Comparison of Oxford Agar, PALCAM and Listeria monocytogenes Blood Agar for the recovery of L. monocytogenes from foods and environmental samples

This work had as the main objective a comparison between Listeria monocytogenes Blood Agar (LMBA) and the conventional selective agar media, Oxford and PALCAM, relative to its efficacy in the detection of L. monocytogenes in naturally contaminated food and environmental samples. 173 environmental samples and 272 samples of foods were analysed. A higher sensitivity for detection of L. monocytogenes was verified for LMBA than for PALCAM and Oxford. In LMBA L. monocytogenes could be distinguished from other Listeria spp. by detection of hemolysis. In Oxford and PALCAM this distinction was not possible. The higher growth rate of L. innocua cf. L. monocytogenes in selective liquid media could result in a high number of false negatives (non-detection of the target organism on plates, although its presence was observed by other tests, eg. mini-VIDAS LMO). The need for specific media for the detection of L. monocytogenes in food was confirmed. LMBA could be an alternative medium to use together with PALCAM or Oxford.     

Survival of Listeria monocytogenes inoculated in retail soymilk products

The consumption of soymilk products has been growing; because these foods contain proteins and isoflavone, and lack of lactose and cholesterol. The survival and growth of Listeria monocytogenes in the soymilk products were investigated by inoculating Listeria monocytogenes into various retail soymilk products which claimed to improve or fortify the functional properties. The results show that the refrigeration cannot successfully prevent the growth of Listeria monocytogenes in soymilk products. The addition of fiber content and higher viscosity in soymilk may significantly inhibit the growth of L. monocytogenes at 4–7 °C; however, this phenomenon was not observed when the incubation temperature was elevated to 22 °C. The change of pH value and TTS (total soluble solids) in soymilk products owing to L. monocytogenes, especially those with rich fiber content, might be negligible for the public. Thus the possible contamination of L. monocytogenes to soymilk products should be of crucial concern.     

Survival and growth of Listeria monocytogenes on sausage formulated with inoculated and stored rework product

To assess the effect of including contaminated rework on survival and growth of Listeria monocytogenes, two sausage formulations (one American, Bologna sausage; and one Bulgarian, Stranja sausage) were inoculated with the pathogen and stored for 4 days at 10 °C plus 15 h at 30 °C. After storage, both rework types were included (at 20% and 40%) in corresponding fresh sausage emulsions and heated to 68, 70 and 71.7 °C; fresh Bologna and Stranja emulsions served as controls and were inoculated with 24 h broth cultures of the same 10-strain mixture of L. monocytogenes and thermally treated to the same temperatures. The results showed that heating to 68 and 70 °C inactivated 3–4 log CFU/g of the initial concentration of L. monocytogenes cells (>7 log CFU/g), while heat treatment to 71.7 °C in the center of experimental samples reduced counts by 6 log CFU/g. Survival of L. monocytogenes in samples heated to 68 and 70 °C was higher in controls. Control samples of Stranja emulsion heated to 71.7 °C allowed higher growth (P < 0.05) during storage (5 days at 10 °C) as compared to other control and experimental rework samples. The Stranja emulsion had a higher fat content (20.2%) compared to the Bologna emulsion (11%). This study provides evidence about the possible danger when potentially contaminated rework is stored and then introduced into fresh product formulations.     

Prevalence of Listeria monocytogenes in delicatessen food products in China

This study was to investigate the prevalence of Listeria monocytogenes in delicatessen food, raw materials and environmental samples in food processing chain. Two hundred and forty-five samples of delicatessen foods in markets, 98 samples of cooked foods from restaurants and hotels, 154 samples of food product contact surfaces such as counters, iceboxes and chopping blocks etc. from processing and selling sites, 51 environmental samples from the food-cooking rooms of restaurants and hotels were collected and detected for the prevalence of L. monocytogenes. The results showed that there were 32 strains isolated from delicatessen foods in the market and the average prevalence was 13.06% which is significantly higher (p < 0.01) than those isolated from cooked foods in hotels and restaurants (1.02%) suggesting that the delicatessen food products may be contaminated during the delivery from hotels and restaurants to the markets. Ten strains were isolated from 335 raw materials and 21 strains were isolated from 154 processing equipments in selling and processing sites. No strains were isolated from 51 samples of equipments in cooked food rooms of hotels and restaurants. The study shows that the prevalence of L. monocytogenes in delicatessen foods in the market was significantly higher than those in the cooked foods of hotels and restaurants, and therefore, the critical control points were: (1) to establish relative closed selling rooms; (2) to establish the sterilizing measures to keep the delicatessens from being contaminated with L. monocytogenes during the delivery from restaurants or hotels to the retail markets.     

The direct enumeration of Listeria monocytogenes in a food with a low background microflora

Concentrated yoghurt (labneh) with an inherently low count of non-lactic acid bacteria was inoculated with various levels of Listeria monocytogenes, and direct plating onto Listeria Selective Agar or PALCAM Agar proved effective for monitoring high counts. At levels of < 10 cfu/g, an MPN count using UVM I/Fraser Broth was more reliable, and a ‘presumptive’ count, taking the colour change in Fraser Broth from yellow to black as being ‘positive’, had a failure rate of < 2%. Given that such low counts are below the likely infective dose for humans, it is suggested that the direct MPN count could be employed for routine quality control examinations.     

Flow cytometric assessment of the antimicrobial activity of essential oils against Listeria monocytogenes

Flow cytometry was applied to assess the antimicrobial activity of oregano, thyme and cinnamon essential oils against Listeria monocytogenes ATCC19114, using combined staining with propidium iodide (PI) for membrane damage evaluation and carboxyfluorescein diacetate (cFDA) for esterase activity detection. The antimicrobial activity of the essential oils was also tested at different NaCl concentrations.Significant differences were observed between plate count results and flow cytometric data, which suggested the presence of a sublethally stressed subpopulation, not able to form colonies on agar plates.Following treatments, flow cytometric assessment clearly discriminated three different subpopulations: viable, dead and injured cells. Cinnamon essential oil exerted a different impact on the cellular subpopulations, with a lower overall activity and a large percentage of cells having minimally damaged membranes. On the contrary, membrane disintegration seemed to be the primary inactivation mechanism of oregano and thyme essential oils.The antimicrobial activity of the essential oils increased with NaCl concentration increase, but higher NaCl concentrations were necessary following treatments with cinnamon essential oil.Our findings suggest differences in the mode of action of cinnamon essential oil against L. monocytogenes, in comparison with thyme and oregano essential oils.     

Growth of acid-adapted Listeria monocytogenes in orange juice and in minimally processed orange slices

The aim of this work was to study the growth/survival of acid-adapted cells of Listeria monocytogenes, in orange juice and in minimally processed orange slices. The L. monocytogenes OML 45 behaviour into TSB (Tryptic Soy Broth) medium was evaluated at different pH values (between 3.7 and 6.7). The acid-adapted cells were obtained maintaining L. monocytogenes in TSB at pH 5.7 for 3 h. The obtained cells were then inoculated into a diluted orange juice with a pH of 2.6. Moreover, the acid-adapted cells were inoculated into minimally processed orange slices. The growth was evaluated during storage at different temperatures. The study confirms that orange juice and minimally processed orange slices can support the acid-adapted pathogen growth.     

The antimicrobial effect of wine on Listeria innocua in a model stomach system

A model stomach, containing a food matrix and a synthetic gastric fluid, was used to study the bactericidal effect of ingested wine on Listeria innocua. Volumes of wine equivalent to the ingestion of one glass and half a bottle, led, over a period of less than 2 h, to a reduction of 3 and 4 logarithmic cycles of the initial population respectively. The influence of ethanol and organic acids, wine constituents with known antimicrobial properties, was investigated. Ethanol exhibited a higher bactericidal effect than the mixture of the main wine organic acids. When testing the organic acids separately, malic and lactic acids were found to have the strongest effect. The combination of ethanol with the organic acids acted synergistically but to a lesser extent than wine itself. The results suggest that the ingestion of wine during a meal may diminish the quantity of Listeria persisting further in the alimentary tract.     

Survival of Listeria innocua and Listeria monocytogenes in muscle of cod (Gadus morhua L.) during salt-curing and growth during chilled storage of rehydrated product

The aim of this work was to study survival of Listeria innocua and Listeria monocytogenes in muscle of cod during salt-curing and growth during chilled storage of the rehydrated product. Fresh cod was inoculated with L. innocua and L. monocytogenes at different levels before salt-curing. After salt-curing and rehydration, the levels were within 1 log₁₀ CFU/g lower than prior to salt-curing in all experiments. During the first 5 days of storage after rehydration, growth of L. innocua was observed in 1 out of 5 experiments at 4 °C, but a 10–100-fold increase were observed in all experiments from day 5 to day 10. The growth started earlier and was more rapid when samples were stored at 7 °C. Growth of L. monocytogenes at 4 °C appeared to start earlier than for L. innocua, but a 10–100-fold increase was observed also for this bacterium. The lag phases in rehydrated products were longer than in experiments with cod muscle juice. The differences could be explained by a different level of salt stress. This work demonstrates that long term exposure to very high salt concentrations does not eliminate Listeria spp., and that Listeria being present in the fish prior to salt-curing can recover and grow in rehydrated salt-cured cod during chilled storage.     

Comparison of antibiotic resistance patterns in Listeria monocytogenes and Salmonella enterica strains pre-exposed and exposed to poultry decontaminants

Recent opinions expressed by European Union Scientific Committees suggest there is a potential for poultry decontaminants to increase resistance to antibiotics. At this moment there is no scientific information available to estimate this risk accurately. Four strains (Listeria monocytogenes serovar 1/2a, Listeria monocytogenes serovar 4b, Salmonella enterica serotype Typhimurium and Salmonella enterica serotype Enteritidis) were repeatedly exposed to increasing sub-inhibitory concentrations of decontaminants (trisodium phosphate, acidified sodium chlorite, citric acid, chlorine dioxide or peroxyacetic acid) and tested against 15 antibiotics by means of a standard disc-diffusion technique (NCCLS). The antibiotic resistance patterns of strains were compared before and after exposure to decontaminants. Intra-specific differences in antibiotic resistance patterns were found among strains. Increases in resistance to various antibiotics were observed in L. monocytogenes and S. enterica strains after exposure to chemicals (especially ASC). These results raise concerns over the application of certain poultry decontaminants, since they could contribute to the development of microbial resistance mechanisms. However, these are preliminary results derived from laboratory-based experiments. Additional studies under practical field conditions would be needed to substantiate these findings.     

Survival of Listeria innocua in Minas Traditional Serro cheese during ripening

Cheese produced with raw milk can be a risk to consumer health. It is known that lactic acid bacteria present in raw milk and in natural starters can produce antimicrobial compounds against some foodborne bacteria. This work aimed to evaluate the survival of Listeria innocua in Minas Traditional Serro cheese during cheese ripening. The cheeses were inoculated with 10¹, 10² or 10³ CFU mL⁻¹ of the bacterium and were analyzed for 60 days of ripening at 30 °C. It was observed that the time and the dose of bacteria inoculated affected (p < 0.05) the survival of L. innocua. Even when the lowest dose was inoculated, at the end of the 60 days, approximately 10² CFU mL⁻¹ of L. innocua was detected in the cheese. The lactic acid bacteria present in the milk and in the natural starter were not sufficient to guarantee the absence of L. innocua in Minas Traditional Serro cheese even after 60 days of storage, as is required by Brazilian legislation.     

Sigmoidal thermal inactivation kinetics of Listeria innocua in broth: Influence of strain and growth phase

Listeria innocua inactivation was studied within the temperature range 52.5–65.0 °C, comparing two different strains (10528 and 2030c) and two growth phases (exponential and stationary). Survival curves may present a sigmoidal behaviour, with an initial shoulder (L), followed by a maximum inactivation rate (k_max) period and a final tailing tendency. A Gompertz-inspired model was used to fit experimental data, and kinetic parameters (L, k_max and tail) were estimated by non-linear regression analysis. The influence of temperature, growth phase and strain on kinetic parameters was studied using a 2³ factorial experimental design. Results showed that temperature and growth phase were the most significant variables affecting the kinetic parameters. Listeria thermal inactivation varied from a log-linear tendency till a pronounced sigmoidal behaviour, depending on the studied factors.     

Survival of Listeria innocua in thermally processed orange juice as affected by vanillin addition

Presence of Listeria monocytogenes could seriously affect the safety of fruit juices. Addition of natural antimicrobials may be an alternative to enhance microbial inactivation in fruit juice thermal preservation. The response of L. innocua, surrogate for L. monocytogenes, to combined treatments involving moderate temperatures (57–61 °C) and addition of different levels of vanillin (0–1100 ppm) was assessed to find the most effective inactivation treatment in orange juice. The presence of vanillin greatly increased the bactericidal effect of the mild heat treatment. This effect considerably depended on the amount of added vanillin when working at the lowest temperature (57 °C), while at higher temperatures (60 or 61 °C) the increase in vanillin concentration did not produce a clear change in the response. Nonlinear semilogarithmic survival curves were successfully fitted using a modified version of Gompertz model and by the Weibullian model.     

Effect of weak acids on Listeria monocytogenes survival: Evidence for a viable but nonculturable state in response to low pH

Listeria monocytogenes is capable of surviving environmental conditions that are normally fatal to many other bacteria, enabling this pathogen to remain on foods and eventually establish infection after consumption. This study investigated the extent to which L. monocytogenes is killed by several weak acids, with particular emphasis on the commonly used food preservative, potassium sorbate. The effects of potassium sorbate were found to be highly pH dependant, with significant killing only observed at a pH of 4.0. At this pH, the order of effectiveness of the acids tested was Na benzoate > Na propionate > acetic acid > K sorbate. Temperature had a significant impact on sensitivity, with K sorbate being effective at 50 mM and elevated (37 °C) temperature, but failing to affect survival when cells were at 4 °C or 21 °C. Finally, we found that cells grown in the presence of K sorbate at pH 4.0 entered a viable but nonculturable (“VBNC”) state, but became nonviable by 24 h. In contrast, cells incubated under these conditions in the absence of K sorbate entered the VBNC state and maintained viability throughout the 24 h study period.     

Adherence kinetics, resistance to benzalkonium chloride and microscopic analysis of mixed biofilms formed by Listeria monocytogenes and Pseudomonas putida

Comparison between the resistance to BAC and the microscopic structure between mixed-species biofilms formed by different strains of Listeria monocytogenes and Pseudomonas putida CECT 845 under different scenarios and that obtained by the corresponding monospecies L. monocytogenes biofilm was carried out. The association of P. putida with L. monocytogenes quickens biofilm formation and increases significantly (p < 0.05) the BAC-resistance of the biofilm after 4 days of incubation at 25 °C respecting to that formed by monospecies biofilms. According with the adherence profiles of P. putida, two different patterns of association between both species (A and B) were identified, being type A pattern found in the mixed biofilms much more resistant to BAC. After 11 days of incubation, a destructuration of mixed biofilms occurred in all experimental assays, being in 2 out of 5 experimental cases (4032 and BAC-adapted 5873 on polypropylene) accompanied by a sharp decrease in the number of adhered cells. Microscopic analyses demonstrated that complex three-dimensional microscopic structure showed the highest resistance to BAC (4032-SS). Obtained results clearly highlight that to improve disinfection protocols for assuring food safety, it is necessary to mimick those bacterial association that occur in nature.     

Viability of Listeria monocytogenes in an experimental model of nigiri sushi of halibut (Hippoglossus hippoglossus) and salmon (Salmo salar)

Sushi is a traditional Japanese food, mostly consisting of raw seafood in combination with rice. However, eating sushi has become popular among consumers in many countries outside Japan. Sushi is not free from health risks and foodborne illness linked to this product has been caused by Listeria monocytogenes. The aim of our work was to study viability of L. monocytogenes in emulated nigiri sushi comprised of halibut and salmon. Raw samples of halibut and salmon were inoculated with the pathogen and subsequently put on top of vinegar marinated sushi rice followed by storage for 7 days at 4 and 8 °C. As controls, inoculated seafood without rice was used. The experiments demonstrated that the level of L. monocytogenes in nigiri sushi was significant lower during storage over 7 days at 4 and 8 °C compared to controls, in case of inoculated seafood only. The pH drop in the fish muscle, caused by the sushi rice, is believed to be the reason for the decrease in viability of L. monocytogenes in nigiri sushi.     

Antibacterial activity of two Phlomis essential oils against food pathogens

Phlomis species from the Lamiaceae family are widely distributed in Turkey. In this study, the essential oils of Phlomis russeliana (Sims.) Bentham and Phlomis grandiflora H.S. Thompson var. grandiflora collected from North and Southern parts of Turkey, were obtained by hydrodistillation of the aerial parts. The essential oils were subsequently analysed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC/MS). The major constituents of P. russeliana essential oil were identified as sesquiterpenes β-caryophyllene (23%), germacrene-D (15%), and caryophyllene oxide (8%). Analysis of P. grandiflora var. grandiflora oil also showed oxygenated sesquiterpenes such as β-eudesmol (42%) and α-eudesmol (16%) as major constituents.Furthermore, essential oils were tested in vitro against common food borne bacteria such as Aeromonas hydrophila, Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Yersinia enterocolitica, and the anaerobic pathogen Clostridium perfringens using the micro-broth dilution assay. When compared with antimicrobial standards weak to moderate (125 to >1000 μg/ml) minimum inhibitory concentrations (MIC) were observed. The results show that Phlomis essential oils might be an alternative to conventional antimicrobials in various foods.     

Inhibition of food poisoning and pathogenic bacteria by Lactobacillus plantarum strain 2.9 isolated from ben saalga, both in a culture medium and in food

The bacteriocinogenic strain Lactobacillus plantarum 2.9 isolated from the traditional pearl millet-based African fermented food ben saalga was tested for inhibition of food poisoning and pathogenic bacteria in MRS broth and in a malted millet flour slurry. In MRS broth, strain 2.9 completely eliminated Bacillus cereus, Escherichia coli O157:H7 and Salmonella enterica cells within 48 h incubation at 22–30 °C. A much lower inhibition was observed at 15 °C. The inhibitory effect of strain 2.9 on the above-mentioned target bacteria was corroborated in the malted millet flour slurry, reducing viable cell counts below detection levels after 8 h storage for B. cereus or after 24 h for S. enterica and 48 h for E. coli. An Enterobacter aerogenes strain was only moderately inhibited in the slurry. Results from the present study suggest that strain 2.9 could be used as starter culture to improve the microbiological safety of fermented ben saalga.     

Fate of Listeria monocytogenes and Escherichia coli O157:H7 in the presence of natural background microbiota on conventional and organic lettuce

Foodborne illnesses due to the consumption of contaminated raw vegetables is a continuing food safety concern. The limited efficacy of chlorine products to disinfect in fresh-cut industries, has led to study other methods or strategies to improve the safety of processed fresh-cut products. It has been reported that the presence of competing microorganisms on the surfaces of fresh produce can contribute to the reduction of pathogens. The aim of this study was to evaluate the interactions between the natural background microbiota of shredded conventional and organic lettuce and Listeria monocytogenes and Escherichia coli O157:H7. The effect of different initial load of background microbiota (‘low’, ‘medium’ and ‘high’) was tested for its ability to reduce L. monocytogenes and E. coli O157:H7 populations on shredded lettuce during storage at 10 ± 1 °C for 8 days. After the different pre-conditioning steps in order to obtain different initial loads of microbiota, in general, we observed that varying its size had no effect on L. monocytogenes and E. coli O157:H7 survival/growth during the storage period. Only differences on the survival/growth of L. monocytogenes and E. coli O157:H7 inoculated onto organic and conventional lettuce, respectively at the end of storage period at 10 °C were found. These results highlight the necessity for corrective measures to avoid contamination of fresh-cut vegetables with foodborne pathogens.     

Comparison of intense pulsed light- and ultraviolet (UVC)-induced cell damage in Listeria monocytogenes and Escherichia coli O157:H7

The purpose of this study was to compare the degree of microbial inactivation and cell damage induced by intense pulsed light (IPL) and short-wavelength ultraviolet (UVC) in Listeria monocytogenes and Escherichia coli O157:H7. The viability of the food-borne pathogens treated with IPL and UVC (254 nm) decreased exponentially with treatment time. Particularly dramatic reductions in L. monocytogenes and E. coli O157:H7 were observed for IPL treatments at energy densities of 376 and 455 W/m², with an approximately 7-log reduction for a treatment time of 60-180 s. Also, a 4-log reduction of L. monocytogenes and a 5-log reduction of E. coli O157:H7 were achieved with UVC irradiation for 1200 s. The types and amounts of IPL- and UVC-induced DNA damage in both microorganisms were determined and compared. DNAs from cells irradiated with either IPL or UVC accumulated double-strand breaks (DSBs), single-strand breaks, and cyclobutane pyrimidine dimers, and with a similar pattern; however, more DSBs were detected following UVC than following IPL in both types of microorganism. Transmission electron microscopy observations of IPL- and UVC-induced cell damage clearly indicate that bacterial cell structures were destroyed by IPL treatment but not by UVC treatment.