OXYRASETM ENZYME AND MOTILITY ENRICHMENT FUNG-YU TUBE FOR RAPID DETECTION OF LISTERIA MONOCYTOGENES AND LISTERIA SPECIES1

A rapid and simple method using a U-shaped glass apparatus (Fung-Yu tube) for early determination of the presence of Listeria monocytogenes and Listeria species in mixed cultures and inoculated meat samples has been developed. This system utilizes unique biochemical and physical properties of Listeria for selective enrichment. Fraser broth was used as a selective enrichment broth especially for observation of esculin hydrolysis (blackening of broth), and semisolid Modified Oxford agar was used for selective detection of motility of Listeria. When Fung-Yu tubes containing 0.1 unit/mL of Oxyrase^TM (membrane fractions of Escherichia coli) were inoculated with L. monocytogenes, an enhanced early growth of L. monocytogenes occurred. A presumptive positive result for low numbers of L. monocytogenes (1–100 CFU/g) in the presence of large numbers of competitive microflora in pre-enriched (24 h) ground beef samples using the Fung-Yu tube method with the aid of Oxyrase^TMwas obtainable within 10 h. Using this system, isolation of Listeria in the presence of mixed bacterial flora (44 species), such as Bacillus, Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Staphylococcus, and Streptococcus, and in inoculated ground beef was successful in 24–48 h. The Fung-Yu tube procedure is a highly sensitive, selective, and easy-to-use method to separate and isolate L. monocytogenes and other Listeria spp. from other contaminating microorganisms in meats.     

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) .     

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.     

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 .     

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 .     

INHIBITION OF LISTERIA MONOCYTOGENES BY LACTOCOCCUS LACTIS SUBSP. LACTIS ISOLATED FROM ITALIAN RAW HAM

A total of 168 strains of lactic acid bacteria were isolated from Italian raw ham and screened for antagonistic activity against Listeria monocytogenes by using an agar spot assay. Only one strain of Lactococcus lactis subsp . lactis produced antagonistic effects other than inhibition by low pH and hydrogen peroxide. The proteinaceous nature of the compound produced by L. lactis B10 was demonstrated by its inactivation by proteolytic enzymes. The cell-free culture super-natants (filtered and heat-treated) showed a bactericidal mode of action. Bacteriocin produced by L. lactis B10 was inhibitory to other lactic acid bacteria and one strain of Staphylococcus aureus, but not to the gram-negative bacteria tested .     

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.     

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.     

THERMAL RESISTANCE OF YERSINIA ENTEROCOLITICA AND LISTERIA MONOCYTOGENES IN MEAT AND POTATO SUBSTRATES

The heat resistance of a wild type and nalidixic acid resistant strain of Yersinia enterocolitica, and Listeria monocytogenes, was measured in meat (minced beef and minced beef homogenate) and potato substrates over the temperature range 50–60C. Comparisons of heat resistance were determined using D-values calculated using a linear survival model. The results showed that the wild-type strain of Y. enterocolitica was more heat resistant than the mutant (p<0.05). Under most conditions, the use of a nonselective/overlay recovery medium resulted in higher D-values compared to a selective recovery medium (p<0.05). Analysis of the data using a nonlinear survival model (D₁ and D₂ -values) suggested the presence of heat resistant subpopulations and was particularly evident for the mutant strain, and in potatoes compared to minced beef.     

BACTERIOCIN EXPOSURE AND FOOD INGREDIENTS INFLUENCE ON GROWTH AND VIRULENCE OF LISTERIA MONOCYTOGENES IN A MODEL MEAT GRAVY SYSTEM

The antilisterial activity of bacteriocin-producing Lactobacillus sakei 1 bac⁺ alone and combined with food ingredients (sodium chloride, D-glucose, oregano, black pepper) was studied in a model meat gravy (1.8% proteose peptone, 1.2% meat extract, 0.6% yeast extract, 2.0% corn starch) kept under refrigeration for 10 days. Two strains of L. monocytogenes (serotypes 4b and 1/2a) were employed in coinoculation experiments and Lactobacillus sakei ATCC 15521 was used as a negative control for bacteriocin production. The LAB bac⁺ strain was more effective in inhibiting both L. monocytogenes serotypes than the LAB bac⁻ strain. The serotype 4b was more sensitive to bacteriocin than serotype 1/2a. The effect of the ingredients on inhibition of L. monocytogenes was serotype dependent. Bacteriocin exposure did not affect sensitivity to ampicilin and rifampicin. However, L. monocytogenes partially lost their hemolytic activity after exposure to bacteriocin-producing Lb. sakei 1 and food ingredients.     

USE OF MEAT ISOLATE, LACTOBACILLUS BAVARICUS MN, TO INHIBIT LISTERIA MONOCYTOGENES GROWTH IN A MODEL MEAT GRAVY SYSTEM

The ability of Lactococcus lactis 11454 , Pediococcus pentosaceus 43200 and Lactobacillus bavaricus MN, originally isolated from dairy, vegetable, and meat products, respectively, to inhibit growth of Listeria monocytogenes Scott A in a model beef gravy was examined. In the first series of experiments, where the lactic acid bacteria and L. monocytogenes were inoculated at levels of 10⁵ CFU/mL and 10³ CFU/mL, respectively only L. bavaricus inhibited listerial growth at 10C. Subsequent experiments using L. bavaricus MN confirmed that the inhibition was caused by a bacteriocin, occurred at temperatures at low as 4C, and could be initiated by 10³ CFU/mL L. bavaricus in the presence of L. monocytogenes at levels 10-fold higher. Although the inhibitory agent was protease-sensitive and inhibition occurred in the absence of a fermentable carbohydrate, the presence of acid enhanced efficacy of the bacteriocin .     

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.     

FATE OF LISTERIA MONOCYTOGENES AND SALMONELLA SPP. IN THERMALLY PROCESSED COLOR-MODIFIED TURKEY MEAT

The growth/survival of Listeria monocytogenes and Salmonella spp. was evaluated in two thermally-processed turkey raw materials. Flaked turkey (FT) was prepared from boneless, skinless thighs. Portions were washed with sodium phosphate buffers to lighten tissue color and produce color-modified turkey (CMT). Raw materials were cooked, inoculated with pathogens, and held at 4 and 20C. Salmonella numbers declined in cooked samples stored at 4C for 21 days, but increased by approximately 6 logs in 2 days for the FT and CMT held at 20C. L. monocytogenes increased by approximately 5 logs in samples held for 14 days at 4C. By 2 days at 20C, L. monocytogenes increased more than 5 logs in both materials. Growth/survival of these pathogens did not differ (p > 0.05) between FT and CMT. The data suggest that CMT does not present any more health hazards than conventional turkey raw materials.     

A QUANTITATIVE MICROTITER PLATE HEMOLYSIS ASSAY FOR LISTERIA MONOCYTOGENES

All virulent strains of L. monocytogenes produce the extracellular SH-activated hemolysin, listeriolysin O, while nonhemolytic strains of L. monocytogenes are avirulent suggesting that the expression of this hemolysin is necessary for virulence of L. monocytogenes. Hence, testing for hemolysis becomes clinically relevant for an isolate identified as L. monocytogenes. However, the quantification and interpretation of this characteristic on blood agar poses several problems. Hence we have proposed a simple quantitative microtiter plate hemolysis assay. The assay is made of SRBC (3% SRBC in PBS) in microtiter plates to which CASO-cultured cell-free supernatants of L. monocytogenes or other test cultures are added. After mixing, the plates are incubated at 37C for 15–30 min and the hemolysis is visually read as CHU and MHU, as well as by a 620 nm scan for absorbancy. Percent hemolysis is calculated. We feel that this assay should prove to be of significance in a microbiology laboratory in which routine hemolysis assays are conducted.