GROWTH RESPONSE OF A SALMONELLA TYPHIMURIUM POULTRY ISOLATE TO PROPIONIC ACID UNDER AEROBIC AND ANAEROBIC CONDITIONS

Organic acids have long been used as additives in poultry feed to reduce microbial populations, including Salmonella spp. Propionic acid addition to poultry feed has a potential role in reducing Salmonella spp. in the chicken intestine. In this study , in vitro growth response of S. typhimurium isolated from poultry to propionic acid under aerobic and anaerobic conditions was determined. When grown in tryptic soy broth (TSB) containing buffered propionic acid (BPA), the growth rate of S. typhimurium gradually decreased as the level of BPA increased and broth pH decreased. No growth was detected with BPA concentrations greater than 3% (volume/volume) of the broth. When the growth rates of S. typhimurium in aerobic and anaerobic TSB were compared at two pH levels (pH 5.0 and 7.0), the growth inhibition of S. typhimurium by propionic acid was markedly suppressed by anaerobiosis at both pHs, as indicated by significantly (p<0.05) higher half-inhibition constants (K°). Also, the growth rates of S. typhimurium in the presence of propionic acid were dramatically reduced by the decrease in pH from 7.0 to 5.0. The results of this study indicated that the growth inhibitory effect of propionic acid against S. typhimurium strains was enhanced by pH decrease and suppressed by anaerobiosis, suggesting that the growth response of S. typhimurium to propionic acid in the chicken intestine might be affected by the environmental conditions such as pH and anaerobiosis.     

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 .     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

D VALUES OF COMPOSITE SALMONELLA ENTERICA SEROTYPES TYPHIMURIUM AND ENTERITIDIS IN PHILIPPINE NATIVE ORANGE JUICE

ABSTRACT

This study established the decimal reduction times (D values) of a composite inoculum of Salmonella enterica serotypes Typhimurium (American Type Culture Collection 13311 and E 1366) and Enteritidis (BIOTECH 1963) in Philippine native orange juice (pH 3.0, 0.84 % citric acid, 8.4°Brix). Acid‐adapted cells (10 log₁₀ cfu/g) were heated to 50–70C for 0–1.5 min and recovered by surface plating onto a selective (bismuth sulfite agar) and a nonselective medium (nutrient agar [NA]). Results showed that in both enumerating media and in all heating temperatures, survivor populations significantly decreased (P < 0.05) with increasing treatment time. The greater populations enumerated on NA in all treatment times were attributed to the sublethal injuries imparted by the acidity of the suspending medium and the heat treatments. The D values calculated from the survivor populations on both media were not significantly different (P > 0.05). The D values ranged from 0.16 to 0.40 min.

PRACTICAL APPLICATIONS

This study established the thermal inactivation rates of a composite Salmonella spp. inoculum in a Philippine orange juice medium. Salmonella is a target organism pertinent in the assuring citrus juices. Hence, appropriate thermal process schedules for citrus juices with similar physicochemical properties as the test medium, against the test organisms may be calculated using the results obtained from this study.

     

METABOLIC RESPONSES OF BAKER'S YEAST TO GLUCOSE ADDITION DURING AEROBIC STARVATION

The kinetics and extent of glucose uptake, ethanol and acetate production, glucose-induced medium acidification and endogenous respiration were studied in baker's yeast starved aerobically for 0, 28, 54 and 96 h in saline (9 g litre^?1 NaCI) at 35°C. Except for the last interval, prolonged starvation brought about an overall increase in most parameters; the transient increase in endogenous respiration observed after glucose addition was lowered with proceeding starvation. A 96 h starvation brought about a change in the kinetics of ethanol production, sharp increase in acetate production and a strong reduction of both the extent and the rate of glucose-induced acidification and of endogenous metabolism.     

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.     

EFFECT OF ELECTRICAL STIMULATION ON KILLING SALMONELLA TYPHIMURIUM IN VARIOUS SALT SOLUTIONS

Electrical stimulation was evaluated as a method to kill Salmonella typhimurium in various salt solutions at different concentration . Salmonella typhimurium at 2 × 10⁵ CFU/ml was treated at 22–24C for 60 min in each salt solution using electricity at 10 mA/cm² current, 1 kHz frequency, and 50% duty cycle. Samples taken at various times were serially diluted, plated on tryptic soy agar and xylose lysine desoxycholate agar, and incubated at 37C for 18–24 h. To detect injured cells, samples were also pre-enriched in buffered peptone water at 37C for 4–5h before being plated. Results indicated all salmonellae were electrically killed at 5 min in NaCl, at 30 min in NaNO₃, and at 45 min in NaC₂H₃O₂ at 0.15 and 0.015 M concentrations. Salmonellae were also killed at 45 min in Na₃PO₄ and at 60 min in Na₂CO₃ at 0.0015 M concentration by electricity in combination with high pH .