Isolation from food sources, of lactic acid bacteria that produced antimicrobials

The potential of lactic acid bacteria, isolated from a variety of foods, to inhibit indicators representative of spoilage and pathogenic bacteria associated with food products was examined. Fruit and vegetables were a poor source of lactic acid bacteria but large numbers were readily isolated on MRS agar from cheese, milk and meat samples. Approximately 1000 isolates from each of the food samples were examined by the deferred antagonism procedure to determine their ability to inhibit Staphylococcus aureus, Listeria innocua and Pseudomonas fragi. Listeria innocua was the bacterium predominantly inhibited by isolates from the cheese, milk and meats, but antagonism was also observed to a lesser extent against the other indicators. The only inhibition observed for isolates from vegetable material was directed against Staph. aureus. The majority of inhibitor producers were effective against only one of the indicators but a small number were isolated which inhibited two or three.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods.

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Evaluation of selective direct plating media for their suitability to recover uninjured, heat-injured, and freeze-injured Listeria monocytogenes from foods

Six direct plating media were evaluated for their suitability to recover uninjured, heat-injured, and freeze-injured cells of four strains of Listeria monocytogenes from four foods. Cells were inoculated into foods to achieve ca. 10(2) to 10(3), 10(4) to 10(5), or 10(5) to 10(6) viable cells per ml or g (low, medium, and high populations, respectively). No appreciable differences in recovery of the four test strains within a treatment were observed. Generally, recovery on all test media was similar and not markedly affected by freeze treatment. Modified Despierres agar and modified McBride Listeria agar yielded poorer recovery of heat-injured cells than did McBride Listeria agar and gum base-nalidixic acid-tryptone soya agar. Overall, gum base-nalidixic acid-tryptone soya agar was best for recovering L. monocytogenes from pasteurized milk and chocolate ice cream mix. Enumeration was complicated by the growth of background microflora present in Brie cheese and cabbage, especially at the low inoculum. Dominguez Rodriguez isolation agar was superior for recovering L. monocytogenes from Brie cheese, whereas modified Despierres agar was best for recovering the organism from cabbage. Direct plating procedures can successfully be utilized for recovering healthy and injured L. monocytogenes from foods containing low populations of background microflora.     

Quantitative isolation efficiency of O26, O103, O111, O145 and O157 STEC serotypes from artificially contaminated food and cattle faeces samples using a new isolation protocol

Aims:  A range of new differential and confirmation plating media for some non-O157 Shiga toxin producing Escherichia coli (STEC) serotypes (O26, O103, O111, O145) and both sorbitol-positive and -negative O157 were evaluated using artificially contaminated samples.
Methods and Results:  Dairy products (raw milk, cheese made from pasteurized milk and raw milk), meat (ground beef, fermented meat) and cattle faeces were artificially contaminated using clinical STEC strains. Isolation efficiency was 100%, 82·3%, 88·5%, 65·9%, 64·3% and 15·8%, respectively, for an inoculum size of ≤100 CFU 25 g⁻¹. The consecutive use of differential and confirmation media limited the incidence of false positive isolates from 0% for raw milk samples, cheese made from pasteurized milk and for fermented meat to 2·1% for cheese made from raw milk, and to 8·9% for ground beef.
Conclusions:  Data presented in this paper indicated that the efficiency of the applied isolation method was dependent on sample-to-sample variation but not on the inoculum size.
Significance and Impact of Study:  Data in this paper indicated that isolation of low levels of non-O157 and sorbitol-positive O157 STEC from food samples is possible.     

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity

AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.     

Evaluation of four protocols for the detection and isolation of thermophilic Campylobacter from different matrices

Aims: To identify the optimal method for detection of thermophilic Campylobacter at various stages in the food chain, three culture‐dependent (direct plating, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n = 38), neck skin (n = 38) and packed fresh meat (n = 38). Methods and Results: Direct plating was compared to enrichment with either Bolton broth (ISO 10272:2006‐1) or Preston broth, followed by culture on two selective agars: modified charcoal cefoperazone desoxycholate agar (mCCDA) and Campyfood agar (CFA). Direct plating on CFA provided the highest number of positive samples for faeces and neck skin samples. Enrichment of meat samples in Preston followed by plating on mCCDA gave significantly higher number of positives than the recommended ISO method. Real‐time qPCR yielded the highest number of positive samples. Conclusion: Direct plating on CFA is optimal for Campylobacter isolation from highly contaminated samples such as faeces or neck skin. When enrichment is required for less‐contaminated samples such as poultry meat, Preston broth is the best choice. The maximum of detectable cells predicted by qPCR is a sensitive and powerful evaluation tool. Significance and impact of the study: The recommended ISO protocol had the least sensitivity, and application of this method could result in underreporting. We detected a high prevalence of Campylobacter on packed meat to be distributed, which suggests this is still a significant risk for consumers.     

Sensitive in situ monitoring of a recombinant bioluminescent Yersinia enterocolitica reporter mutant in real time on Camembert cheese

Bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promoterless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens, and their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10 degrees C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm(2). Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a "real-product" status, and at a low temperature.     

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.     

Comparison of lithium chloride-phenylethanol-moxalactam and modified Vogel Johnson agars for detection of Listeria spp. in retail-level meats, poultry, and seafood.

The effectiveness of Modified Vogel Johnson agar and lithium chloride-phenylethanol-moxalactam agar for detection of Listeria spp. in foods was compared by using the media to analyze retail-level meat, poultry, and seafood both by direct plating and in conjunction with a three-tube most-probable-number enrichment. The most-probable-number protocol detected Listeria species, including Listeria monocytogenes, in a substantial portion of the fresh meat and seafood samples. In most instances the Listeria levels were less than 2 CFU/g, which precluded detection by direct plating. Modified Vogel Johnson agar performed as well as did lithium chloride-phenylethanol-moxalactam agar and was considerably easier to use because of its ability to differentiate Listeria spp. from other microorganisms.     

The clones of Listeria monocytogenes detected in food depend on the method used

S. LONCAREVIC, W. THAM AND M.-L. DANIELSSON-THAM. 1996. Restriction enzyme analysis (REA) with pulsed-field gel electrophoresis (PFGE) has been used to characterize and compare Listeria monocytogenes strains isolated from foods by two methods, an enrichment procedure and a direct plating procedure. In total 151 isolates from nine foods were investigated. In six of the foods (101 strains investigated) only one clone of L. monocytogenes was found irrespective of the method used. In three foods (50 strains investigated) the direct plating procedure yielded more clones than the enrichment procedure. At the most, five clones were detected in the same food. The results presented here indicate that direct plating from the food reveals more L. monocytogenes clones than revealed by an enrichment procedure.     

Virulent Bacteriophage for Efficient Biocontrol of Listeria monocytogenes in Ready-To-Eat Foods

Food-borne Listeria monocytogenes is a serious threat to human health, and new strategies to combat this opportunistic pathogen in foods are needed. Bacteriophages are natural enemies of bacteria and are suitable candidates for the environmentally friendly biocontrol of these pathogens. In a comprehensive set of experiments, we have evaluated the virulent, broad-host-range phages A511 and P100 for control of L. monocytogenes strains Scott A (serovar 4b) and WSLC 1001 (serovar 1/2a) in different ready-to-eat (RTE) foods known to frequently carry the pathogen. Food samples were spiked with bacteria (1 × 10³ CFU/g), phage added thereafter (3 × 10⁶ to 3 × 10⁸ PFU/g), and samples stored at 6°C for 6 days. In liquid foods, such as chocolate milk and mozzarella cheese brine, bacterial counts rapidly dropped below the level of direct detection. On solid foods (hot dogs, sliced turkey meat, smoked salmon, seafood, sliced cabbage, and lettuce leaves), phages could reduce bacterial counts by up to 5 log units. Variation of the experimental conditions (extended storage over 13 days or storage at 20°C) yielded similar results. In general, the application of more phage particles (3 × 10⁸ PFU/g) was more effective than lower doses. The added phages retained most of their infectivity during storage in foods of animal origin, whereas plant material caused inactivation by more than 1 log₁₀. In conclusion, our data demonstrate that virulent broad-host-range phages, such as A511 and P100, can be very effective for specific biocontrol of L. monocytogenes in contamination-sensitive RTE foods.     

A new method for direct detection of Listeria monocytogenes from foods by PCR.

The preparation of DNA by alcohol precipitation in the presence of Na1 was used to enable the direct detection of Listeria monocytogenes in the amount of 10(3) CFU/0.5 g of sample. This procedure produces PCR-quality DNA directly from foods, such as soft cheese and minced meat.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

DETECTION OF LISTERIA IN FOOD AND ENVIRONMENTAL SAMPLES BY IMMUNOMAGNETIC BEAD CAPTURE AND BY CULTURAL METHODS

Detection of Listeria by capture on antibody-coated magnetic beads has been shown to decrease test time and improve sensitivity, relative to cultural methods, in a study of spiked environmental samples (Mitchell et al. 1993). In this study, immunomagnetic capture was compared to standard cultural methods for detection of Listeria in a broad range of spiked and naturally contaminated food and environmental samples. Immunomagnetic capture was at least as sensitive as cultural methods for detection of Listeria in seafood, meats, dairy foods, and environmental samples. It was possible to determine the number of Listeria present in a sample, because immunomagnetic capture was carried out directly from the sample, without enrichment. These quantitative results were produced within 24 h, while cultural methods required 6–14 days to produce a qualitative result. Immunomagnetic capture was thus more rapid and as sensitive as standard cultural methods for detection of Listeria in the food and environmental samples tested.     

Prevalence and lineages of Listeria monocytogenes in Chinese food products

AIMS: This study was undertaken to investigate the prevalence and lineages of Listeria monocytogenes in different kinds of food products in local Chinese markets. METHODS AND RESULTS: A total of 2686 food samples and 645 water samples were collected and L. monocytogenes was isolated from 2.28% (76 of 3331) of all samples. The prevalence of L. monocytogenes (14 of 290, 4.83%) in raw meat products was significantly higher than that in other raw food products (P < 0.05). Among 844 ready-to-eat (RTE) food samples, 21 samples were positive for L. monocytogenes. RTE packaged food products from two supermarkets had a prevalence ranging from 0.00% to 25.00%. The prevalence of L. monocytogenes in meat products of freshly slaughtered hogs was 0.95% (four of 420), significantly lower than that in raw meat products in the retail markets (P < 0.05). Ten isolates were recovered from 645 water samples, which were collected after hands washing by shopkeepers or waiters. A total of 38 isolates were randomly selected for lineage classification based on the nucleotide variation of actA gene. Eighty percentage of isolates from RTE food products belonged to Lineage II while only 20% belonged to Lineage I. CONCLUSIONS: Food products in Chinese markets are contaminated with L. monocytogenes. Raw meat products have the highest contamination rates among all the raw food samples. RTE food products are more likely to be contaminated with Lineage II strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here show the main contamination sources of L. monocytogenes in Chinese food products.     

Automated measurements of antilisterial activities of lactate and diacetate in ready-to-eat meat

Standard procedures to enumerate Listeria organisms rely on plating food samples on selective agar media. The procedures are labor-intensive, and because of the limited sensitivity, pre-enrichment step is required for the detection of low numbers of the pathogen. In the present study, an automated rapid optic procedure and the standard procedure were used to determine the behavior of the pathogen in ready-to-eat (RTE) meat and to test the effect of antilisterial agents. Listeria monocytogenes strain Scott A or a six-strain mixture of Listeria was studied using lactate (2.5%), diacetate (0.2%) and their combination in beef bologna and in sterile beef emulsion. Samples stored for up to 60 days at 5 and 10 degrees C were tested at time intervals during storage. Using the plate count method, each of the salts caused a delay in growth of the pathogen, and the salt combination was most effective causing listeriostatic effects and decline in growth of the pathogen at 5 degrees C. High negative correlation (r), ranging from 0.92 to 0.99, was obtained between the detection time (DT) recorded by the optic procedure (BioSys instrument) and cell numbers determined by the plate count procedure. The rapid (< 24 h) optic procedure was reliable in assessing the efficacy of antimicrobials and in rapid detection of low levels of listeriae that are undetectable by direct plating procedure.     

Evaluation of ALOA plating medium for its suitability to recover high pressure-injured Listeria monocytogenes from ground chicken meat

AIMS: To evaluate a chromogenic plating medium for the isolation of sublethally injured cells of Listeria monocytogenes from processed foods. METHODS AND RESULTS: The inactivation of L. monocytogenes at pressures up to 400 MPa and 12 degrees C in ground chicken meat was employed to examine the recovery of high-pressure injured cells. Before and after different repair incubation periods at 30 degrees C in a nonselective broth, samples were plated onto a selective and differential agar [Agar Listeria according to Ottaviani and Agosti (ALOA)] and in the same medium supplemented with 4% sodium chloride (ALOA-S), and incubated at 37 degrees C. Sublethally injured cells were able to grow when directly plated onto the ALOA medium, without a previous repair incubation period. However, only uninjured cells grew on the ALOA-S medium. CONCLUSIONS: Sublethally injured cells of L. monocytogenes can be quantified by subtracting counts on ALOA-S medium from counts on ALOA medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Possible applications include direct enumeration on ALOA of stressed cells of L. monocytogenes in foods with more than 100 colony forming units per gram.     

Rapid detection of low levels of Listeria in foods and next-day confirmation of L. monocytogenes

Outbreaks of foodborne listeriosis caused by Listeria monocytogenes in recent years, and the high mortality rate associated with listeriosis, have raised the need for reliable and rapid detection of the pathogen. A simple, automated method was developed for the detection of Listeria organisms in foods. It consists of a 6-h pre-enrichment step followed by overnight incubation in selective broth at 35 degrees C. Changes in light transmittance in the selective broth are registered continuously by an optical sensor of the BioSys instrument (MicroSys, Ann Arbor, MI), and recorded in the computer. Esculin hydrolysis by listeriae results in black coloration of the media that causes a sharp drop in light transmittance, whereas negative samples remain colorless. Confirmation of L. monocytogenes is carried out only on esculin-positive samples and is completed within 6 h. Detection of 10-50 cells of Listeria inoculated into 25 g of food was confirmed in shell eggs, milk and ground beef. Naturally contaminated raw and ready-to-eat foods were further screened to validate the procedure.     

Production of monoclonal antibody against Listeria monocytogenes and its application to immunochromatography strip test

An immunochromatography (ICG) strip test based on a monoclonal antibody for the rapid detection of L. monocytogenes in meat and processed-meat samples was developed in this study. A monoclonal antibody (MAb) specific to L. monocytogenes was produced from cloned hybridoma cells (FKLM-3B12-37) and used to develop an ICG strip test. The antibody showed a stronger binding to L. monocytogenes than other Listeria species, and a weak cross-reaction to S. aureus based on an ELISA. The detection limit of the ICG strip test was 10(5) cell/ml. In total, 116 meat and processed-meat samples were collected and analyzed using both the ICG strip test and a PCR. The ICG strip test and PCR indicated L. monocytogenes contamination in 34 and 27 meat samples, respectively. The 7 meat samples not identified as L. monocytogenes positive by the PCR were also tested using an API kit and found to be contaminated by Listeria species. In conclusion, the ICG strip test results agreed well with those obtained using the PCR and API kit. Thus, the developed ICG has potential use as a primary screening tool for L. monocytogenes in various foods and agricultural products, generating results within 20 min without complicated steps.     

Use of polymerase chain reaction for detection of Listeria monocytogenes in food.

A previously described polymerase chain reaction (PCR) assay (B. Furrer, U. Candrian, C. H?felein, and J. Lüthy, J. Appl. Bacteriol. 70:372-379, 1991) was used to analyze food for the presence of Listeria monocytogenes. Food samples were artificially contaminated to develop two procedures to detect the organism following enrichment steps. Procedure A was based on dilution of the enrichment broth followed by lysis of the bacteria and direct analysis of the lysate with PCR. With procedure A and artificially contaminated food samples, it was possible to detect fewer than 10 bacteria per 10 g of food. In procedure B, centrifugation was used to concentrate bacteria before lysis and PCR. With procedure A, 330 naturally contaminated food samples of several types were analyzed. Twenty samples were found to be positive for L. monocytogenes, which was in agreement with the classical culture technique. By using procedure B on a subset of 100 food samples, 14 were found to be positive by PCR whereas the classical culture method detected only 13. Analysis times, including enrichment steps, were 56 and 32 h with procedures A and B, respectively.