Douglas L. Marshall

Adaptation of Escherichia coli O157:H7 to pH Alters Membrane Lipid Composition, Verotoxin Secretion, and Resistance to Simulated Gastric Fluid Acid

ABSTRACT The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37°C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1ω7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 108 CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.

Ready-to-Eat Shrimp as an International Vehicle of Antibiotic-Resistant Bacteria†

The occurrence of antibiotic-resistant bacteria in foods of animal origin is a potential health threat because resistance can be transferred among bacteria, and antibiotic-resistant pathogens may not respond to antibiotic treatments. Thirteen brands of ready-to-eat shrimp representing four countries of origin were obtained from local grocery stores. Total heterotrophic plate counts were determined, and antibiotic-resistant bacteria were isolated. Total heterotrophic colony counts ranged from 3.3 to 5.6 log CFU/g, which was within approved quality limits. A total of 1,564 isolates representing 162 bacterial species were recovered during screening of resistance to 10 antibiotics: ampicillin, ceftriaxone, chloramphenicol, clindamycin, erythromycin, nalidixic acid, streptomycin, tetracycline, trimethoprim, and vancomycin. Six hundred fifty-seven (42%) of the isolates and 131 (81%) of the species had acquired resistance to antibiotics. Numerous resistant human pathogens were isolated, including Escherichia coli, Enterococcus spp., Salmonella, Shigella flexneri, Staphylococcus spp., and Vibrio spp. Nonresistant Yersinia spp. also were isolated. Ready-to-eat shrimp is sold with instructions to thaw the product before serving, which may result in consumer exposure to antibiotic-resistant bacteria. Widespread trade of this product provides an avenue for international dissemination of antibiotic-resistant pathogens.

Heat Adaptation Alters Escherichia coli O157:H7 Membrane Lipid Composition and Verotoxin Production

ABSTRACT The influence of heat adaptation (growth at 42 and 45°C) on changes in membrane lipid composition and verotoxin concentration of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant of ATCC 43895 (FRIK 816-3), a verotoxin mutant E. coli O157:H7 (B6-914), and nonpathogenic E. coli (ATCC 25922) was investigated. D values (57°C) of heat-adapted cells were up to 3.9 min longer than those of control cells for all four strains. Heat adaptation increased the amounts of palmitic acid (16:0) and cis-vaccenic acid (18:1ω7c) in membrane lipids of ATCC 43895 and the rpoS mutant, whereas there was a reduction and no change in the amount of cis-vaccenic acid in nonpathogenic and verotoxin mutant E. coli, respectively. The ratio of palmitic to cis-vaccenic acids decreased in ATCC 43895 and in the rpoS mutant, whereas the ratio increased in nonpathogenic E. coli and was not different in the verotoxin mutant with elevated growth temperature. Total verotoxin concentration decreased due to a reduction in intracellular verotoxin amount in heat-adapted ATCC 43895 and rpoS mutant strains. However, extracellular verotoxin concentration increased in heat-adapted cells. The rpoS gene did not influence membrane lipid composition changes although it did affect heat resistance. Results suggest that increased membrane fluidity may have caused increased verotoxin secretion.

Growth of Listeria monocytogenes at 10 degrees C in milk preincubated with selected pseudomonads

Preliminary studies involving co-inoculation of Listeria monocytogenes with Pseudomonas fragi into whole or skim milk demonstrated that neither inhibition nor stimulation of growth occurred for either organism. Additional investigations involved preincubation of whole milk, skim milk, and 10% reconstituted nonfat dry milk (NDM) for 3 d at 10°C with P. fragi , Pseudomonas fluorescens P26, P. fluorescens T25, or P. fluorescens B52, followed by inoculation with L. monocytogenes and further incubation at 10°C. Growth curves of L. monocytogenes were constructed for each treatment combination and generation times were statistically compared for differences. Results indicated that L. monocytogenes did not affect growth or survival of the preincubated Pseudomonas spp. However, growth rates of L. monocytogenes were significantly (P<0.05) enhanced in milks preincubated with pseudomonads. Doubling times of L. monocytogenes were reduced by up to 3 h when grown in milk preincubated with Pseudomonas spp. Three strains of P. fluorescens showed more stimulation of the growth rate of L. monocytogenes compared to P. fragi in preincubated whole or skim milk but not in preincubated NDM. Milk composition had little effect on growth of either genus when incubated alone. Results of this study indicate that L. monocytogenes can grow in the presence of Pseudomonas spp. either as a co-inoculant or following preincubation in milk at 10°C. Furthermore, data suggest that the presence of the pseudomonads may enhance growth of L. monocytogenes in milk.

Antacid increases survival of Vibrio vulnificus and Vibrio vulnificus phage in a gastrointestinal model.

ABSTRACT Viable counts of three strains of Vibrio vulnificus and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37°C.V. vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5). ViableV. vulnificus cells delivered from the gastric compartment during the first 30 min of exposure reached 106 to 108 CFU/ml in the intestinal compartment after 9 h (pH 7.0). Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5). Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0). When the gastric compartment contained antacid V. vulnificus counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 107 to 109 CFU/ml. Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h. Presence of antacid in the gastric compartment of the model greatly increased the ability of both V. vulnificus and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness.

Monolaurin and acetic acid inactivation of Listeria monocytogenes attached to stainless steel.

Individual and combined antimicrobial effects of monolaurin and acetic acid on Listeria monocytogenes planktonic cells or stainless-steel-adherent cells were determined in order to evaluate cell viability during a 25-min exposure period at 25 degrees C. A 10(7)-colony-forming units (CFU)/ml population of planktonic cells was completely inactivated by the synergistic combination of 1% acetic acid with 50 or 100 microg/ml of monolaurin within 25 or 20 min, respectively. Either compound alone caused partial but incomplete inactivation within the same time periods. A population of 10(5) CFU/cm2 of 1-day adherent cells on stainless steel was completely inactivated within 25 min, but with the highest concentrations of the combined chemicals, i.e., 1% acetic acid and 100 microg/ml of monolaurin. The combined chemical treatment again synergistically produced greater inhibition. A 10(6)-CFU/cm2 population of 7-day adherent cells was not completely inactivated within 25 min of exposure, although counts did decline. The results demonstrate increased resistance of attached L. monocytogenes to acetic acid and monolaurin and show that resistance increased with culture age. Combinations of organic acids and monolaurin might be considered as sanitizers of food contact surfaces, but activities of such combinations are likely to be less than other commonly used sanitizers.

Effect of pH on the Minimum Inhibitory Concentration of Monolaurin Against Listeria monocytogenes1

The effect of pH on the minimum inhibitory concentration (MIC) of glycerol monolaurate (monolaurin) against four strains of Listeria monocytogenes at 35°C in tryptic soy broth supplemented with 0.6% yeast extract was investigated. Our results demonstrate that the MIC of monolaurin was lower than MIC values reported for other common food antimicrobials such as potassium sorbate, tertiary butylhydroquinone, propyl paraben, and butylated hydroxyanisole. The MIC of monolaurin was reduced by decreasing the pH value of the medium. A 3-fold MIC reduction occurred when the pH decreased from pH 7.0 (10 μg/ml) to pH 5.0 (3 μg/ml).

Low temperature growth and thermal inactivation of Listeria monocytogenes in precooked crawfish tail meat

Growth of Listeria monocytogenes in precooked crawfish tail meat at 0, 6, and 12°C was determined. Thermal death times were also determined. Growth curves for L. monocytogenes revealed that little multiplication was observable for the entire storage time of 20 d at 0°C. At 6 and 12°C, exponential growth began immediately with no observed lag phase. Generation times of 72.2, 17.0, and 6.9 h were calculated at 0, 6, and 12°C, respectively. Observed D values at 55, 60, and 65°C were 10.23, 1.98, and 0.19 min, respectively. The z value for L. monocytogenes in precooked crawfish tail meat was calculated to be 5.5°C. Results from this study indicate that a refrigeration temperature of 6°C (42.8°F) will support growth of L. monocytogenes and short-term temperature abuse at 12°C will induce very rapid growth of the organism on crawfish tail meat. Thermal treatment values from this study can be used to establish postpicking heat treatments that would eliminate L. monocytogenes from packaged crawfish tail meat prior to retail sale.

Influence of Acetic, Citric, and Lactic Acids on Escherichia coli O157:H7 Membrane Lipid Composition, Verotoxin Secretion, and Acid Resistance in Simulated Gastric Fluid

The effect of organic acid (acetic, citric, and lactic acids) adaptation at equivalent initial pH values (6.4 and 5.4) on changes in membrane lipid composition, verotoxin concentration, and acid resistance in simulated gastric fluid (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 ATCC 43895 (HEC) and an rpoS mutant of E. coli O157:H7 ATCC 43895 (RM, FRIK 816-3). For HEC, lactic acid-adapted (pH 5.4) cells had the greatest D-value (32.2 min) and acetic acid-adapted (pH 5.4) cells had the smallest D-value (16.6 min) in simulated gastric fluid. For RM, D-values of citric and acetic acid-adapted cells were similar to those for nonadapted cells grown at pH 7.3, but D-values increased from 13.1 to 27.9 min in lactic acid-adapted cells (from pH 7.3 to pH 5.4). For both strains, the ratio of cis-vaccenic to palmitic acids decreased for citric and lactic acid-adapted cells, but the ratio increased for acetic acid-adapted cells at pH 5.4. Organic acid-adapted cells produced less total verotoxin than did nonadapted cells at approximately 10(8) CFU/ml. Extracellular verotoxin concentration proportionally decreased with decreasing pH for both HEC and RM. Changes in membrane lipid composition, verotoxin concentration, and acid resistance in HEC and RM were dependent on both pH and organic acid. Deletion of the rpoS gene did not affect these changes but did decrease acid resistance in citric acid-adapted cells. Results indicate that decreased membrane fluidity may have caused increased acid resistance and decreased verotoxin secretion.

Extending shelf life of refrigerated catfish fillets using sodium acetate and monopotassium phosphate

The effects of sodium acetate (SA) and monopotassium phosphate (MKP) on total aerobic plate counts (APC), pH, odor, and appearance of catfish fillets during storage at 4°C were determined. Use of 0.75% and 1.0% SA lowered (P < 0.05) initial APC by 0.6 to 0.7 log units compared to the control. Microbial counts of SA-treated fillets remained lower than the control during storage, resulting in a 6-day shelf-life increase. MKP alone had no effect on APC values, but it did influence the activity of SA. The results indicate that a combination of SA and MKP could prolong the microbiological shelf life of catfish to 12 days at 4°C. Fillets treated with 1% SA alone or SA-MKP combinations had pH values and odor scores that were similar to fresh controls for up to 9 days; however, appearance scores were lower after 3 days, probably due to a brownish and watery appearance. MKP alone is not recommended for shelf-life extension of catfish fillets. Conversely, SA alone or combined with MKP is recommended to extend the microbiological shelf life of refrigerated catfish fillets.