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.     

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.     

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.     

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.     

A MODEL FOR PREDICTING THE GROWTH OF LISTERIA MONOCYTOGENES IN PACKAGED WHOLE MILK

Experiments were conducted to determine growth characteristics of Listeria monocytogenes in sterilized whole milk at nine temperatures in the range of 277.15 to 308.15K (4 to 35C). Based on these data, the parameter values of the Baranyi dynamic growth model were statistically determined. Finite element software, ANSYS, was used to determine temperature distributions in milk cartons subject to a time‐varying ambient temperature profile. The space‐time‐temperature data were input to the Baranyi dynamic growth model, to predict the microbial population density distribution and the average population density in the milk carton. The Baranyi dynamic growth model and the finite element model were integrated and validated using experimental results from inoculated sterilized whole milk in half‐gallon laminated paper cartons. In all experiments, the milk cartons were subjected to the same temperature profile as the Baranyi dynamic growth model. Experimental microbial counts were within predicted upper and lower bounds obtained using the integrated Baranyi dynamic growth and finite element models. In addition, the growth curve at the mean value of initial physiological state parameter for L. monocytogenes underpredicted the microbial growth (standard error = 0.54 log (cfu/mL) and maximum relative difference = 15.49%).     

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.     

BEHAVIOR OF LISTERIA MONOCYTOGENES IN RECONSTITUTED INFANT CEREALS

Listeria monocytogenes may contaminate and grow in reconstituted infant cereals refrigerated or temperature abused before consumption. To assess this risk, a 5-strain composite of the pathogen was inoculated in rice, oatmeal, and wheat-rice-oatmeal infant cereals, hydrated (1/6, w/v) with apple juice, pasteurized milk (2% fat) or water, and stored at 4, 15, or 25C for 0, 8 and 24 h to simulate potential advance home preparation and abuse. L. monocytogenes survived with minimal (<0.6 log cfu per mL) or no growth in all cereal preparations at 4C, but it increased in cereals hydrated with water or milk by 2.4–3.3 and 5.3–6.4 log cfu per mL in 24 h at 15 and 25C, respectively. Reconstitution of cereals with water or milk supported growth (P < 0.05) even at 8 h of abuse. In cereals hydrated with apple juice and stored at 15 or 25C, populations of L. monocytogenes were maintained below 3.0 log cfu per mL at 24 h. These results indicate that refrigeration and/or reconstitution with an acid fruit juice may inhibit growth of L. monocytogenes in infant cereal meal preparations. However, in order to ensure prevention of growth, such meals should preferably be consumed soon after preparation or held at 4C for less than 8 h.     

PREVALENCE OF LISTERIA MONOCYTOGENES IN SOME TURKISH FOODSTUFFS

In this study, the presence of Listeria monocytogenes was evaluated in basic groups of foods in Ankara, Turkey between 2001 and 2002. Eighty prepared ground meat, 100 white cheese and 100 parsley samples were obtained from different markets where they were offered for consumption. L. monocytogenes was detected according to the method of ISO Draft International Standard. Fraser Broth was used for enrichment process, and isolation of Listeria spp. was carried out by Oxford Agar. L. monocytogenes was then identified by biochemical and serological tests. L. monocytogenes was isolated from 13.75, 4 and 2% of 80 prepared ground beef, 100 white cheese and 100 parsley samples, respectively. The results indicate that these foodstuffs have risk of L. monocytogenes. Contamination of L. monocytogenes was not detected in packaged ground beef and white cheese samples. High incidence rate of L. monocytogenes was noted for the samples from open and local market.     

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 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.     

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。     

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.     

THE INCIDENCE AND LEVEL OF LISTERIA SPP. AND LISTERIA MONOCYTOGENES CONTAMINATION IN PROCESSED POULTRY AT A POULTRY PROCESSING PLANT

To estimate the incidence and levels of Listeria spp. in an industrial poultry processing plant, samples of chicken breast meat, livers, surfaces of saws and tables, hands and gloves were analyzed. Forty percent of the breast samples presented Listeria: L. monocytogenes, L. innocua and L. grayi. Liver samples were contaminated by L. innocua and L. grayi. High levels of Listeria monocytogenes were found on saws (<30–2400 MPN/equipment) and tables (<30–11,000 MPN/equipment). Hands were contaminated by L. monocytogenes, L. innocua and L. grayi and gloves with L. innocua and L. grayi. The levels of Listeria on hands and gloves were low (<110 MPN/hand). L. monocytogenes serotypes were 1/2a, 1/2b, 1/2c and 4b. Overall, the study demonstrated the high prevalence of Listeria spp. and specifically L. monocytogenes in chicken breast meat, equipment and hands. Improvements and innovations at the poultry processing plant may effectively reduce final production contamination with Listeria.     

INFLUENCE OF FOOD ENVIRONMENT ON LISTERIA MONOCYTOGENES INFECTION IN THE GUINEA PIG MODEL

Listeria monocytogenes is a widespread foodborne pathogen associated with severe illness in humans. Food composition, processing, storage, distribution and handling conditions are all factors that may individually or collectively contribute to the virulence of a pathogen. Using the guinea pig as a human surrogate, we investigated the impact of carrier vehicle and improper food handling practices on the infectivity of L. monocytogenes. Chocolate milk was artificially contaminated with L. monocytogenes prior to exposure to improper handling conditions. Improper handling of chocolate milk included leaving the milk at an elevated temperature (37C) and then placing it in a refrigerator (4C). Guinea pigs were orally challenged with either 10² or 10⁸ cfu of L. monocytogenes in 1 mL of nutrient broth or chocolate milk that had been exposed to improper handling conditions. A third group was challenged with L. monocytogenes suspended in water. On days 2 and 4, upon enrichment of organ samples, the presence of L. monocytogenes was detected in 27% of the animals receiving the low dose regardless of the carrier vehicle and improper handling conditions. Animals fed the high dose (10⁸ cfu) of L. monocytogenes had similar populations of the pathogen in the spleen and liver, regardless of the carrier vehicle. These results are significant in that a low dose (10² cfu) could lead to listerial infection. Under the conditions tested in this study, the carrier vehicle and exposure of the pathogen to improper handling practices had no detectable effect on the establishment of listerial infection in the guinea pig model.     

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.     

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 .     

INFLUENCE OF FAT CONTENT AND PRESERVATIVES ON THE BEHAVIOR OF LISTERIA MONOCYTOGENES IN BEAKER SAUSAGE

Behavior of Listeria monocytogenes Scott A in pork liver sausage containing 22–67% fat, and antilisterial activities of sodium lactate, sorbic acid, potassium sorbate and sodium propionate were studied during storage at 4C and 10C. Commercial pork liver sausage batter (22% fat), alone and with additions of lard (15, 30, and 45% by weight) were tested. Concentrations of 1.8% sodium lactate, 0.1% sorbate as the acid or the potassium salt, and 0.2% sodium propionate were tested in heat sterilized sausage inoculated with a 24 h culture of the organism (10⁴ CFU/g). Fat content alone caused small reductions in cell numbers by the end of the storage periods: from log CFU/g of 9.9 to 9.4 after 14 days at 10C, and from 7.3 to 6.5 after 50 days at 4C in the basic sausage formulation and with 45% added fat, respectively. The inhibitory activities of lactate and propionate increased with increase in fat content, and were more pronounced at 4C, where the effects were listericidal. Inhibition by sorbic acid was least influenced by the fat content, and the potassium salt was less antilisterial than the acid.