Development of a surface adhesion immunofluorescent technique for the rapid isolation of Listeria monocytogenes and Listeria innocua from meat

The use of a novel surface adhesion technique to isolate Listeria monocytogenes and Listeria innocua from an enrichment meat system was developed. Minced beef samples inoculated with L. monocytogenes (10 cfu g(-1)) were incubated at 30 degrees C for 14-18 h in a suitable enrichment broth. Listeria monocytogenes cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane which was attached to a glass microscope slide. The Listeria cells on the membrane were subsequently visualized using an immunofluorescent microscopy procedure. The antibody used in this technique reacts with L. monocytogenes and L. innocua. The technique was demonstrated to have a detection level of log10 3.11 cfu ml(-1). There was excellent correlation (r2 = 0.98) between the counts obtained by this surface adhesion immunofluorescent (SAIF) technique and counts obtained using traditional methods, i.e. plate counts on PALCAM. When the regression equation relating the rapid and standard methods was validated using the data from 50 retail beef mince samples, an rsd value of +/- 0.25 was obtained. No false-negative or false-positive results were recorded for L. monocytogenes or L. innocua species using the SAIF technique.     

RAPID DETECTION OF SALMONELLA TYPHIMURIUM IN FOOD SAMPLES USING A BIENZYME ELECTROCHEMICAL BIOSENSOR WITH FLOW INJECTION

A bienzyme (tyrosinase and horseradish peroxidase) electrochemical biosensor was developed for detection of Salmonella typhimurium, and evaluated for application in a flow injection system coupled with immunomagnetic separation for food samples. Parameters for immunomagnetic separation, enzymatic reaction, flow injection and electrochemical detection were determined using pure culture samples. The selectivity was tested in the presence of Listeria monocytogenes, Campylobacter jejuni and E. coli 0157:H7. The results showed a linear relationship for logarithmic values between peak current ratio and the cell number of S. typhimurium in the range of 10³ 10⁵ cfu/mL, with R²= 0.99. The detection limit of this method was 1.09 × 10³ cfu/mL for S. typhimurium and the detection time was 2.5 h. Samples of chicken carcass wash water and ground beef were used to evaluate the biosensor. The results demonstrated that this biosensor has a potential for rapid detection of different pathogens in various food samples.     

Thermal inactivation of Listeria monocytogenes during rapid and slow heating in sous vide cooked beef

T.B. HANSEN AND S. KNØCHEL. 1996. Heating at slowly rising temperatures is suspected to enhance thermotolerance in Listeria monocytogenes and, since anaerobic environments have been shown to facilitate resuscitation of heat-injured cells of this micro-organism, concern may arise about the possibility of L. monocytogenes surviving in minimally preserved products. The effect of rapid (> 10°C min^-1) and slow (0.3 and 0.6°C min^-1) heating on survival of L. monocytogenes in sous vide cooked beef was therefore examined at mild processing temperatures of 56, 60 and 64°C. No statistically significant difference ( P = 0.70) was observed between the tested heating regimes. Since the average pH of beef was low (5.6), and little or no effect was observed, a pH-dependency of heat shock-induced thermotolerance in L. monocytogenes is suggested to account for this result.     

Specific detection of cytopathogenic Listeria monocytogenes using a two-step method of immunoseparation and cytotoxicity analysis

The development of rapid methods for detection of viable Listeria monocytogenes is crucial to prevent listeriosis and product recalls. While immunomagnetic separation has been used for isolating Listeria spp., lack of specificity and pathogenicity determination render this method unsatisfactory. A two-step method using Protein A agarose beads (Immunobeads) coated with a more specific antibody, monoclonal antibody (MAb)-C11E9 for L. monocytogenes was developed. Immunobeads were allowed to capture Listeria cells from a variety of samples and tested for cytopathogenic action on a murine hybridoma B-lymphocyte, Ped-2E9 cell line by Trypan blue staining, and by an alkaline phosphatase (AP)-based cytotoxicity assay. The two-step method was used to test uninoculated hotdogs, bologna, and raw beef, chicken, and pork samples, following selective enrichment in half-Fraser broth. Pure culture studies proved the assay to be specific for L. monocytogenes, while a similar assay with Dynal Anti-Listeria immunomagnetic beads was positive for L. monocytogenes, L. ivanovii, and L. seeligeri. Detection and confirmation of cytopathogenicity of Listeria cells from food samples after 24-h selective enrichment were completed in 2-4 h. Isolates were further analyzed by the CAMP test for hemolytic activity and RiboPrinter for genomic patterns. Using immunoseparation and cytotoxicity as a two-step rapid method, viable L. monocytogenes could be isolated, detected, and confirmed as cytopathogenic in 28 h or less.     

Rapid detection of Listeria species by selective impedimetric assay

A selective medium was developed for the detection of Listeria monocytogenes, L. innocua and L. welshimeri by impedimetric assay. Addition of 21 mg l^-1 proflavine hydrochloride and 20 mg l^-1 moxalactam to glucose-enriched nutrient broth suppressed the impedimetric responses of Gram-negative and other Gram-positive test micro-organisms, permitting the quantitative detection of the three Listeria species within 36 h.     

Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese

The polymerase chain reaction (PCR) amplification technique was investigated as a tool for direct detection of Listeria monocytogenes in soft cheeses. Different sets of oligonucleotide primers were used, and parts of the L. monocytogenes Dth18-gene could be amplified specifically when either a plasmid vector carrying the cloned gene or chromosomal DNA was used as a template. The detection limit for L. monocytogenes in dilutions of pure cultures was between 1 and 10 colony-forming units. In extracts from soft cheeses containing L. monocytogenes DNA, the amplification was strongly inhibited. This inhibition could be reduced by an additional purification step. Despite this the detection limit showed a large variation, depending on the brand of cheese used. In some cheeses 10³ cfu/0.5 g could be visualized whereas in others the presence of 10⁸ cfu/0.5 g did not yield a detectable quantity of amplified product.     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

Reduction of caecal Listeria monocytogenes in Leghorn chicks following treatment with a competitive exclusion culture (PREEMPT™)

Day-of-hatch Leghorn chicks were treated by oral gavage with PREEMPT^™, a continuous-flow competitive exclusion culture containing broiler caecal bacteria, followed by an oral challenge with Listeria monocytogenes , to determine the effects of PREEMPT^™ on L. monocytogenes caecal colonization. Increased ( P < 0·001) concentrations of caecal propionic acid in control chicks compared with PREEMPT^™-treated chicks at 3 days of age were indicative of the establishment of the PREEMPT^™ bacteria. Caeca from control chicks at 7 days after the oral challenge with L. monocytogenes contained mean 3·4 ± 1·4 log cfu g⁻¹ of caecal content, while caeca from PREEMPT^™-treated chicks contained no detectable Listeria. Enrichment for L. monocytogenes resulted in 100 % of caeca from control chicks testing culture-positive for L. monocytogenes , while none of the caeca from PREEMPT^™-treated chicks were culture-positive. The results indicated that prophylactic treatment of newly hatched chicks with PREEMPT^™ significantly reduced caecal colonization by L. monocytogenes.     

Use of a microcolony technique combined with an indirect immunofluorescence test for the rapid detection of Listeria in raw meat

A rapid technique for the detection of Listeria was developed by combining a microcolony technique with an immunofluorescence test. Listeria was detected in artificially contaminated beef homogenates within 5 h. The detection level was log₁₀5·0. Listeria was detected in 1/4 naturally contaminated beef samples using the rapid method.     

F0F1-ATPase分子马达技术对单核细胞增生李斯特菌检测的研究

利用载色体chromatophore上的F₀F₁-ATPase分子马达生物传感器,建立了应用在食品检测中快速检测方法。首先从嗜热菌中提取载色体后,标记荧光探针F-DHPE,合成生物素化单增李氏菌prfA探针,在已标记F-DHPE的载色体ATP合酶的ε亚基上连接ε亚基抗体-生物素-链霉亲和素-生物素-prfA探针,将待测单核细胞增生李斯特菌标准菌株和阴性对照分别与此生物传感器结合,通过环境H+量测定ATP产生量,进而对单核细胞增生李斯特菌DNA进行检测。结果表明,chro prfA(连接在载色体chro上的prfA探针)的浓度在0.052 mg/mL,单核细胞增生李斯特菌DNA浓度在40 ng/mL为最适检测条件。通过传统检测方法及PCR检测方法对照,本方法具有良好的检测符合性。     

Comparison of selective and non-selective enrichment media in the detection of Listeria monocytogenes from meat containing Listeria innocua

AIMS: This study investigated whether the higher incidence of recovery from meat of Listeria innocua compared with L. monocytogenes could be due to the laboratory media used, leading to an artificially lower detection of the pathogenic species, L. monocytogenes. METHODS AND RESULTS: Minced beef was inoculated with L. monocytogenes, L. innocua, or a mixture of these species, and stored at 0 or 10 degrees C under vacuum or aerobic conditions for up to 28 days. Listeria were recovered from the minced beef using selective (University of Vermont Medium, UVM) and non-selective (Buffered Peptone Water, BPW) enrichment broths after 0, 14, and 28 days of storage. In general, there were no significant differences (P < 0.05) between the numbers of L. monocytogenes recovered from minced beef samples after 24 h enrichment in BPW and the numbers recovered using UVM. In addition, the presence of L. innocua in meat samples containing L. monocytogenes did not significantly (P < 0.05) affect the numbers of L. monocytogenes recovered using either enrichment broth. In most cases there were no significant differences (P < 0.05) between the numbers of L. innocua recovered from minced beef samples after 24 h enrichment in BPW compared with numbers recovered using UVM. CONCLUSION: Listeria innocua was found to have no significant competitive advantage over L. monocytogenes in selective or non-selective enrichment media. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that, in some instances, the use of a selective enrichment broth offers no advantage over a non-selective enrichment broth for the recovery of Listeria species from minced beef.     

Surfactin reduces the adhesion of food-borne pathogenic bacteria to solid surfaces

Aims:  To investigate the effect of the biosurfactants surfactin and rhamnolipids on the adhesion of the food pathogens Listeria monocytogenes , Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces.
Methods and Results:  Quantification of bacterial adhesion was performed using the crystal violet staining technique. Preconditioning of surfaces with surfactin caused a reduction on the number of adhered cells of Ent. sakazakii and L. monocytogenes on stainless steel. The most significant result was obtained with L. monocytogenes where number of adhered cells was reduced by 10² CFU cm⁻². On polypropylene, surfactin showed a significant decrease on the adhesion of all strains. The adsorption of surfactin on polystyrene also reduces the adhesion of L. monocytogenes and Salm. Enteritidis growing cells. For short contact periods using nongrowing cells or longer contact periods with growing cells, surfactin was able to delay bacterial adhesion.
Conclusions:  The prior adsorption of surfactin to solid surfaces contributes on reducing colonization of the pathogenic bacteria.
Significance and Impact of the Study:  This is the first work investigating the effect of surfactin on the adhesion of the food pathogens L. monocytogenes , Ent. sakazakii and Salm. Enteritidis to polypropylene and stainless steel surfaces.     

Thermal inactivation of Listeria monocytogenes during a process simulating temperatures achieved during microwave heating

Conventional heating was used to expose cells of Listeria monocytogenes , either in broth or in situ on chicken skin, to the mean times and temperatures that are achieved during a 28 min period of microwave cooking of a whole chicken. Heating L. monocytogenes by this method in culture broth resulted in a reduction in viable cell numbers by a factor of greater than 10⁶ upon reaching 70°C. Simulated microwave cooking of L. monocytogenes in situ , on chicken skin, resulted in more variability in the numbers of survivors. Heating for the full cook time of 28 min, however, resulted in a mean measured temperature of 85°C and no surviving listerias were detected. This indicated a reduction in viable numbers of greater than 10⁶. To reduce temperature variation, cells were heated on skin in a submerged system in which exposure to 70°C for 2 min resulted in a reduction in viable cell numbers of all strains of listerias tested of between 10⁶ and 10⁸. These results show that when a temperature of 70°C is reached and maintained for at least 2 min throughout a food there is a substantial reduction in the numbers of L. monocytogenes . The survival of this organism during microwave heating when temperatures of over 70°C are reported is probably due to uneven heating by microwave ovens resulting in the presence of cold spots in the product. The heat resistance of L. monocytogenes is comparable with that of many other non-sporing mesophilic bacteria.     

Rapid detection of Listeria monocytogenes in ham samples using immunomagnetic separation followed by polymerase chain reaction

AIMS: To develop a 24-h system for the detection of Listeria monocytogenes in ham. METHODS AND RESULTS: An immunomagnetic separation (IMS) of bacteria directly from ham followed by extraction of DNA and detection using a new multiplex polymerase chain reaction (PCR) was used. The PCR method used one primer pair targeted at the listeriolysin O gene of L. monocytogenes and the other pair for a region of the 23S rRNA genes of Listeria, giving products of 706 and 239 bp, respectively. The combined IMS/PCR was calculated to be capable of detecting as few as 1.1 L. monocytogenes cells g-1 in a 25-g ham sample. CONCLUSION: The process produced acceptable results, but the IMS step is the main barrier to further improvement of sensitivity. The DNA isolation was the most time-consuming step in the process. SIGNIFICANCE AND IMPACT OF THE STUDY: A 24-h test for the presence of L. monocytogenes will be useful to the food industry and significantly assist in the timely investigation of outbreaks.     

Research note: In vitro inhibition of Listeria monocytogenes growth by veillonellae cultures grown on tartrate media

Veillonellae cultures were grown on agar media supplemented with tartrate and examined for inhibitory effects on the growth of Listeria monocytogenes . Veillonellae cultures grown on media supplemented with 0 or 50 mmol l⁻¹ of tartrate did not inhibit the growth of L. monocytogenes ; however, veillonellae grown on media supplemented with 100, 150 or 200 mmol l⁻¹ of tartrate did inhibit the growth of L. monocytogenes . The inhibition of the growth of L. monocytogenes by the veillonellae was correlated with the increased production of acetate and propionate from tartrate by veillonellae and with the reduction of the pH of the media by L. monocytogenes .     

Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive

The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10³ cfu g^-1 than on leaves inoculated with about 10⁵ cfu g^-1, but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested.     

Response of Listeria monocytogenes to liquid smoke

Aims:  To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes.
Methods and Results:  Growth and survival curves were recorded in brain–heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 μg ml⁻¹ phenol while a rapid decrease in cell viability occurred in the presence of 30 μg ml⁻¹ phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 μg ml⁻¹ phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly gene expression, showing a potential inhibition of protein activity or stability.
Conclusions:  The presence of liquid smoke in a rich medium strongly affected growth and survival of L. monocytogenes . Brief smoke stress affected the metabolic pathways and inhibited the haemolytic activity of L. monocytogenes .
Significance and Impact of Study:  This study is a first step in the investigation of the influence of a smoked product on L. monocytogenes strains.     

A 20–24 H MICROCOLONY-IMMUNOBLOT TECHNIQUE to DIRECTLY DETECT and ENUMERATE LISTERIA MONOCYTOGENES INOCULATED INTO FOODS

A microcolony-immunoblot technique (MCIBI) was developed to directly enumerate, in less than 24 h, very low numbers of Listeria monocytogenes (8–12 colony forming units: CFU/g or mL) inoculated into foods. Four meat and poultry and two dairy products were artificially inoculated with L. monocytogenes V7 diluted and plated on Oxford agar medium. Each plate was overlaid with an Immobilon-P membrane and incubated for 18–20 h at 37C. Blot-transferred colonies on these membranes were probed with C11E9 monoclonal antibody (MAb) and developed using peroxidase conjugated goat antimo use Ig G and a water insoluble substrate (3,3-diaminobenzidin tetrahydmchloride; (DAB-HCI), Nickel chloride and H₂O₂). the MCIBT gave L. monocytogenes counts that were not significantly lower than direct colony counts on selective agars. This technique allowed the recovery of 94–100% of L. monocytogenes cells inoculated into foods containing natural background flora counts of 3 × 10⁴ to 8 × 10⁶ CFU/g or mL. Using a 2 h resuscitation period on nonselective agar before overlay with Oxford media, the MCIBT allowed detection of sublethally heat injured cells of strain V7.     

Incidence of Listeria spp. in Breton live shellfish

Live shellfish samples (120) were collected from nine littoral sites in Brittany (western France). They were screened for Listeria spp. and a count of faecal coliforms was carried out. Analysis of the results revealed Listeria spp. in 55% of samples, a much higher rate than the previous, infrequent, recorded data. Furthermore, the study demonstrated that the frequency of Listeria spp. in winter was more important than in summer ( P < 0·001), and underlined a significant relationship between the occurrence of these bacteria and the concentration of faecal coliforms ( P < 0·001). Finally, comparison of the official and Gen-Probe^® methods revealed the limits of the standardized technique in the search for L. monocytogenes in shellfish.