IN VITRO COMPARISON OF ANAEROBIC AND AEROBIC GROWTH RESPONSE OF SALMONELLA TYPHIMURIUM TO ZINC ADDITION

Zinc supplemented diets have been used to provide zinc as a nutrient and higher concentrations have been used to induce molt in laying hens. It is not known if the zinc in these diets would inhibit Salmonella spp growth. This study examines the effects of zinc compounds on the growth of S. typhimurium poultry isolate under aerobic and anaerobic conditions. The aerobic growth response of S. typhimurium was determined either in tryptic soy broth (TSB) or minimal (M9) broth containing five different concentrations (0.67, 2.01, 3.35, 4.69, and 6.03% [wt/vol]) of either Zn acetate [Zn(C₂H₂O₂)₂2H₂O] or Zn sulfate [ZnSO₄7H₂O] while anaerobic growth response was determined in M9 broth with or without reductants (L-cysteine hydrochloride [C₃H₇NO₂SHCl], and sodium sulfide [Na₂S 9H₂O]). Aerobic growth rates inhibited (P < 0.05) by Zn acetate than by Zn sulfate in TSB medium. The Zn source and concentration decreased (P < 0.05) aerobic growth response of S. typhimurium poultry isolate in M9 medium. The growth rates of S. typhimurium under anaerobic growth conditions were less responsive to Zn salts but were generally lower (P < 0.05) in the presence of reductant than in the absence of reductants at each concentration of Zn compound. The results in this study provide evidence that Zn may inhibit S. typhimurium under in vitro aerobic or anaerobic atmospheric conditions and S. typhimurium grows less optimally under anaerobic growth conditions.     

SURVIVAL OF A SALMONELLA TYPHIMURIUM POULTRY MARKER STRAIN ADDED AS A DRY INOCULUM TO ZINC AND SODIUM ORGANIC ACID AMENDED FEEDS

The effects of different organic acid compounds on the survival of a dry inoculum of Salmonella typhimurium marker strain added to poultry feed were determined. Organic acids were added as 1% Zn or Na salts (w/w) to poultry feeds and stored at room temperature for 9 days. Reduction of S. typhimurium populations from 0 to day 9 was not substantially different between poultry layer ration nonamended feed (96.76%) and Zn acetate (99.72%) or Zn propionate (99.60%) amended feed. However, over 90% reduction of S. typhimurium populations occurred in Zn acetate and Zn propionate amended feed by day 3 while poultry layer ration populations reached 90% reduction by day 5. S. typhimurium populations after 9 days were nearly 40% more reduced in Na propionate amended feed than Na acetate amended feed. The results of this study indicated 1% Zn compounds may have potential to reduce survival of S. typhimurium in a poultry diet during the early stages of storage.     

LATE LOGARITHMIC SALMONELLA TYPHIMURIUM HEp-2 CELL ASSOCIATION AND INVASION RESPONSE TO SHORT-CHAIN FATTY ACID ADDITION

The entry of Salmonella into the intestinal epithelial cells is essential for its pathogenesis. In the present stuay, late logarithmic growth (12 h) in Luria Bertani broth supplemented (25, 50, or 100 mM) with acetate, propionate, butyrate, or a mixture of the three short-chain fatty acids (SCFA), influenced the ability ofS. typhimurium to associate and invade cultured HEp-2 cells. The response to SCFA addition was dependent on the concentration and the pH of the medium. Cell-association was decreased 4 to 100-fold at pH 6 and 2 to 20-fold at pH 7. At pH 7, invasion was increased 2-fold when bacteria were cultured in the presence of all concentrations of acetate and 25 mM propionate or butyrate; however, a 1.5 to 163-fold decrease in invasion occurred at pH 6 for all SCFA. The SCFA concentration also affected cell-association and invasion of S. typhimurium, especially at pH 6. In general, for each of the SCFA at pH 6 a lower percentage of cell-association and invasion was observed as the SCFA concentration was increased while at pH 7 decreases did not consistently occur as SCFA concentration was increased. These differences among the SCFA along with the pH effects suggest that the concentration of SCFA and the pH of the intestinal lumen may influence the expression of the invasion phenotype of S. typhimurium.     

LACTIC ACID INACTIVATION OF SALMONELLA TYPHIMURIUM ATTACHED TO CATFISH SKIN

This study was conducted to test sanitizer efficacy of lactic acid on Salmonella typhimurium populations that were firmly attached or loosely attached on catfish skin with or without mucus. For both cell types, counts of S. typhimurium were reduced from 5.9 log¹⁰ CFU/skin to below the limit of detection of 2.0 log¹⁰ CFU/skin by a 5 min exposure to 2% lactic acid. Exposure for 1 min reduced a 10⁴ CFU/skin inoculum to below the limit of detection. Catfish skin mucus slightly decreased the antimicrobial effect of lactic acid against firmly attached S. typhimurium.     

VIRULENCE RESPONSE OF A SALMONELLA TYPHIMURIUM HILA:LACZY FUSION STRAIN TO SPENT MEDIA FROM PURE CULTURES OF SELECTED BACTERIA AND POULTRY CECAL MIXED CULTURE

Virulence gene expression in Salmonella is triggered by a variety of environmental factors including changes in the gastrointestinal environment of birds during different dietary regimes. The objective of this study was to determine if growth of specific microorganisms alters the environmental conditions sufficiently to signal Salmonella Typhimurium virulence response. Spent media was obtained from a Salmonella Typhimurium hilA:lacZY fusion strain, a poultry Salmonella Typhimurium strain , Eschcrichia coli K12, and Lactobacillus caseii Spent media samples were collected after 2, 4, 8 and 24 h of growth in brain heart infusion broth (BHI) and M9 media, β-galactosidase assays were performed on the samples to determine virulence expression. Virulence response to Salmonella, spent media was 2-fold greater than Lactobacillus spent media at 4, 8 and 24 h growth ( P < 0.05). Virulence expression almost doubled when exposed to Salmonella Typhimurium (NONA) spent media compared to mixed culture spent media at 4 h, and Salmonella Typhimurium (NONA) was significantly higher than mixed culture spent media at 24 h ( P < 0.05). Based on these results, it appears that growth of similar bacterial species may alter the composition of rich media sufficiently to influence virulence.     

INHIBITION OF SALMONELLA TYPHIMURIUM ON POULTRY BY THE LACTOPEROXIDASE SYSTEM1

The antibacterial activity of the lactoperoxidase (LP) system on the growth and survival of a naladixic acid resistant strain of Salmonella typhimurium on poultry was determined. The LP system treatment, which consisted of lactoperoxidase (1 μg/ml), potassium thiocyanate (5.9 mM), and hydrogen peroxide (2.5 mM) (final concentrations) in water, reduced the level of Salmonella on inoculated chicken legs. The magnitude of reduction was dependent on temperature and time. For a water temperature of 25C and a time of 30 min, a 13.2% reduction was seen, as compared to a water temperature of 60C and a time of 15 min which showed a 80.6% reduction. In a 48 h shelf-life study, the LP system controlled growth of psychrotrophic bacteria on chicken legs. Hunterlab color values (L, a, b) for chicken thigh skin and oxidative deterioration as measured by TBA values for chicken thigh meat did not significantly differ (p < 0.01 and p < 0.05, respectively) between control and treated thighs.     

ANTIFUNGAL EFFECTS OF SORBIC ACID AND PROPIONIC ACID AT DIFFERENT pH AND NaCl CONDITIONS

The effect of pH (7, 5.6, 3.5), NaCl concentration (3 to 20% w/v) and EDTA on the antifungal activity of sorbic acid and propionic acid (Ca salt) against Candida spp. , Sporothrix sp. , Fusarium sp. , Penicillium spp. , Paecilomyces sp. and Aspergillus spp. was investigated by the Spiral Gradient End Point Test. Sorbic acid was active against most yeasts and molds, and in the presence of EDTA, which enhanced the effect of sorbic acid, all the organisms tested were inhibited. Sorbic acid was most effective at low pH. The inhibitory effect of sorbic acid increased with increasing NaCl concentration for all test organisms. Propionic acid was not as active as sorbic acid, but when combined with EDTA the inhibition was improved for some of the molds tested. Increasing or decreasing the pH did not change the antifungal activity of propionic acid. Synergistic effects of NaCl occurred at concentrations higher than 10%, but when EDTA was added, the inhibition was seen even at the lower NaCl concentrations.     

INHIBITION OF AEROBIC AND ANAEROBIC GROWTH OF STAPHYLOCOCCUS AUREUS IN A MODEL SAUSAGE SYSTEM

Sodium nitrite, potassium sorbate, and glycerol monolaurate all inhibited anaerobic growth of Staphylococcus aureus more than its aerobic growth in an agar-meat model sausage system, and all were more inhibitory when lactic acid was added. Whereas anaerobic growth of S. aureus was inhibited by concentrations of 100, 2500, and 2500 ppm, respectively, of nitrite, potassium sorbate, and glycerol monolaurate, corresponding concentrations of 150, 5000, and 5000 ppm were required to inhibit aerobic growth ((20 meq lactic acid added).
Sorbic acid, a 3:1 mixture of sorbic acid and glycerol monolaurate by weight, and butylated hydroxyanisole (BHA), however, did not show differential inhibitory effects toward aerobic and anaerobic growth of S. aureus and also were more effective inhibitors with addition of lactic acid. Sorbic acid suppressed staphylococcal growth in the model sausage system at 500 ppm, the mixture of sorbic acid and glycerol monolaurate at 750 ppm, and BHA at 10,000 ppm (20 meq lactic acid added).     

EFFECTS OF TRISODIUM PHOSPHATE ON POPULATIONS OF SALMONELLA TYPHIMURIUM AND SALMONELLA ENTERITIDIS IN VITRO

This study determined the bactericidal activity of different concentrations of trisodium phosphate (TSP) against Salmonella typhimurium and Salmonella enteritidis in vitro. One milliliter of each TSP solution (0.5, 1, 1.5 and 2% w/v) was mixed with 1 mL of each bacterial culture and left to stand at room temperature (18  ±  1C) for 0, 15, 30, 45, 60 or 90 min before enumeration of the surviving pathogens.
The effects of 1.5 and 2% TSP were apparent on both bacterial species from 0 min and bacterial counts reached undetectable ( < 1.0  ×  10¹ cfu/mL) levels immediately. The 1% TSP solution effected close to 4.20–5.72 log reduction after 15 min. However, the reduction effect of 0.5% TSP was a function of treatment time, and some culturable bacteria remained even after 90 min of treatment. These results suggest that the bactericidal effect of TSP solution on these pathogens depends on its concentration and on the duration of treatment.

PRACTICAL APPLICATIONS

Poultry has been identified as one of the most important reservoirs of Salmonella typhimurium and Salmonella enteritidis in food chain. Efforts to eliminate or reduce Salmonella spp. and other pathogens attached to poultry carcasses during processing have been made for many years. In 1992, the U.S. Department of Agriculture approved the use of trisodium phosphate (TSP) as a processing aid to reduce Salmonella spp. on raw poultry carcasses. The aim of the present study was to determine the bactericidal effects of different concentrations of TSP on pure cultures of these pathogens in vitro .     

CAFFEINE IS NON-MUTAGENIC TO SALMONELLA TYPHIMURIUM AND HUMAN CELLS IN CULTURE

Caffeine (1,3,7-trimethylxanthine) was inactive as a mutagen at the hgprt locus in two human lymphoblast lines at concentrations up to 20 mM. In addition, caffeine did not induce 8-azaguanine-resistant mutants in Salmonella typhimurium at similar levels.     

EFFECT OF GAMMA RADIATION ON TOXICITY AND IMMUNOGENICITY OF SALMONELLA TYPHIMURIUM

Salmonella typhimurium H391 neurotoxin was sensitive to gamma radiation under in vivo conditions but resistant under in vitro conditions. Cells were detoxified after exposure to a radiation dose of 500 krad while isolated neurotoxin retained toxicity up to a dose of 3,000 krad. Detoxification of S. typhimurium H391 cells after exposure to 500 krad was accompanied by loss of antigenic properties which may be attributed to changes in the physico-chemical behavior of the neurotoxin moiety after in vivo radiation.     

GROWTH, SURVIVAL AND HEAT RESISTANCE OF SALMONELLA TYPHIMURIUM AND ESCHERICHIA COLI IN REGULAR AND OMEGA-3 HENS EGG PRODUCTS

Omega‐3 enriched eggs are currently produced by hens fed a flax diet. Since omega‐3 polyunsaturatedfatty acids are prone to oxidation, the addition of vitamin E is supplemented into the hen's diet as an antioxidant. Typically these eggs contain about 2 and 4 times more omega‐3 fatty acids and vitamin E, respectively compared to regular eggs. Since table eggs have a long history of association with foodborne salmonellosis, it was of interest to compare the growth and/or survival of several bacterial pathogens in omega‐3 and regular eggs. In this respect nalidixic‐resistant Escherichia coli and Salmonella Typhimurium were inoculated into regular and omega‐3 hen's egg products (whole eggs, albumen, yolk) and incubated at 22, 8 and ?20C. Time‐course studies indicated that by 72 h both Salmonella and E. coli levels increased by 7 logs at 22C in both types of whole egg. At 8C population levels for these bacteria both increased by approximately 3 logs at 6 weeks. In regular and omega‐3 yolks, salmonellae maintained at 22 and 8C for 48 h and 6 weeks increased by approximately 6 and 1.5 logs, respectively. E. coli levels were higher in egg yolk compared to Salmonella at both temperatures. Regardless of the yolk source, however, no significant (P > 0.05) differences in population levels were observed. Survival patterns of E. coli at ?20C were not significantly (P > 0.05) different between whole egg sources. This trend was also observed in the yolks. For Salmonella no significant (P > 0.05) differences in survival were observed between yolk preparations maintained at ?20C. Increasing the level of α‐tocopherol from c. 63 to 240 ppm in regular egg yolk, resulted in no significant (P > 0.05) differences in the growth of Salmonella at 22C. In addition, when the bacterium was heated in regular egg yolk amended with vitamin E at 56.5C, no significant (P > 0.05) difference was observed in resistance regardless of α‐tocopherol (55‐713 ppm) or total tochopherol (92–1238 ppm) level.     

EFFECT OF SPICES ON GROWTH AND SURVIVAL OF SALMONELLA TYPHIMURIUM DT 104 IN GROUND BEEF STORED AT 4 AND 8C

Few studies have addressed the use of spices against pathogens associated with meat. The effects of garlic, ginger and turmeric were evaluated against Salmonella Typhimurium DT 104 that were inoculated either in spice paste or in buffered peptone water (BPW) or in heat‐treated ground beef and stored at 4 and 8C for 10 days. Data from the spice pastes study showed a decrease in Salmonella Typhimurium DT 104 counts, and the greatest reduction (3.39 log) was observed in garlic paste stored at 4C. Garlic in BPW data showed a reduction of 1.5 and 1.0 log in Salmonella Typhimurium counts at 4 and 8C, respectively. Ground beef stored at 4C showed no growth or a slight reduction in growth in samples with spice, while all samples at 8C showed an increase in Salmonella Typhimurium counts. Results show that the spices inhibit or inactivate Salmonella Typhimurium DT 104 when they are in direct contact. However, when spices are added to a complex food system such as ground beef, the inhibitory activity of these spices considerably decreases.     

DETECTION OF SALMONELLA TYPHIMURIUM IN OYSTERS BY PCR AND MOLECULAR HYBRIDIZATION

Because shellfish (oysters, clams and mussels) are filter feeders, i.e., able to concentrate pathogens from the surrounding waters within their tissues, they have been widely associated with outbreaks illness. The incidence of salmonellosis caused by the consumption of raw or undercooked shellfish, is a primary concern of public health agencies. Then, in recent years, more rapid and specific methods based on the DNA sequence of salmonella genes have been developed to detect low levels of pathogens in environmental and food samples. In this study, we developed a sensitive method to detect low levels of Salmonella typhimurium in oyster tissues (0.1 cfu/g). This methodology consisted of dissection of the gastrointestinal oyster tract, pre‐enrichment of the samples in nonselective medium, DNA extraction and polymerase chain reaction followed by molecular hybridization using a digoxygenin‐labeled amplicon‐derived probe. These results can benefit the public health agencies and shellfish producers concerning microbiological and quality aspects of the commercial oyster production.     

RESPONSE SURFACE MODELS FOR THE EFFECTS OF TEMPERATURE,pH, SODIUM CHLORIDE,AND SODIUM NITRITE ON THE AEROBIC AND ANAEROBIC GROWTH OF STAPHYLOCOCCUS AUREUS 196E1

The effects and interactions of temperature (12–45C), initial pH (4.5–9.0), NaCl (0.5–16.5%), and sodium nitrite (1–200 μg/ml) on the aerobic and anaerobic growth of Staphylococcus aureus 196E were studied using 50 ml portions of Brain Heart Infusion Broth in 250ml unsealed and sealed trypsinizing flasks, respectively. The flasks were inoculated to a level of approximately 10³ cfu/ml, incubated on a rotary shaker, sampled periodically, and enumerated on Tryptic Soy Agar. Growth curves were generated by fitting the data to the Gompertz function using nonlinear regression analysis. The general growth characteristics of S. aureus in response to the five environmental variables were similar to those observed by other investigators including (1) enhanced growth in the presence of oxygen, (3) ability to grow at high sodium chloride concentrations, and (3) dependence of the bacteriostatic activity of sodium nitrite on pH and oxygen availability. Supplemental studies indicated that growth kinetics were independent of inoculum size, which allowed the Gompertz A term to be treated as a constant. However, the maximum population density (MPD) achieved by the cultures was dependent on the independent variables, requiring that it be modeled in addition to the Gompertz B and M terms. The MPD was then used to calculate the Gompertz C term. Quadratic and cubic response surface models were generated using various data transformations. Quadratic models using and LN-transformation provided reasonable predictions of the effects of the four variables on the growth kinetics of S. aureus, and should prove useful for providing initial estimates of the behavior of S. aureus in foods.     

ATTACHMENT AND REMOVAL OF SALMONELLA SPP. ON MEAT AND POULTRY TISSUES

Muscle pieces from beef, pork, and chicken were exposed to Salmonella strains in various aqueous solutions to determine mechanisms of microbial attachment and release. Binding was measured by scanning electron microscopy and by bacteriological methods. Bacteria appear to attach preferentially to connective tissue fibers, rather than to myofibrils. Muscle fiber swelling and shrinkage during processing permits some microbial entrapment between muscle bundles. Mannose and salt solutions were examined as potential inhibitors of attachment or as removal agents. Mannose inhibited attachment slightly and isotonic saline rinses removed some attached cells, but either method effected only about a one log reduction (90%). Application of 41 rinsings only effected a 4 log reduction. The apparent variety of attachment mechanisms by Salmonella hinders complete removal from meat tissues by simple rinsing procedures.