Fate of salmonellas in the alimentary tract of chicks pre-treated with a mature caecal microflora to increase colonization resistance

Oral challenge with a strain of Salmonella kedougou , resistant to nalidixic acid, gave a time-course for salmonella survival in the the alimentary tract of chicks pre-treated with a caecal culture from an eight-week-old bird to increase colonization resistance. In untreated, control chicks, salmonella colonization of the caeca resulted in counts of > 10⁶/g within 48 h and a mean generation time of 1·6 h. With treated birds, however, the salmonellas failed to multiply in the caeca and decreased to a low level over a 48-h period, thus suggesting a mainly bacteriostatic effect. Pretreatment of chicks with the caecal culture also reduced the proportion of salmonella-positive crop samples. Growth of the salmonella in a feed-slurry system resembling moistened crop contents occurred rapidly at 37°C but was prevented by addition of the treatment culture, a decline in numbers coinciding with Lactobacillus populations of ca 10⁹/g and a pH value of 5·5. Incorporation of a feed decontaminant, 1% formic acid (pH 4·0), rapidly eliminated both salmonellas and organisms added from the treatment culture.     

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin^® pre-production to the milk. Each batch of cheese was inoculated with 10²–10³ cfu g⁻¹ of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l⁻¹ could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss.     

The anti-inflammatory drug Diclofenac retains anti-listerial activity in vivo

Aims:  The interactions between nonsteroidal anti-inflammatory drugs (NSAID) and Listeria monocytogenes have not been sufficiently documented to date. The aim of this study was to investigate the possible effects of Diclofenac (Dc) in a murine listerial infection model.
Methods and Results:  Dc was administered orally at 2·5 μg g⁻¹ to female albino strain of laboratory mouse (BALB/c) thrice postinfection (1 × 10⁸ CFU ml⁻¹ oral challenge with L. monocytogenes ATCC 51774), which resulted in significantly ( P  < 0·01) reduced bacterial counts in liver and spleen, decreased (10-fold, P  < 0·05) hepatic colonization and necrosis, and caused up-regulation of the expression of inflammatory cytokines (interferon-γ, interleukin-1β, tumour necrosis factor-α), compared with drug-free control.
Conclusions:  Dc may be useful as a promising adjuvant to the existing therapies in controlling systemic listerial infection. Further, quantitative structure–activity relationship studies might contribute in manipulating it as a lead compound for the synthesis of new, more effective nonantibiotics, perhaps, devoid of side-effects that could be recommended as a compassionate therapy for listeriosis.
Significance and Impact of the study:  This is the first in vivo study designed to evaluate the antilisterial effect of the NSAID Dc with special emphasis on the immunological mechanism of action of the drug.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Growth and reducing capacity of Listeria monocytogenes under different initial redox potential

Aims:  To determine the reducing capacity of Listeria monocytogenes and to highlight the effect of redox potential on its growth parameters.
Methods and Results:  The reducing capacity of L. monocytogenes was monitored in Brain Heart Infusion Broth media at different initial redox potential (Eh) and pH at 37°C. The effect of Eh obtained by gas flushing (air, N₂ and N₂-H₂) or by adding potassium ferricyanide and dithiotreitol in concentration from 1 to 10 mmol l⁻¹on L. monocytogenes growth parameters at pH 6·0, 7·0 and 8·0 was investigated. A total change of 539 mV (±44 mV) from an initial redox value of +330 ± 8 mV to a more negative potential in redox curves was observed resulting from L. monocytogenes growth at pH 7·0 at 37°C. A significant influence of pH and redox potential on L. monocytogenes lag phase of growth was shown ( P  < 0·05).
Conclusions:  Listeria monocytogenes exhibited longer lag phase in reducing conditions and at pH 6·0. The method used to modify the redox potential was shown to have no effect on growth parameters at pH 7·0.
Significance and Impact of the Study:  The provided information on the extending lag time and the possible delayed growth of this major pathogen in reducing conditions might be useful for its control in foods.     

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.     

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.     

Microbiological composition of raw milk from selected farms in the Camembert region of Normandy

Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens , yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas , coliforms, Escherichia coli , enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml⁻¹), lactococci (760 cfu ml⁻¹) and Micrococcaceae (720 cfu ml⁻¹) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts <100 cfu ml⁻¹. Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml⁻¹. About 80% of supplies had ≤10 E. coli cfu ml⁻¹ and all samples had 1 Cl. perfringens cfu ml⁻¹. Two of the tested milks were positive for salmonellas (2·9%), four were positive for Listeria monocytogenes (5·8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1·4%).     

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.     

Efficacy of sodium hypochlorite and peracetic acid in sanitizing green coconuts

Aims:  To evaluate the efficacy of sanitizing green coconuts ( Cocos nucifera L.) through the treatment applied by juice industries using sodium hypochlorite and peracetic acid.
Methods and Results:  The surface of the fruits was inoculated with a mixture of five Listeria monocytogenes strains. The treatments consisted in immersing the fruits for 2 min at room temperature in sodium hypochlorite solution containing 200 mg l⁻¹ residual chlorine at pH 6·5, and 80 mg l⁻¹ solution of peracetic acid or sterile water. Bacterial populations were quantified by culturing on trypticase soy agar supplemented with yeast extract and Oxford selective culture medium; however, recovery was higher on the nonselective medium. Immersion in water produced a reduction in the L. monocytogenes population of 1·7 log₁₀ CFU per fruit, while immersion in sodium hypochlorite and peracetic acid solutions resulted in population reductions of 2·7 and 4·7  log₁₀ CFU per fruit respectively.
Conclusions:  The treatments studied are efficient to green coconuts.
Significance and Impact of the Study:  Sanitation of green coconut is one of the most important control measures to prevent the contamination of coconut water. This article provides information that shows the adequacy of sanitizing treatments applied by the juice industries.     

Survival of Listeria monocytogenes in sea water and effect of exposure on thermal resistance

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes , Scott A and an environmental strain KM, on exposure to sea water at 12·8 or 20·8 °C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T₉₀ values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0·6% yeast extract) at 12·8 and 20·8 °C were 61·7 and 69·2 h for L. monocytogenes Scott A, and 103·0 and 67·0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T₉₀ values at 12·8 and 20·8 °C were 60·6 and 56·9 h for L. monocytogenes Scott A, and 83·0 and 65·9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12·8 and 20·8 °C was 1·7 and 17·7%, respectively, while for KM the values were 19·0 and 1·6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 °C showed an apparent increase in heat resistance after exposure to sea water at 20·8 °C for 7 d ( D ₅₈= 2·64 min) compared with before exposure ( D ₅₈= 1·24). This increase in thermal resistance was not apparent at temperatures greater than 63 °C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different ( P > 0·05) over the temperature range tested (58–62 °C).     

Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 in fresh pork

Aims:  To evaluate the effectiveness of organic acids and supercritical carbon dioxide (SC-CO₂) treatments as well as their combined effect for the reduction of nonpathogenic Escherichia coli and three pathogenic bacteria in fresh pork.
Methods and Results:  The different treatment conditions were as follows: (i) treatment with acetic (1%, 2% or 3%) or lactic acid (1%, 2% or 3%) only, (ii) treatment with SC-CO₂ at 12 MPa and 35°C for 30 min only and (iii) treatment with 3% acetic or lactic acid followed by treatment with SC-CO₂. Within the same organic acid concentration, the lactic and acetic acid treatments had similar reductions. For the combined treatment of lactic acid and SC-CO₂, micro-organism levels were maximally reduced, ranging from 2·10 to 2·60 log CFU cm⁻² ( E. coli , 2·58 log CFU cm⁻²; Listeria monocytogenes , 2·60 log CFU cm⁻²; Salmonella typhimurium , 2·33 log CFU cm⁻²; E. coli O157:H7, 2·10 log CFU cm⁻²).
Conclusions:  The results of this study indicate that the combined treatments of SC-CO₂ and organic acids were more effective at destroying foodborne pathogens than the treatments of SC-CO₂ or organic acids alone.
Significance and Impact of the Study:  The combination treatment of SC-CO₂ and organic acids may be useful in the meat industry to help increase microbial safety.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.     

Multidrug resistance gene deficient (mdr1a–/–) mice have an altered caecal microbiota that precedes the onset of intestinal inflammation

Aim:  To compare caecal microbiota from mdr1a ^–/– and wild type (FVB) mice to identify differences in the bacterial community that could influence the intestinal inflammation.
Methods and Results:  Caecal microbiota of mdr1a ^–/– and FVB mice were evaluated at 12 and 25 weeks of age using denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR. DGGE fingerprints of FVB and mdr1a ^–/– mice (with no intestinal inflammation) at 12 weeks revealed differences in the presence of DNA fragments identified as Bacteroides fragilis , B. thetaiotaomicron , B. vulgatus and an uncultured alphaproteobacterium. Escherichia coli and Acinetobacter sp. were only identified in DGGE profiles of mdr1a ^–/– mice at 25 weeks (with severe intestinal inflammation), which also had a lower number of total bacteria in the caecum compared with FVB mice at same age.
Conclusions:  Differences found in the caecal microbiota of FVB and mdr1a ^–/– mice (12 weeks) suggest that the lack of Abcb1 transporters in intestinal cells due to the disruption of the mdr1a gene might lead to changes in the caecal microbiota. The altered microbiota along with the genetic defect could contribute to the development of intestinal inflammation in mdr1a ^–/– mice.
Significance and Impact of the Study:  Differences in caecal microbiota of mdr1a ^–/– and FVB mice (12 weeks) suggest genotype specific colonization. The results provide evidence that Abcb1 transporters may regulate host interactions with commensal bacteria. Future work is needed to identify the mechanisms involved in this possible cross-talk between the host intestinal cells and microbiota.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

Survival of Listeria monocytogenes in yogurt during storage at 4°C

Cows' milk was inoculated with ca 10³and 10⁷cfu/ml Listeria monocytogenes. After fermentation at 42°C for 0–5 h, the yogurt was stored at 4°C. Low and high inocula survived for 48 h and 7 d, respectively; L. monocytogenes cells were not detectable by direct plating or cold-enrichment after 5 and 15 d, respectively. In low inoculum samples, initial pH at the time of refrigeration was 4·9; the final pH at the time of last sampling was 4·2. In the samples with high inoculum the pH decreased from 5·0 to 4·2.     

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 .     

Effect of rising temperature on the heat resistance of Listeria monocytogenes in meat emulsion

The heat resistance of a strain of L. monocytogenes was determined both in broth and in meat emulsion. The D -values for meat emulsion were approximately two to three times higher than those for broth and also the z -value increased significantly. The micro-organism proved to be more resistant when the cells were heated up slowly (0·5°C/min) to constant temperatures of 60, 63 and 66°C in meat emulsion. The D ₆₀, D ₆₃ and D ₆₆ were, respectively 12·95, 5·4 and 2·3 min. Results may have implications in the survival of Listeria monocytogenes in particular food preparations.     

Listeria monocytogenes in poultry and poultry products: epidemiological investigations in seven Danish abattoirs

Listeria monocytogenes was isolated from 11/236 (4·7%) caecal samples from parent flocks, providing broilers to the abattoirs investigated. Caecal samples from 2078 broilers representing 90 randomly selected broiler flocks were negative for L. monocytogenes. A total of 3080 samples from seven abattoirs including poultry processing line samples, and final products were also examined for L. monocytogenes. Listeria monocytogenes was isolated in 0·3% to 18·7% of the samples collected in the different abattoirs. Epidemiological typing of 247 L. monocytogenes isolates, including serotyping, phage typing, pulsed-field gel electrophoresis and ribotyping revealed 62 different clones. Based upon typability and discriminatory power, DNA typing methods used were found equally suitable as epidemiological markers. Serotyping and phage typing were not found useful as epidemiological markers for poultry isolates of L. monocytogenes since only 120/247 (48·6%) isolates were typable by phage typing and 230/247 (93·1%) L. monocytogenes belonged to serotype 01 while 6/247 (2·4%) belonged to 04. The discovery of a few dominating clones in each abattoir might indicate an endemic occurrence of L. monocytogenes. It is concluded that L. monocytogenes in the broiler production is primarily localized to the abattoirs. The incidence of L. monocytogenes may be reduced by improving the hygiene.