EFFECT OF OXYRASETM ENZYME ON LISTERIA MONOCYTOGENES AND OTHER FACULTATIVE ANAEROBES1

The use of an oxygen-reducing membrane fraction to stimulate growth of some anaerobic bacteria has been reported. In this study, stimulated recovery of Escherichia coli 0157:H7, Salmonella typhimurium, Streptococcus faecalis, and heat-injured Listeria monocytogenes was achieved by using broth media supplemented with Oxyrase^TM (membrane-bound enzyme system derived from E. coli). The Oxyrase^TMenzyme allowed rapid growth of these facultatively anaerobic organisms in a nonselective broth. The activity of Oxyrase^TMin four different selective enrichment broths to support growth of L. monocytogenes also was evaluated. Among tested broths, FDA-approved Listeria Enrichment Broth containing Oxyrase^TMwas found to be substantially superior for facilitating the growth of L. monocytogenes. Oxyrase^TMpromoted a significantly greater number of L. monocytogenes than commonly used but nonspecific reducing agents, such as L-cysteine HCl and sodium thioglycolate. These findings indicated that all organisms tested can be grown well in liquid media using the oxygen-consuming membrane fragments to achieve and maintain oxygen-limited conditions. The Oxyrase ^TM enzyme system has potential applications in some selective media and other diagnostic tests specified for rapid detection of these facultatively anaerobic pathogens.     

ENRICHMENT OF SEVERELY AND MODERATELY HEAT-INJURED LISTERIA MONOCYTOGENES CELLS

Recovery of injured cells of a 90% heat kill of Listeria monocytogenes strain Lm82 in Trypticase soy yeast extract broth (TSBYE) at 30C was determined in enrichment broth and modified enrichment broth. Although the surviving population was heterogeneous with respect to degree of damage, two fractions of surviving cells defined as moderately and severely damaged were considered. Progeny of moderately damaged survivors (NaCl-sensitive but not enrichment medium-sensitive) increased about 100-fold in 5 h; severely damaged cells (enrichment medium- and NaCl-sensitive) did not grow in this time period. Most of the severely damaged cells required 20 h or longer to recover in TSBYE and even longer in TSBYE plus selective agents. Recovery was accelerated either by adding sodium pyruvate or by reducing the oxygen level. The results were used to design a Mark I preenrichment/enrichment protocol based on the U.S. Food and Drug Administration's selective enrichment broth.     

A STUDY ON SUITABILITY OF FOUR ENRICHMENT BROTHS FOR PCR-BASED DETECTION OF LISTERIA MONOCYTOGENES FROM RAW MEAT

Four enrichment broths were evaluated for their compatibility with the polymerase chain reaction (PCR) for detection of Listeria monocytogenes from raw meat after single‐step enrichment. Standardized PCR protocols for listeriolysin O (hlyA) gene were used for the species‐specific identification of L. monocytogenes. Four broths, namely, modified University of Vermont broth (MUVM), Listeria enrichment broth (LEB), Fraser broth (FB) and polymyxin, acriflavin, lithium chloride, ceftazidime, aesculin, mannitol, egg yolk broth (PALCAM) , were inoculated with L. monocytogenes. The enriched cultures were subjected for PCR. Similarly, meat samples were artificially spiked with various concentrations of L. monocytogenes, these spiked samples were enriched in the above‐mentioned four broths and subjected to PCR to determine the medium that was most compatible for PCR‐based detection of L. monocytogenes. The aliquots taken during different incubation periods were subjected to three different procedures for the concentration of the target organism for use in PCR. Results revealed that MUVM was better than other broths for the detection of L. monocytogenes by both PCR and cultural method; moreover, it was able to support the growth of as low as 10 cfu/g of meat. Concentration of the target organisms by centrifugation and washing with PCR buffer was the most suitable method for improving PCR performance for detection of L. monocytogenes. Goat (n = 67) and buffalo (n = 45) meat samples from local markets were also screened by both PCR and cultural method to validate the results obtained from the spiking studies. Both results were in agreement in spiking studies as well as screening of market meat samples.     

SIMPLE oPSU BROTH-BAX® PCR-PICOGREEN® SYSTEM FOR RAPID DETECTION OF LISTERIA MONOCYTOGENES IN PASTEURIZED MILK AND HOT DOGS

Factors that significantly affect BAX® PCR detection of Listeria monocytogenes from optimized Penn State University (oPSU) broth were identified and optimized. Concentration of PCR product was significantly reduced by BAX™ protease and significantly increased by eliminating the lysis step and directly diluting oPSU broth prior to PCR. A simple oPSU broth-BAX® PCR-PicoGreen® (PSU-BAX-PicoGreen) system was developed and compared with current methods for detecting low levels of L. monocytogenes in commercial milk and hot dogs. All 30 milk samples inoculated with 10–20 CFU per mL L. monocytogenes were positive by FDA, BAX® and PSU-BAX-PicoGreen methods and all 42 uninoculated milk samples were negative by all of the above methods. All 30 hot dog samples inoculated with 10-20 CFU/g L. monocytogenes were positive by the USDA and PSU-BAX-PicoGreen methods, however, 2 hot dog samples gave indeterminate results with the standard BAX® method. All 42 uninoculated hot dog samples were negative by USDA, 9 were indeterminate by BAX® and 2 were positive by PSU-BAX-PicoGreen. The PSU-BAX-PicoGreen system may provide a simple and accurate method for rapidly screening pasteurized foods for both injured and noninjured L. monocytogenes.     

A REVIEW OF METHODS FOR DETECTION OF THE PSYCHROTROPHIC FOODBORNE PATHOGENS LISTERIA MONOCYTOGENES AND AEROMONAS HYDROPHILA1

The detection of the psychrotrophic foodborne pathogens Listeria monocy-togenes and Aeromonas hydrophila in food depends on the use of various selective media designed specifically for their isolation. These selective media, which contain combinations of dyes, antibiotics, and other inhibitory substances, restrict the background microflora while permitting the desired organism (either L. monocytogenes or A. hydrophila) to form characteristic colonies. Since the selective media are not completely specific, confirmation tests specific to L. monocytogenes or A. hydrophila are used to verify the identity of the respective isolates. It has been observed that the inhibitory substances used will not permit injured (stressed) cells to form colonies and special techniques are needed to recover injured cells. The present techniques, while not ideal, do allow for a reasonably quantitative estimate of any L. monocytogenes or A. hydrophila present in a food.     

DEVELOPMENT OF NEW ENRICHMENT BROTHS AND PLATING AGARS FOR ISOLATION OF HEMOLYTIC SPECIES OF LISTERIA

A new basal broth medium was formulated for optimal growth of Listeria monocytogenes, which occurred at 37°C when the initial pH was 6.8. This formulation was used as the basal medium for development of a highly selective plating agar which proved suitable for direct culture of vaginal and rectal swabs for L. monocytogenes. A modification with slightly lower selectivity was necessary for recovery of hemolytic strains of Listeria ivanovii and Listeria seeligeri. The same basal medium was used as a pre-enrichment broth and for the development of a selective enrichment broth which were incorporated into a two-step enrichment procedure for the isolation of L. monocytogenes from foods. These new media were compared with several others that have been proposed by comparing recoveries of Listeria from laboratory seeded foods (100% positive), raw milk (50 samples, all negative) and comminuted meat products (75% positive) .     

ANTAGONISM OF BACTERIAL COMPETITORS TOWARD LISTERIA MONOCYTOGENES IN ENRICHMENT CULTURE

A study was made of the competitiveness toward Listeria monocytogenes ( Lm82 ) in Listeria enrichment broth (LEB) by bacteria isolated from foods and by strains of Enterococcus and other Gram-positive bacteria. Competitive (i.e., able to mask during enrichment in LEB for 24 h) and noncompetitive bacteria were tested for production ofanti-Lm82 agents in diffusion zone assays on deMann-Rogosa-Sharpe (MRS) agar with added beta-glycero-phosphate (MRSB) and in Listeria enrichment agar (LEA). Enterococci were the most active competitors. The presence of small (2–6 mm diameter) inhibitory zones on MRSB correlated significantly with competitive activity in LEB; however, the correlation was not due to the metabolic activity that produced inhibitory zones on MRSB. Zone-producing bacteria were more likely to be competitors than were nonzone producers, but not all zone producers were competitors. Similarly, about 15% of bacteria that did not produce zones were competitive. The few inhibitory zones on LEA indicated that competitor activity in the selective enrichment broth may only rarely be due to the production of diffusible inhibitors. The most important factor in competitiveness was the ability of enterococci and some other bacteria to maintain superior numbers in the presence of prolisterial selective agents in LEB. With their superior numbers, competitors significantly decreased the pH of LEB. faster than did noncompetitors. Diffusible inhibitors produced in LEA by bacteria may also contribute significantly to competitiveness .     

COMPETITION OF FOOD BACTERIA WITH LISTERIA MONOCYTOGENES DURING ENRICHMENT CULTURE

Thirty-two foodborne bacterial isolates were tested as potential competitors of Listeria monocytogenes strain LM82 during enrichment because of their resistance to the selective agents in Listeria enrichment and isolation media. Competitive ability of each isolate was classified as weak, moderate, or strong by determining the ratio at which it masked identification of LM82 at an inoculation concentration of 10 colony forming units (CFU)/10 mL of Listeria enrichment broth. Of the competitive isolates identified, six were Enterococcus spp., two were Staphylococcus spp., and one was a Corynebacterium sp. Although several strains of Enterococcus faecium were examined, not all were competitive. Of six other bacterial strains associated with food fermentations and tested for competitiveness with LM82, one, a Gram-positive tetrad, was competitive. This study showed that although food microfloral strains that are able to survive in enrichment and isolation environments are fairly common, they do not necessarily compete with Listeria. Not all strains in a competitive species are necessarily competitive .     

RECOVERY LIMITS OF HEAT-INJURED LISTERIA MONOCYTOGENES FROM ENRICHMENT BROTH AND ENRICHED CULTURES OF INOCULATED FOODS

Recovery of heat-injured Listeria monocytogenes strain LM82 was evaluated quantitatively in Listeria enrichment broth (LEB) and in enriched cultures of cooked shrimp and Brie cheese. LM82 cells [10⁸ colony forming units (CFU)/ml] were heated for 60 min at 52C in phosphate-buffered saline. After 24 and 48 h enrichment, injured LM82 (6 replicates at each of 5 inoculation levels) were isolated on 3 selective media: lithium chloride-phenylethanol-moxalactam agar (LPMA), modified McBride agar (MMA) and Oxford agar (OXA). The recovery limit was expressed as a 50% end point value (RL₅₀), which is the calculated inoculation value necessary to recover LM82 on half of the replicates of each type of isolation agar plate after streaking from the enrichment of measured inoculum. The RL₅₀ values for injured cells were comparable to those of uninjured cells after 48 h enrichment in LEB without food. The type of isolation agar did not affect the RL₅₀ value, although with food, MMA gave consistently but not significantly higher values, i.e., recovery inferior to that of LPMA and OXA. RL₅₀ values were higher in Brie and cooked shrimp, presumably because of the competitive microflora in those foods. Addition of lactose or pyruvate to LEB improved recovery but had little or no effect when foods were present .     

COMPARISON OF SELECTIVE AND NONSELECTIVE ENRICHMENT MEDIA FOR THE ISOLATION OF LISTERIA SPECIES FROM RETAIL FOODS

Enrichment in a nonselective medium, Buffered Peptone Water (BPW) was compared with selective enrichment in University of Vermont Medium (UVMI and UVMII) for the isolation of Listeria spp. from foods. The selectivity of the 2 types of media for the pathogenic strain, Listeria monocytogenes, was also compared. In total, 221 food samples including beef burgers, ham, turkey, lettuce, broccoli, carrots, coleslaw, salads, fish, and ice cream, were purchased from local retail outlets and examined for the presence of Listeria species and L. monocytogenes using both enrichment media Listeria species were detected in 57 (25.8%) samples using UVM, and 56 (25.3%) using BPW. L. monocytogenes was present in 33(14.9%) samples enriched in UVM and in 29(13.1%) samples enriched in BPW. The advantages and disadvantages of selective and nonselective enrichment for detection of Listeria species from a range of foods are discussed.     

EXTINCTION OF LISTERIA MONOCYTOGENES IN SINGLE-STRENGTH ORANGE JUICE: COMPARISON OF METHODS FOR DETECTION IN MIXED POPULATIONS1

The FDA method of selective enrichment followed by selective plating on modified McBride agar was capable of detecting the presence of L. monocytogenes inoculated into a typical, commerical, reconstituted, single-strength orange juice at the 10⁰ cfu/mL level. The KOH shock treatment, formerly recommended in the FDA protocol, negatively affected recovery of Listeria at 10⁰ and 10¹ levels in juice which also had a low background microflora (10² cfu/mL total count); however, KOH treatment was required for Listeria detection in juice which had high background microflora (10⁸ cfu/mL). Two other protocols for detection of Listeria which utilized cold enrichment or different selective enrichment media were less effective than the FDA procedure due to heavy growth of background microflora. No Listeria was detected in 100 commerical orange juice samples from dairy and nondairy processors in geographically distinct areas of North America using the FDA method.     

METHODS FOR THE DETECTION OF FOODBORNE LISTERIA MONOCYTOGENES IN THE U.S.

A review of methods for the isolation and detection of Listeria monocytogenes used in the United States is presented. Methods reviewed include the cold enrichment technique, the FDA Method, the USDA Method, and two rapid techniques, the Listeria-Tek enzyme-linked immunosorbent assay and the Gene-Trak Listeria Assay. Comparisons of new rapid biochemical test kits, the MICRO-ID LISTERIA System, API Listeria, and the Rosco system vs. conventional tests of identification are also reviewed. Contemporary isolation methods detect all Listeria species so confirmation of L. monocytogenes is still necessary after isolation. The USDA method is the most practical of the cultural methods due to the rapid reporting of negative samples. Rapid methods (Listeria-Tek and the Gene-Trak Listeria Assay) are faster and more objective than cultural procedures but still depend on sample enrichment for detection of Listeria. These rapid techniques are best utilized when screening large numbers of food samples. All the rapid biochemical test kits reviewed provide fast reliable identification of Listeria species when compared to classical techniques. However, the API Listeria system identifies the test strains without a complementary CAMP test. Refinements are still needed in both cultural and rapid methods. Future Listeria methodology must emphasize molecular techniques not requiring enrichment which would be both rapid and specific for L. monocytogenes.     

EFFICACY OF PULSED ELECTRIC FIELDS FOR LISTERIA MONOCYTOGENES INACTIVATION AND CONTROL IN HORCHATA

Although Listeria monocytogenes is readily destroyed by thermal treatment, the factor that makes it particularly difficult to control in nonpasteurized foods is its ability to grow at refrigeration temperatures. In heat‐sensitive products, nonthermal technologies such as pulsed electric fields (PEF) as part of hurdle technology could minimize the presence of foodborne pathogens. The influence of PEF‐treatment conditions, inoculum size and substrate conditions on the inactivation and recovery of L. monocytogenes in a traditional low‐acid, vegetable beverage was investigated. The combined effect of PEF, low temperature (5C) and low inoculum level contributed to slow down the recovery of sublethally injured cells. However, at 12 or 16C, this elongation of the lag phases after PEF treatment observed for low inoculum levels of cells was not achieved. Therefore, to prevent the development of L. monocytogenes in low‐acid products by PEF, it may be necessary to combine it with low refrigeration temperatures during distribution and storage, as well as to achieve a very low initial contamination by pathogens in the raw ingredients.     

OPTIMIZATION OF LISTERIOLYSIN O PRODUCTION BY LISTERIA MONOCYTOGENES1

Listeria monocytogenes serotype 1 and Scott A strains were examined to determine the effect of culture conditions onproduction of listeriolysin O (LLO). The hemolytic activity was assayed by hemolysis of sheep red blood cells. The optimum pH range for LLO production in serotype 1 and Scott A were pH 6.2–6.0 and pH 5.8–6.0, respectively. Production of LLO by serotype 1 was 20–fold higher than by Scott A. Addition of more than 1.0% glucose significantly reduced production of LLO by the cells, while favoring growth. By adding 0.5% glucose after stationary phase and adjusting pH to 5.5–7.0, maximum LLO was produced after 2 h incubation at 37C by both strains.     

MATHEMATICAL MODEL FOR THE SURVIVAL OF LISTERIA MONOCYTOGENES IN MEXICAN-STYLE SAUSAGE

Survival of Listeria monocytogenes in chorizos (Mexican‐style sausages) was modeled in relation to initial water activity (a_w0) and storage conditions using the Weibull cumulative distribution function. Twenty survival curves were generated from chorizos formulated at a_w0 = 0.85–0.97 then stored under four temperature (T) and air inflow velocity (F) conditions. The Weibull model parameters (α and β) were determined for every curve. Predicted survival curves agreed with experimental curves with R² = 0.945–0.992. Regression models (R² = 0.981–0.984) were developed to relate α and β to operating conditions. The times to one‐ and two‐log reduction in count (t_1D and t_2D) were derived from the Weibull model in terms of α and β. A parametric study revealed that L. monocytogenes survival was most sensitive to a_w0 between 0.90 and 0.95. The inactivation of L. monocytogenes could be maximized with higher T and lower a_w0; however, F did not significantly influence survival.     

INHIBITION OF LISTERIA MONOCYTOGENES AND OTHER BACTERIA BY SODIUM DIACETATE

Growth and survival patterns of Listeria monocytogenes strain Scott A were studied in brain heart infusion broth containing sodium diacetate. Minimum inhibitory concentrations decreased with decrease in temperature, from 35 and 32 mM at 35C and 20C, respectively, to 28 mM at 5C. Broth pH containing 35, 32, and 28 mM sodium diacetate was 5.25, 5.40 and 5.60, respectively. Sodium diacetate was more effective than acetic acid alone in inhibiting the organism over the pH range of 5.0-6.0. Addition of 21 mM (0.3%) sodium diacetate to ground beef or beef slurry suppressed total aerobic counts during refrigerated storage. Although the meat pH decreased from 5.6 to 5.2 by the addition of the compound, a major part of the antimicrobial effect was attributed to the diacetate and not just pH. Sodium diacetate suppressed growth of three additional L. monocytogenes strains and strains of Escherichia coli, Pseudomonas fluorescens, Salmonella enteriditis and Shewanella putrefaciens. P. fragi, Yersinia enterocolitica, Enterococcus faecalis, Lactobacillus fermentis and Staphylococcus aureus were insensitive to the compound. Sodium diacetate has potential for use in controlling growth of listeriae in meat, poultry and fish products and suppressing growth of certain Gram-negative spoilage and pathogenic microorganisms.     

INTERACTIONS BETWEEN pH AND MALIC ACID CONCENTRATION ON THE INACTIVATION OF LISTERIA MONOCYTOGENES1

The effects and interactions of malic acid concentration and pH on the inactivation kinetics of a three strain mixture of Listeria monocytogenes was studied in brain heart infusion broth (BHI). The medium was supplemented with malic acid and monosodium malate to achieve pH levels of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0 in conjunction with concentrations of 0.0, 0.1, 0.5, 1.0 and 2.0 M. Duplicate 20-mL portions of each pH/malate level were inoculated (ca. 10⁸ cfu/mL), stored aerobically at 28C, and assayed periodically for viable counts by plating on BHI agar. Survivor curves were generated by fitting data to a linear model that includes a lag term and used to calculate D-values and “times to 4-D inactivation”. Inactivation rates were dependent on both the pH and malic acid concentration. At the higher pH levels, malic acid appeared to provide some degree of protection compared to control cultures where the pH was adjusted with HCl. At lower pH values and at higher malic acid concentrations, a concentration-dependent anion effect was observed. The results indicate that malic acid is a relatively benign organic acid. Its antimicrobial characteristics are similar to those of citric acid and is substantially less bactericidal than lactic or acetic acids.