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) .     

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 .     

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.     

THE EFFECT OF AEROBIC MESOPHILIC MICROFLORAL LEVELS ON THE ISOLATION OF INOCULATED LISTERIA MONOCYTOGENES STRAIN LM82 FROM SELECTED FOODS

The effect of aerobic mesophilic microfloral concentration on the isolation of Listeria monocytogenes LM82 was studied in 31 (18 cheeses and 7 noncheese) retail foods having standard plate counts of 10¹ to 10⁸ colony forming units (CFU)/g. Foods were spiked with L. monocytogenes and enriched at 30°C for 24 h in a selective enrichment broth used in a U.S. Food and Drug Administration method. Inoculum levels for isolation on modified McBride agar ranged from 0.1 to > 5 × 10³ with a geometric mean value of 5 inoculated CFU/g or 1.4 CFU/g. Pure Enterococcus (Streptococcus) faecalis ( 0 to 6 × 10⁶ inoculated CFU/mL ) in the absence of food matrix had no effect on the enrichment of L. monocytogenes. Ease of isolation of LM82 was independent of the food microflora concentration both generally and in the specific food type of 9 Brie cheeses. Competition, when it occurs, therefore, may be due to specific bacterial competitors rather than bacterial numbers .     

A COMPARISON OF MICROBIOLOGICAL AND RADIOIMMUNOASSAY METHODS FOR THE DETERMINATION OF PANTOTHENIC ACID IN FOODS

Seventy-five foods were analyzed for the vitamin pantothenic acid using a microbiological assay and a new radioimmunoassay (RIA). The food sample extract used in both assays was the result of dialysis after enzyme hydrolysis. In the method used previously, the food-enzyme mixture had been filtered.
A very high correlation (r²=.94) between the results from the RIA and the microbiological assay was found. There was a statistically significant difference between the two assay results for all foods and for the subgroups meats, breads and cereals, and fruits and vegetables at p=.05. At p = .01 breads and cereals and fruits and vegetables did not have significantly different results between the two assay methods. For all foods and all subgroups, the microbiological assay produced a higher mean result than the RIA.
The RIA is an acceptable method for assaying pantothenic acid in breads and cereals and fruits and vegetables. Futher study is needed to determine how components of meat interact with one or both assay systems.     

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.     

PREVALENCE AND RESISTANCE TO ANTIBIOTICS FOR AEROMONAS SPECIES ISOLATED FROM RETAIL FISH IN TURKEY

Mesophilic aeromonads are among the most common bacteria in water habitats throughout the world, and these bacteria frequently cause disease in fishes. They are also causative agents of acute diarrheal disease in man following ingestion water. In this study, a total of 132 market fish (64 freshwater and 68 seawater) samples representing were collected in Ankara (Turkey) and investigated for the presence of Aeromonas spp. They were isolated from 106 (80.3%) out of the 132 fish samples tested. The distribution of Aeromonas spp. varied depending on the samples (gill, intestine, liver, kidney) examined. In freshwater samples, the predominant species was A. caviae (66.0%), followed by A. hydrophila (22.6%) and A. veroni bv. sobria (11.6%). In seawater samples, the predominant species was found A. veroni bv. sobria (41.5%), followed by A. hydrophila (30.1%) and A. caviae (28.3%). The 132 isolated Aeromonas spp. strains were further examined for hemolytic, lipolytic and proteolytic activity. Of the present isolates, more than 80% (A. veroni bv. sobria, A. hydrophila) were positive for hemolysin activities. Lipolytic and proteolytic activity of identified strains were found in more lower incidence. All aeromonads (A. hydrophila, A. veroni bv. sobria, A. caviae) strains showed resistance to ampicillin, cephalothin and trimethoprim. The least resistance was found for chloramphenicol (9.0%). In contrast, all the strains were susceptible to ciprofloxacin and ceftriaxone. In Turkey, there have been few studies on Aeromonas and its virulence factors. This study therefore highlights an important incidence of Aeromonas spp., with virulence and antibiotic resistance, isolated from fish intended for human consumption in Turkey.     

COMPARISON OF RECOVERY METHODS FOR CLOSTRIDIUM PERFRINGENS FROM SELECTED FOODS1, 2

Ground beef, extruded soy protein (ESP), and ESP extended ground beef were analyzed for Clostridium perfringens by several isolation techniques. C. perfringens was isolated by one or more of nine procedures from 68% of all units analyzed. One or more of the four isolation temperatures when samples were blended and one or more of the four temperatures when samples were not blended detected C. perfringens in 60 and 59% of the units, respectively. Incubation of fluid thioglycollate media (FTM) at 37 and 46°C resulted in a significantly higher isolation rate than all other methods, with blended samples incubated at 46°C being the most practical. Heat shocking samples at 75 and 95°C yielded only two isolates not isolated at 37 or 46°C. Direct inoculation of sulfite polymyxin sulfadiazine agar pour plates showed only 40% of all units analyzed contained C. perfringens. C. perfringens was isolated from 85, 23, and 94% of the ground beef, ESP, and ESP extended ground beef units, respectively.     

DETERMINATION OF THE EFFECTIVENESS OF NOVOBIOCIN ADDED TO TWO AGAR PLATING MEDIA FOR THE ISOLATION OF SALMONELLA FROM FRESH MEAT PRODUCTS

Five plating media, Hektoen enteric (HE) and xylose lysine deoxycholate (XLD) agars with and without 80 and 5 μg/ml of novobiocin (N), respectively, and brilliant greeen sulfadiazine (BGS) agar with 80 μg/ml of the antimicrobial agent, were analyzed for the recovery of salmonellae from various fresh beef, pork, and poultry meat products. Of the total Samonella positive samples, 50.0% and 82.5% were found on XLD and XLD-N agars, respectively, 75.0% and 85.0% on HE and HE-N agars, respectively and 65.0% on BGS agar. HE-N and BGS media isolated three times more false positives than did XLD-N agar, while XLD and HE agars gave the highest numbers of false positives. The major H₂S producing false positive on XLD and HE agars was Proteus mirabilis. With the addition of N, P. mirabilis was eliminated, and the major H₂S producing false positive was almost exclusively Citrobacter freundii. The false positives on BGS agar were predominately distributed among C. freundii, Enterobacter sp., and Klebsiella sp.     

COMPARISON OF FORCE-DEFORMATION CHARACTERISTICS OF ARTIFICIAL AND SEVERAL NATURAL FOODS FOR CHEWING EXPERIMENTS

The masticatory performance of an individual has been quantified by determining the particle sizes of the comminuted food. Optosil, a silicon rubber, has been used as an artificial test food in clinical studies, because it has reproducible textural properties. A comparison between Optosil and several natural foods was made by measuring textural properties of Optosil, turnip, carrot, Gouda cheese and peanut. Several types of probes were used to imitate the cusp form of the teeth. Force-deformation plots revealed similar maximum firmness for Optosil, turnip and carrot, whereas peanut showed a larger firmness. The force needed to crush Optosil is much larger than for the natural foods, but it is well within the physiological range of healthy subjects. The reproducibility of the textural properties is better for Optosil than for the natural foods. The particle size distribution of Optosil broken by a testing machine was compared with the results obtained during chewing.     

ISOLATION AND CHARACTERIZATION OF ICE STRUCTURING PROTEINS FROM COLD-ACCLIMATED WINTER WHEAT GRASS EXTRACT FOR RECRYSTALLIZATION INHIBITION IN FROZEN FOODS

Antifreeze proteins have shown a great potential in improving the quality of frozen foods, and the isolation of proteins from natural sources that are readily available is deemed to be important. The raw leaf apoplastic extract from cold‐acclimated winter wheat grass ( Triticum aestivum ) containing recrystallization inhibition (RI) proteins was screened for proteolytic and lipolytic activity, and a heat‐stable RI protein was isolated. The enzymes detected could be easily removed by heat treatment without any loss in the RI activity. The RI protein was isolated after heat treatment of the raw extract, alcohol precipitation and separation on a size exclusion chromatographic column. Circular dichroism indicated that the RI protein was mainly β‐sheet and random coil, and these structures were preserved up to 75C. After trypsin digestion, mass fingerprinting and sequencing of the digests revealed that the protein belongs to the thaumatin‐like protein group.     

COMPARISON OF MULLER-KAUFFMANN'S TETRATHIONATE BROTH AND MODIFIED RAPPAPORT'S MEDIUM FOR ISOLATION OF SALMONELLA

Results obtained with tetrathionate broth (TBB) using the standard method and Rappaport-Vassiliadis medium (RV) did not differ markedly with respect to the proportion of positive samples or the sero- and phage types of salmonellae isolated from minced beef and pork. TBB, based on the commercial dehydrated medium of Oxoid (CM 343), gave fewer positive samples while that of Merck (Art. No. 5172) was equivalent to RV. In individual experiments results were sometimes markedly different. Results obtained for reference samples of minced meat, with salmonellae added, demonstrated that the chances of detecting salmonellae increase with increasing numbers present initially. At the same time it was also demonstrated that the presence of minced meat and its competing microflora have an unpredictable effect on the detection of salmonellae.