts, confirming that monitoring of spent irrigation water is a good way to monitor pathogen levels during sprouting.

Use of data mining at the Food and Drug Administration

OBJECTIVES This article summarizes past and current data mining activities at the United States Food and Drug Administration (FDA). TARGET AUDIENCE We address data miners in all sectors, anyone interested in the safety of products regulated by the FDA (predominantly medical products, food, veterinary products and nutrition, and tobacco products), and those interested in FDA activities. SCOPE Topics include routine and developmental data mining activities, short descriptions of mined FDA data, advantages and challenges of data mining at the FDA, and future directions of data mining at the FDA.

Influence of Fruit Variety, Harvest Technique, Quality Sorting, and Storage on the Native Microflora of Unpasteurized Apple Cider

Apple variety, harvest, quality sorting, and storage practices were assessed to determine their impact on the microflora of unpasteurized cider. Seven apple varieties were harvested from the tree or the ground. The apples were used fresh or were stored at 0 to 4 degrees C for < or = 5 months and were pressed with or without quality selection. Cider yield, pH, Brix value, and titratable acidity were measured. Apples, postpressing apple pomace, and cider samples were analyzed for aerobic bacteria, yeasts, and molds. Aerobic bacterial plate counts (APCs) of ciders from fresh ground-picked apples (4.89 log CFU/ml) were higher than those of ciders made from fresh, tree-picked apples (3.45 log CFU/ml). Quality sorting further reduced the average APC to 2.88 log CFU/ml. Differences among all three treatment groups were significant (P < 0.0001). Apple and pomace microbial concentrations revealed harvest and postharvest treatment-dependent differences similar to those found in cider. There were significant differences in APC among apple varieties (P = 0.0001). Lower counts were associated with varieties exhibiting higher Brix values and higher titratable acidity. Differences in APC for stored and fresh apples used for cider production were not significant (P > 0.05). Yeast and mold counts revealed relationships similar to those for APCs. The relationship between initial microbial load found on incoming fruit and final cider microbial population was curvilinear, with the weakest correlations for the lowest apple microflora concentrations. The lack of linearity suggests that processing equipment contributed to cider contamination. Tree-picked quality fruit should be used for unpasteurized cider production, and careful manufacturing practices at cider plants can impact both safety and quality of the final product.

Efficacy of Sanitation and Cleaning Methods in a Small Apple Cider Mill

The efficacy of cleaning and sanitation in a small apple cider processing plant was evaluated by surface swab methods as well as microbiological examination of incoming raw ingredients and of the final product. Surface swabs revealed that hard-to-clean areas such as apple mills or tubing for pomace and juice transfer may continue to harbor contaminants even after cleaning and sanitation. Use of poor quality ingredients and poor sanitation led to an increase of approximately 2 logs in aerobic plate counts of the final product. Reuse of uncleaned press cloths contributed to increased microbiological counts in the finished juice. Finally, using apples inoculated with Escherichia coli K-12 in the plant resulted in an established population within the plant that was not removed during normal cleaning and sanitation. The data presented in this study suggest that current sanitary practices within a typical small cider facility are insufficient to remove potential pathogens.

Interactions in indices of vitamin A, zinc and copper status when these nutrients are fed to rats at adequate and increased levels

The purpose of the present study was to determine the effects of feeding nutritionally adequate and increased levels of vitamin A (retinyl acetate at 1.4, 34.4, and 206.4 mg/kg diet) in combination with adequate or increased Zn (12 and 240 mg/kg) and Cu (5 and 50 mg/kg) on serum and tissue concentrations of retinol and retinyl palmitate and on indices of Cu and Zn status in female Sprague-Dawley rats, and to measure interactive effects of such nutrient imbalances. Rats fed on diets containing 34.4 and 206.4 mg vitamin A/kg had higher feed intakes and relative liver weights than those fed on diets containing 1.4 mg vitamin A/kg. An interaction between dietary Cu and Zn and an independent effect of vitamin A affected serum ceruloplasmin oxidase (EC 1.16.3.1) activity. Rats fed on high Zn, adequate-Cu diets (240 and 5 mg Zn and Cu/kg respectively) had lower serum ceruloplasmin oxidase levels than rats fed on adequate-Zn, adequate-Cu diets (12 and 5 mg Zn and Cu/kg respectively). This effect was not observed in rats fed on high-Zn, high-Cu diets (240 and 50 mg Zn and Cu/kg respectively). Alterations in dietary levels of Cu and vitamin A independently affected haemoglobin levels. Serum cholesterol concentration was affected by interactions between Zn and vitamin A and Cu and vitamin A. Levels of retinol and retinyl palmitate in liver and kidney were significantly higher in rats fed on diets with increased dietary vitamin A than in those fed on diets with adequate vitamin A. Three-way interactions among Cu, Zn, and vitamin A affected levels of retinol in serum and liver. Two-way interactions between Cu and vitamin A affected liver retinyl palmitate and the sum of liver retinol+retinyl palmitate. An independent effect of dietary Zn on these variables was also observed. Interactions between Cu and vitamin A affected levels of Cu in liver and kidney, while Fe and Zn in kidney were affected by interactions between Cu and Zn. This study demonstrates that differing interactions among variables of vitamin A metabolism and mineral status occur with higher dietary levels of vitamin A, Zn and Cu in the rat.