A COMPARISON BETWEEN TWO METHODS FOR THE RECOVERY OF LISTERIA MONOCYTOGENES IN MILK POWDER REFERENCE SAMPLES

Two methods (A and B) for the recovery of Listeria monocytogenes were compared using reference samples based on spray drying of milk containing this pathogen and competitive microflora. For method A, the sample was inoculated in a buffered liquid medium (same as IDF Pre-enrichment Broth) and then passed to IDF Enrichment Broth plus phosphate buffer. For method B, the sample was inoculated in FDA Enrichment Broth (LEB) and then passed to LEB (subenrichment). The solid selective medium used in both cases was Listeria Selective Agar (Oxford formulation). The sensitivity and specificity of method B was 95.2% and 100% vs. 76.1% and 88.8% for method A, respectively.     

EFFECTIVENESS OF CARNOBACTERIUM PISCICOLA LK5 FOR CONTROLLING THE GROWTH OF LISTERIA MONOCYTOGENES SCOTT A IN REFRIGERATED FOODS1

The potential for controlling the growth of Listeria monocytogenes in refrigerated foods using Carnobacterium piscicola LK5, a bacteriocin-producing strain originally isolated from raw ground beef, was studied using co-culture techniques. Eight foods, including UHT milk, canned “all-beef”dog food (cooked meat), raw ground beef, irradiation-sterilized raw ground beef, chicken roll, pasteurized crabmeat, canned creamed corn, and frankfurters, were inoculated with 10³ cfu/g L. monocytogenes Scott A, with and without 10⁴cfu/g LK5, and incubated at 5 and 19C. Samples were removed periodically and assayed for total aerobic plate count using Brain Heart Infusion Agar and L. monocytogenes using Vogel-Johnson Agar or Modified Vogel Johnson Agar. The growth of L. monocytogenes was suppressed in milk, dog food, crabmeat, creamed corn, and frankfurters stored at 5C. The microorganism was less inhibitory at 19C. In sterile raw ground beef, LK5 inactivated the pathogen at 5C and prevented its growth at 19C. No activity attributable to LK5 was observed in refrigerated nonsterile ground beef or chicken roll; however, these products did not support the psychrotrophic growth of the pathogen even in the absence of LK5. LK5 was most effective in products where the background microflora was reduced by either thermal processing or irradiation treatment. The results indicate that C. piscicola LK5 has potential as a means for preventing the growth of L. monocytogenes in a variety of refrigerated food products.     

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.     

GROWTH/SURVIVAL OF NATURAL FLORA AND LISTERIA MONOCYTOGENES ON REFRIGERATED UNCOOKED PORK AND TURKEY PACKAGED UNDER MODIFIED ATMOSPHERES

Listeria monocytogenes was inoculated onto fresh pork and turkey slices. Inoculated and control samples were packaged under modified atmospheres (100% N₂, and 20%/80% and 40%/60% CO₂O₂) or air in plastic bags of low gas permeability. Samples were stored at 1 and 7C. Samples stored in air showed a similar microbiological pattern to that usually observed in fresh meat stored aerobically. Packaging under modified atmospheres extended the meat shelf-life. Bacterial growth was strongly inhibited at 1C, particularly in samples stored under CO₂/O₂ enriched atmospheres. Temperature and pH were critical factors for L. monocytogenes growth. This pathogen grew only on pork (initial pH 5.3) packaged in air and stored at 7C. No L. monocytogenes growth was observed at 1C in any atmosphere assayed. However, growth on turkey (initial pH 6.0) was marked at 7C in all atmospheres tested, while at 1C, this bacterium grew weakly only on samples stored in air .     

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 .     

INCIDENCE, SURVIVAL AND GROWTH OF LISTERIA MONOCYTOGENES IN READY-TO-USE MIXED VEGETABLE SALADS IN SPAIN

A total of 116 commercial samples of mixed vegetable salads, packaged in plastic bags, were examined for the presence of Listeria monocytogenes during storage at 4C. Commercial products, belonging to 4 different batches, were sampled over a period of one year and stored for 300 h at 4C in the laboratory. Different sets of enrichment and plating media were used to recover organisms, with subsequent identification by Pasco System and serotyping techniques. Out of a total of 70 control (noninoculated) samples, 21 (30%) were observed to contain Listeria monocytogenes, belonging to serotypes 3a and 3b. A study of salad inoculated with 10³ cfu/g Listeria monocytogenes showed that the initial inoculum increased less than tenfold over the experimental period (300 h). During storage of the product, CO₂ reached nearly 30% and O₂ was no longer detected at 60 h, due to the respiration of the vegetables. The pH inside the packages remained around 6. The specific growth rate in the salad was calculated using the Dmodel Program, which gave a rate of 0.003h^-1, a lower figure than that reported by other authors. This study found growth patterns different to those previously reported for salads with separate ingredients. Our results agree with previous reports that modified atmosphere does not greatly inhibit Listeria monocytogenes. This reflects a lower specific growth rate in this food compared to media, and illustrates the difficulty of validating, in complex food systems, mathematic models based on culture media .     

食品中单增李斯特菌的存在现状及检测方法研究进展

单核细胞增生性李斯特菌（简称单增李斯特菌）是常见的食源性致病菌,该菌可广泛存在于多种食品中,如肉制品、奶制品、水产品、蔬菜等,该菌在欧美国家曾导致多次的食物中毒的爆发,因此对食品中单增李斯特菌的快速检测非常必要,就单增李斯特菌在食品中的污染情况和检测方法作一综述。     

EFFECT OF pH, ACIDULANT, SODIUM CHLORIDE AND TEMPERATURE ON THE GROWTH OF LISTERIA MONOCYTOGENES

Growth of two Listeria monocytogenes strains in tryptic soy broth containing NaCI or combinations of NaCI and acidulants at different pHs and temperatures was investigated . L. monocytogenes was capable of growing in 10% NaCI at 35°C and 12% NaCI at 25°C and 10°C. The maximum NaCI for growth changed when NaCI and pH, in combination with different acidulants and temperature, were tested. The minimum pH/salt level for initiation of growth of L. monocytogenes ranged from 5.0–5.6/8–10% at 35°C and 25°C and 5.6/8% at 10°C, depending upon the acidulant and the strain. Greatest antimicrobial activity occurred at 35°C. Greatest survival occurred at 10°C. In this study L. monocytogenes appeared to persist and tolerate a combination of low pH, high salt and low temperatures .     

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.     

EFFECT OF GROWTH TEMPERATURE AND MEDIA ON INACTIVATION OF LISTERIA MONOCYTOGENES BY CHLORINE

The objective of this study was to determine the effect of growth environment on the susceptibility of Listeria monocytogenes to inactivation by hypochlorite sanitizer. Cells were grown in tryptic soy broth (TSB) at 35, 21, and 6°C and in 1:15 dilution of TSB (low nutrient medium, LNM) at 35°C. Late exponential phase cells were harvested, washed, and exposed to a 1 ppm sodium hypochlorite solution for periods up to 5 min. After 30 s exposure, cells grown at 35°C in TSB were reduced in number by 2.1 ± .3, at 21°C, 3.1 ± .3 and at 6°C, 3.4 ± .3 log units. After 30 s exposure to 5 ppm free chlorine cells grown at 35°C in TSB were reduced in number by 5.2 ± .2 and in LNM by 3.1 ± .1 log units. These data demonstrate that growth environment has a significant effect on chlorine inactivation of L. monocytogenes.     

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 .     

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.     

SURVIVAL OF LISTERIA MONOCYTOGENES IN YOGURT WITH VARYING LEVELS OF FAT AND SOLIDS

Yogurts with varying levels of fat and solids were fermented with Lactobacillus bulgaricus and Streptococcus thermophilus in the laboratory to a titratable acidity (TA) of .09%. Each yogurt was seeded with one of three strains of Listeria monocytogenes at two levels and survival was monitored at 1–7, 14, 21 and 28 days during storage at 4°C. All strains survived longer in the skim milk/high solids yogurts which had a higher pH than the whole milk/low solids yogurt.
To determine the effects of pH, solids and fat, simulated yogurts, prepared by acidifying milk preparations to pH 4.2, 4.1 and 4.0, were inoculated with L. monocytogenes. Survival was affected by differences in pH and solids content and strain L. monocytogenes while fat content had no apparent effect .     

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.     

TEMPERATURE SHIFT EFFECTS ON INJURY AND DEATH IN LISTERIA MONOCYTOGENES SCOTT A

Exposure of Listeria monocytogenes Scott A grown at 37°C to a 1 h heat treatment at 52°C resulted in little death of the cells (< 0.5 log). However, as the temperature of growth decreased, there was an increase in the extent of death (> 4 logs at 10°C growth temperature). Heat induced injury, however, decreased as the growth temperature decreased. Shifting L. monocytogenes grown at 10, 19, or 28°C to 37°C for periods up to 5 h led to cells with increased heat tolerance. However, there was little effect on injury by the shift-up procedure. Presence of chloramphenicol during the shift-up period inhibited the gain in heat tolerance . L. monocytogenes grown at low temperatures (< 28°C), were more susceptible to killing by heat, but this susceptibility could be lost if cells grown at low temperatures are given a short incubation at 37°C. The data obtained here suggest that if foods containing L. monocytogenes are temperature-abused for even short periods, the organisms will acquire an increased heat tolerance and will require higher inactivation temperatures or longer processing times .     

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.