Microbial ranking of porous packaging materials (exposure chamber method), ASTM method: collaborative study.

A collaborative study involving 11 laboratories was conducted to measure the microbial barrier effectiveness of porous medical packaging. Two randomly cut samples from each of 6 commercially available porous materials and one positive and one negative control were tested by one operator in each of 11 laboratories. Microbial barrier effectiveness was measured in terms of logarithm reduction value (LRV), which reflects the log10 microbial penetration of the material being tested. The logarithm of the final concentration is subtracted from that of the initial concentration to obtain the LRV. Thus the higher the LRV, the better the barrier. Repeatability standard deviations ranged from 6.42 to 16.40; reproducibility standard deviations ranged from 15.50 to 22.70. Materials B(53), C(50), D(CT), and E(45MF) differ significantly from the positive control. The microbial ranking of porous packaging materials (exposure chamber method), ASTM method, has been adopted First Action by AOAC INTERNATIONAL.

Effect of oxytetracycline-medicated feed on antibiotic resistance of gram-negative bacteria in catfish ponds.

The effect of oxytetracycline-medicated feeds on antibiotic resistance in gram-negative bacteria from fish intestines and water in catfish ponds was investigated. In experiments in the fall and spring, using ponds with no previous history of antibiotic usage, percentages of tetracycline-resistant bacteria in catfish intestines obtained from medicated ponds increased significantly after 10 days of treatment. In the fall, resistance of the intestinal and aquatic bacteria returned to pretreatment levels within 21 days after treatment. In the spring, resistance declined after treatment but remained higher than pretreatment levels for at least 21 days in intestinal bacteria and for 5 months in aquatic bacteria. Plesiomonas shigelloides, Aeromonas hydrophila, and Citrobacter freundii were isolated frequently in both spring and fall; Escherichia coli, Klebsiella pneumoniae, Edwardsiella tarda, and Enterobacter spp. were isolated primarily in the spring. Oxytetracycline treatment did not affect the distribution of bacterial species in the fall but may have accelerated a shift toward greater prevalence of members of the family Enterobacteriaceae in the spring. Multiple antibiotic resistance did not appear to be elicited by oxytetracycline treatment.

Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus.

Twenty-four randomly selected clinical and environmental Vibrio vulnificus isolates were tested for virulence in iron-overloaded mice (250 mg of iron dextran per kg of body weight). The log10 50% lethal doses of 17 isolates were lower by greater than or equal to 3.5 log10 units in iron-overloaded mice than in control mice. These isolates were classified as virulent. The 50% lethal doses of these virulent isolates were also lower in mice that were immunosuppressed by treatment with cyclophosphamide (150 mg/kg). Four of the seven isolates initially classified as avirulent were virulent in mice that were simultaneously iron overloaded and immunosuppressed. These isolates were classified as moderately virulent. The remaining three isolates were avirulent under all conditions. The incidence of virulent strains among clinical and environmental isolates did not differ. The virulent isolates produced high titers of hemolysin, were resistant to inactivation by serum complement, produced phenolate siderophore, and utilized transferrin-bound iron. The moderately virulent isolates differed from the virulent isolates only in their increased sensitivity to inactivation by serum complement. The avirulent isolates differed from those of the other two classes in their inability to either produce significant amounts of phenolate siderophore or utilize transferrin-bound iron. A modified agar plate diffusion method for transferrin-bound iron utilization was developed to differentiate the two classes of virulent isolates from the avirulent isolates in vitro.

Thermotolerance of heat-shocked Listeria monocytogenes in milk exposed to high-temperature, short-time pasteurization.

The effect of prior heat shock (48 degrees C for 15 min) on the thermotolerance of Listeria monocytogenes at the minimal high-temperature, short-time (71.7 degrees C for 15 s) parameters required by the Pasteurized Milk Ordinance was examined. The mean D71.7 degrees C value for heat-shocked L. monocytogenes was 4.6 +/- 0.5 s (control D = 3.0 +/- 1.0 s); the ratio of D to control D was 1.5. The increased thermotolerance of heat-shocked Listeria cells was not significant and appeared unlikely to have practical implications, in terms of risk assessment, for the safety of pasteurized milk.

Thermotolerance of Listeria monocytogenes and Salmonella typhimurium after sublethal heat shock.

The effect of prior heat shock on thermotolerance of Listeria monocytogenes and Salmonella typhimurium in broth culture was determined. Bacteria were grown at the permissive temperature of 35 degrees C, sublethally heated at 35 (control), 42, 48, and 52 degrees C (nonpermissive control) for various times, and inactivated at either 57.8 or 52 degrees C. The induction of increased thermotolerance by heat shock, although consistent within each experiment, was generally not significant for L. monocytogenes; the increase was significant for S. typhimurium. Temperature shift experiments with L. monocytogenes suggested that induced thermotolerance was not long lived unless the shock temperature was maintained.

Incidence of Vibrio parahaemolyticus in U.S. coastal waters and oysters.

Oyster and seawater samples were collected seasonally from May 1984 through April 1985 from shellfish-growing areas in Washington, California, Texas, Louisiana, Alabama, Florida, South Carolina, Virginia, and Rhode Island which had been designated as approved or prohibited by the National Shellfish Sanitation Program. Fecal coliforms counts, aerobic plate counts, and Vibrio parahaemolyticus densities were determined for the samples. Mean V. parahaemolyticus density was more than 100 times greater in oysters than in water, whereas density of fecal coliforms was approximately 10 times higher in oysters. Seasonal and geographical distributions of V. parahaemolyticus were related to water temperature, with highest densities in samples collected in the spring and the summer along the Gulf coast. The synthetic DNA probe for thermostable direct hemolysin hybridized with 2 of 50 isolates, 1 of which was positive by the Kanagawa test.

Precision parameters of standard methods of analysis for dairy products.

The available collaborative studies for standard methods of analysis for various constituents of milk and milk products were examined in an attempt to assign specific repeatability and reproducibility precision parameters to these methods. The different collaborative assays for the primary constituents (moisture/solids, fat, protein), the nutritionally important elements (calcium, sodium, potassium, phosphorus), and miscellaneous analytes/physical constants (ash, lactose, salt, freezing point) produced different estimates of the precision parameters for the same method. A suitable summary of the precision estimates from collaborative studies is given by the reproducibility relative standard deviation, RSDg, which is relatively constant within a product and permits comparisons across products. An estimate of the variation of RSDR for an analyte from a number of collaborative studies is presented in terms of the median and 90% interval (the range of the centermost 90% of values). These estimates are only informative when a substantial number of independent studies are available for pooling the independent estimates to form a distribution of RSDR values. The RSDR for the determination of the primary constituents of milk and milk products is characterized by a median RSDR of 1% and a 90% interval of 0.3-3%, with RSDR estimates occasionally occurring below 0.3% and above 4%. These overall estimates appear to be independent of analyte, matrix, and method and apply to concentrations of primary constituents that range from about 2 to 80%. The repeatability relative standard deviation, RSDr, is unstable, although it tends to converge to about 0.5-0.7 X RSDR. Too few collaborative assays are available to characterize RSDR for the determination of certain other constituents (acidity, ash, lactose, salt, and the nutritionally important elements) unless RSDR values for different analytes, methods, and matrixes are pooled on the basis of similar analyte concentrations. When pooled, the RSDR values are generally better than predicted from the Horwitz equation, RSDR (%) = 2 exp (1-0.5 log10C), where C is the concentration expressed as a decimal fraction; all but one of 661 RSDR values are within the upper empirical limit of twice this curve.

Listeria spp. found on fresh market produce.

From October 1987 to August 1988, 1,000 tests were conducted on 10 types of fresh produce from two Minneapolis area supermarkets to detect Listeria spp. The produce included broccoli, cabbage, carrots, cauliflower, cucumbers, lettuce, mushrooms, potatoes, radishes, and tomatoes. The vegetables were tested by the Food and Drug Administration method for isolation of Listeria spp., with the addition of LiCl-phenylethanol-moxalactam agar in the last 280 tests; 8.6 and 11.4% of these tests were positive by modified McBride and LiCl-phenylethanol-moxalactam agars, respectively. Listeria monocytogenes was isolated from cabbage, cucumbers, potatoes, and radishes; L. innocua was isolated from cucumbers, lettuce, mushrooms, potatoes, and radishes; L. seeligeri was isolated from cabbage and radishes; and L. welshimeri was isolated from cucumbers, potatoes, and radishes. The isolates were of various serotypes; however, the L. monocytogenes isolates were predominantly serotype 1 (82%). Only potatoes (25.8% positive) and radishes (30.3% positive) showed significant amounts of L. monocytogenes contamination.

Comparative recovery of uninjured and heat-injured Listeria monocytogenes cells from bovine milk.

The standard selective enrichment protocols of the Food and Drug Administration (FDA) and U.S. Department of Agriculture (USDA) were compared with an experimental nonselective broth enrichment (NSB) protocol and variations of the standard cold-enrichment (CE) protocol for the recovery of heat-injured Listeria monocytogenes. Bacterial cells (10(7)/ml) were suspended in sterile milk and heated at 71.7 degrees C in a slug-flow heat exchanger for holding times ranging from 1 to 30 s. Surviving cells were determined (50% endpoint) by the given protocols, and the following D values were obtained: NSB, D = 2.0 +/- 0.5 s; FDA, D = 1.4 +/- 0.3 s; USDA, D = 0.6 +/- 0.2 s; CE, D less than or equal to 1.2 s. The respective direct-plating media used in these enrichments were also analyzed for recovery, and the following D values were calculated from the enumeration of surviving cells; NSB, D = 2.7 +/- 0.8 s; FDA, D = 1.3 +/- 0.4 s; USDA, D = 0.7 +/- 0.2 s. The low levels of heat-injured L. monocytogenes cells which were detected at inactivation endpoints on the optimal nonselective media (25 degrees C for 7 days) failed to recover and multiply during experimental CEs (4 degrees C for 28 days). Initial inactivation experiments in which raw whole milk was used as the heating menstruum gave much lower recoveries with all protocols. The detectable limits for uninjured cells that were suspended in raw milk were similar (0.35 to 3.2 cells per ml) for the standard CE, FDA, and USDA protocols.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of purified altertoxins I, II, and III in the metabolic communication V79 system.

Purified Alternaria alternata altertoxins I, II, and III were evaluated for comparative cytotoxicity and ability to inhibit gap junction communication in the Chinese hamster lung metabolic cooperation assay. The noncytotoxic test range for each altertoxin was determined for the metabolic communication assays: altertoxin I, 1, 2, 3, 4, 5 micrograms/ml; altertoxin II, 0.02, 0.008, 0.006, 0.004, 0.002, 0.0008 micrograms/ml; and altertoxin III, 0.2, 0.1, 0.08, 0.06, 0.04 micrograms/ml. Altertoxin II was the most cytoxic in the V79 system, followed by altertoxins III and I. The last cytotoxic of the three, altertoxin I, weakly disrupted metabolic communication at two concentrations (4 and 5 micrograms/ml). Altertoxins III and II did not significantly inhibit gap junction communication more than the weak tumor promoter 4-O-methyl ether tetradecanoylphorbol 13-acetate.

Precision Parameters for AOAC Bacillus stearothermophilus Disc Assay Based on FDA Milk Laboratory Quality Assurance 1982-1986 Samples.

Inhibitory substance (antibiotic) test results from State Split Milk Samples were used to estimate precision parameters and to compare antibiotic medium 4 (A4) and PM indicator (PM) agars. Five inhibitory substances (ampicillin, cephapirin, erythromycin, neomycin, and penicillin-G) were tested. Repeatability relative standard deviations (RSDr) ranged from 1.0 to 4.8%, and the reproducibility relative standard deviations (RSDR) ranged from 4.8 to 10.4%. Zone sizes of erythromycin, neomycin, and penicillin-G were significantly larger on PM agar (α = 0.05) than on A4 agar. The reverse was observed for cephapirin. No difference between agars was noted for ampicillin.

Thermal inactivation of Listeria monocytogenes within bovine milk phagocytes.

Thermal resistance of intracellular and freely suspended Listeria monocytogenes that was associated with a milkborne outbreak of listeriosis was studied by using the sealed tube and slug flow heat exchanger methods. Test temperatures for the former method were 57.8, 62.8, 66.1, and 68.9 degrees C (136, 145, 151, and 156 degrees F, respectively); whereas those for the latter method were 66.1, 68.9, 71.7, and 74.4 degrees C (151, 156, 161, and 166 degrees F, respectively). The heating menstruum was sterile, whole milk. The intracellular inoculum was generated from an in vitro phagocytosis reaction by using endotoxin-induced bovine milk phagocytes. The phagocyte population consisted of 88% neutrophils, 8% macrophages, and 4% lymphocytes. Neutrophils harbored the majority of intracellular L. monocytogenes. The mean level of infectivity in the phagocyte population was 43%, and there were 26.1 +/- 19.3 bacteria per cell (10(4) viable cells per ml of test milk). Initial bacterial counts for the freely suspended and intracellular experiments (the latter was based on a sonically disrupted sample) were 10(6) L. monocytogenes cells per ml. Heat-stressed bacteria were recovered by direct plating in parallel with recovery from an enrichment broth; both methods gave comparable results. The predicted D62.8 degrees C (145 degrees F) value for intracellular sealed tube studies was 53.8 s (ZD = 5.6 degrees C [10.0 degrees F]), indicating a safe 33.4 D margin of inactivation for vat pasteurization (62.8 degrees C for 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of C3H/10T1 2 cells and induction of EBV-early antigen in Raji cells by altertoxins I and III.

Moulds of the genus Alternaria are common contaminants of some food crops. Some isolates have been shown to produce mutagenic compounds called altertoxins. Altertoxin I (ATX-I) and altertoxin III (ATX-III) were examined for activity in the Raji cell Epstein-Barr virus early antigen (EBV-EA) induction system and in the C3H/10T1 2 murine fibroblast cell transformation system. Exposure of Raji cells to ATX-I or ATX-III activated EBV-EA expression by 8- and 9.5-fold, respectively. A single exposure of C3H/10T1 2 cultures to ATX-I or ATX-III resulted in significant increases in cell transformation, and the response to ATX-I was stronger. Both altertoxins enhanced the transformation of C3H/10T1 2 cells, and chronic exposure of non-initiated C3H/10T1 2 cells to ATX-I and ATX-III, starting 6 days after cells were plated, resulted in cell transformation in 8 59 and 12 37 dishes, respectively, compared with transformation in only 2 63 control dishes. Since activation of EBV-EA in Raji cells has been positively correlated with tumour promoters, these data together indicate that ATX-I and ATX-III are not just mutagens but have a potential role in cell transformation.

Comparison of diethyl ether and ethyl acetate as extracting agents for recovery of Ascaris spp. and Trichuris spp. eggs.

Ethyl acetate and diethyl ether were compared for their ability to recover Ascaris spp. and Trichuris spp. eggs from seeded milorganite, liquid sludge, and cabbage. Concentrations of 10, 100, and 1000 eggs/10 g test sample were prepared for 20 replicates of each product. The use of diethyl ether yielded fewer eggs/10 g than did ethyl acetate in 5 of 6 sets of data. For Ascaris spp., recovery from cabbage was 10 times higher with ethyl acetate at the higher concentration than with diethyl ether. For Trichuris spp., recovery from liquid sludge was slightly higher with diethyl ether for all egg concentrations. The other results ranged from 0 to 23% difference in recovery for the 2 agents. Depending on the parasites in question and the products to be screened, the substitution of ethyl acetate for diethyl ether may be significant.

Evaluation of precision estimates for fiber-dimensional and electrical hygrometers for water activity determinations.

The precision of instruments used in 3 collaborative studies conducted within the Food and Drug Administration over a 4-year period (1981, 1982, 1984) for water activity (aw) determinations according to the official AOAC method is evaluated. Calibration responses of the instruments were tested for linearity over the aw range from 0.75 to 0.97. Average absolute percent difference between predicted and assigned aw values for the linear model ranged from 0.3 to 0.7% for a fiber-dimensional hygrometer (Abbeon) and 3 electrical hygrometers (Beckman, Rotronics, and Weather Measure). The calibration responses for another electrical hygrometer (Hygrodynamics) were nonlinear. The fiber-dimensional hygrometer yielded mean aw values and precision estimates that did not differ significantly from those obtained with the electrical hygrometers for (NH4)2SO4slush, KNO3 slush, sweetened condensed milk, pancake syrup, and cheese spread. However, the mean aw value for a soy sauce was 0.838 for the electrical hygrometers compared with 0.911 for the fiber-dimensional hygrometer. The fiber-dimensional hygrometer was affected by a volatile component(s) in the soy sauce that caused an erroneously high aw value. Pooled estimates of reproducibility (Sx) in the 3 studies were 0.008 for the fiber-dimensional hygrometer and 0.010 for the electrical hygrometers; these values were not significantly different from those reported in the study that verified the current official AOAC method.

Pathogenicity test for Listeria monocytogenes using immunocompromised mice.

The lethality of Listeria isolates was determined with normal adult mice and mice that were immunocompromised by treatment with 20 mg of carrageenan per kg. The mean 50% lethal doses (LD50s) of the pathogenic isolates were significantly lower (alpha = 0.05) in the immunocompromised mice than in the untreated mice, with an average reduction of 5.8 log10 units. In contrast, the mean LD50s of the nonpathogenic isolates were lower in the immunocompromised mice by an average of only 0.4 log10 unit, a difference that was not significant (alpha = 0.05). When immunocompromised mice were used, the LD50s of pathogenic Listeria monocytogenes isolates were lower than those of nonpathogenic L. innocua and L. seeligeri isolates by greater than or equal to 6 log10 units and lower than those of nonpathogenic L. ivanovii isolates by greater than or equal to 4 log10 units. Pathogenic L. monocytogenes isolates could be distinguished from nonpathogenic isolates by their ability to cause deaths in immunocompromised mice in 3 days at a dose of approximately 10(4) CFU per mouse. An alternative procedure using iron-overloaded mice failed to effectively differentiate pathogenic Listeria isolates.

Thermal Resistance of Disease-Associated Salmonella typhimurium in Milk.

The thermal resistance of Salmonella typhimurium cultures that had been associated with a major milkborne oubreak of salmonellosis was determined in raw whole milk. Thirteen patient stool isolates and 24 implicated pasteurized milk isolates at concentrations of 1 × 105/ml were screened for heat resistance at 51.8°C. A representative milk strain was heated in replicate at four temperatures from 51.8 to 68.3°C. The zD value was calculated to be 5.3°C. Mean D-value estimates at 51.8°C were 24.0 and 22.8 min for patient and milk isolates, respectively. Extrapolated D71.7°C values were 0.24 and 0.22 s, and did not differ significantly (α = 0.05). These isolates would not survive proper pasteurization.

Thermal Resistance of Listeria monocytogenes in Dairy Products.

The thermal resistance of Listeria monocytogenes strain Scott A that had been associated with a recent milkborne outbreak of listeriosis was determined in whole and skim milk, heavy cream, and ice cream mix. L. monocytogenes suspended at concentrations of approximately 1 × 105 cells/ml was heated at temperatures ranging from 52.2 to 79.4°C at various contact times. The D71.7°C values computed for milk samples ranged from 0.9 to 2.7 s. The D7.94°C value in ice cream mix was 0.5 s. The zD value for fluid products ranged from 5.8 to 7.1°C; the zF value for ice cream mix was 7.0°C. The L. monocytogenes suspensions would not survive a proper pasteurization process given to raw dairy products.

Thermal resistance of intracellular Listeria monocytogenes cells suspended in raw bovine milk.

The thermal resistance of Listeria monocytogenes associated with a milk-borne outbreak of listeriosis was determined in parallel experiments by using freely suspended bacteria and bacteria internalized by phagocytes. The latter inoculum was generated by an in vitro phagocytosis reaction with immune-antigen-elicited murine peritoneal phagocytes. The heat suspension medium was raw whole bovine milk. Both suspensions were heated at temperatures ranging from 52.2 to 71.7 degrees C for various periods of time. Mean D values for each temperature and condition of heated suspension revealed no significant differences. The extrapolated D71.7 degrees C (161 degrees F) value for bacteria internalized by phagocytes was 1.9 s. Combined tube and slug-flow heat exchanger results yielded an estimated D71.7 degrees C value of 1.6 s for freely suspended bacteria. The intracellular position did not protect L. monocytogenes from thermal inactivation.