Prevalence,genetic diversity and antimicrobial susceptibility of Listeria monocytogenes isolated from open-air food markets in Greece

A total of 210 food samples originating from milk products, ready-to-eat salads, raw meat and raw meat products purchased in ten open-air market places in Thessaloniki, Greece, were analyzed for the presence of Listeria monocytogenes. Thirty (14.3%) contained L. monocytogenes with the highest prevalence in raw meat (27.5%), raw meat products (18%) and cheese (8%). The strains were susceptible to 16 antimicrobials as determined by microbroth dilution, except one strain which displayed resistance to tetracycline (MIC > 32 μg/ml). This strain carried the tetracycline resistance gene tet(M). Pulsed-field gel electrophoresis (PFGE) revealed a low genetic diversity among the isolates, irrespective of their origin. This suggests that dominant L. monocytogenes clones are widespread in different food product types in open-air food markets in Greece. The high prevalence of L. monocytogenes in these products indicates that appropriate hygienic measures and periodic bacteriological controls are also necessary in open-air food markets to reduce contamination with food-borne pathogens. Greek specialties made with raw meat and raw milk may contain L. monocytogenes and should not be consumed by persons at risk.     

Incidence of pathogenic psychrotrophs in ice creams sold in some retail outlets in Mumbai,India

With a view to determine the microbial quality of ice creams, 30 samples of commercial brands of three flavours sold in the `open' and `packaged, (cone, cup)' forms were analyzed for their total bacterial counts (TBC), yeast and mold counts (YMC), coliforms and pathogenic psychrotrophs; Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Yersinia enterocolitica and Salmonella spp. In general in both the types of ice creams bacterial load (2.3 × 10⁴–8.5 × 10⁶ cfu/ml) was higher; particularly coliform levels were 10–100 fold higher (3.0 × 10²–5.8 × 10⁴ cfu/ml) than the safety limits prescribed by Indian Standards Institute (ISI). Staph. aureus was detected in both the types of ice creams but occurrence of B. cereus was more frequent in open samples (40%) than in packed ones (26.6%). Salmonella was not detected in any of the 30 samples tested. While 53% of the packed and 100% of the open ice creams exhibited Listeria contamination, Yersinia were detected in 33% of packed and 40% of open ones. L. monocytogenes and/or Y. enterocolitica was detected only in one of the open ice cream samples. Growth profile of Y. enterocolitica 5692 and L. monocytogenes 036 at simulated temperature abuse conditions during commercial frozen storage showed that after 10 days L. monocytogenes could grow to >1 log and 1 log cycle at 8–10°C and 2–4°C, respectively and Y. enterocolitica grew 2 log cycles at both the temperatures. Results are discussed in the context of present microbiological specifications and the need for its implementation by regulatory agencies to ward off possible health hazards arising from pathogens.     

Survival and growth of Listeria monocytogenes in lubricants used in the food industry

The survival and growth of three Listeria monocytogenes strains in 10 lubricants (synthetic and mineral-oil based) used in the food industry, and rapeseed oil, was investigated at room temperature (20 °C) and refrigerated (5 °C). Additionally, the transfer of L. monocytogenes from lubricants to stainless steel surfaces and vice versa was investigated. Though the amount of L. monocytogenes in most lubricants, both pure and soiled, decreased significantly (p < 0.05) during the 14 d test period, lubricants may act as sources of contamination on the basis of the results obtained on the survival of L. monocytogenes. In general, temperature had significant effect (p < 0.05) on listericidal effect of lubricants contrary to soiling (p > 0.05), however the effect of both factors was dependent on lubricant (p < 0.05). The results clearly showed that L. monocytogenes survived in synthetic conveyer belt lubricant diluted in water. In addition, L. monocytogenes was transferred significantly (p < 0.05) from stainless steel surfaces into conveyer-belt lubricants and into mineral-oil based hydraulic oil.     

Occurrence of Listeria spp. in Brazilian fresh sausage and control of Listeria monocytogenes using bacteriophage P100

Since the 1980s, an increase in outbreaks of human listeriosis linked to contaminated food has been a concern of health authorities. Intensively manipulated foods, such as Brazilian fresh sausage, are frequently responsible for food-borne diseases. In this work the occurrence of Listeria monocytogenes and the efficacy of bacteriophage P100 (LISTEX?) to control the microorganism was evaluated in Brazilian fresh sausage. Eighty samples were analyzed, 40 each of swine and chicken Brazilian fresh sausage. Listeria spp. were isolated from 12 samples (15%), of which three (3.75%) were positive for L. monocytogenes. L. monocytogenes strains isolated belonged to serotype 1/2a. L. monocytogenes 1/2a was inoculated in Brazilian fresh sausage (2.1 × 10⁴ cfu/g) with the bacteriophage added thereafter (3.0 × 10⁷ pfu/g). Samples were analysed immediately (day zero) and then stored at 4 °C for 10 days. The bacteriophage P100 reduced L. monocytogenes counts by 2.5 log units at both 0 and 10 days compared to controls without bacteriophage. In spite of this, the populations of L. moncytogenes increased over the 10 day storage. Our data demonstrate that in one of the samples the use of the bacteriophage dropped the bacteria count below the level of direct detection. This study demonstrates a new alternative for pathogen control in the food industry, especially in the processes used to produce Brazilian fresh sausage.     

Detection of Listeria monocytogenes in raw whole milk for human consumption in Colombia by real-time PCR

The aim of our work was to detect Listeria monocytogenes in raw whole milk from five municipalities of Boyacá-Colombia using real-time PCR. A PCR hybridization probe format was used to analyze the presence of L. monocytogenes, using specific primers to amplify a 149 bp fragment from the metalloprotease gene (mpl). In this study on a total number of 81 samples, 21 gave positive results for L. monocytogenes by real-time PCR and 13 samples were positive by conventional method. These results indicate a high presence of this pathogen in this area of the country and that this method is considerably faster than current standard methods.     

Lactic acid bacteria in an alginate film inhibit Listeria monocytogenes growth on smoked salmon

Antimicrobial packaging with lactic acid bacteria incorporated into the film matrix is a novel approach for controlling the growth of food-borne pathogens in ready-to-eat food. The overall objective of this study was to assess the effect of two strains of lactic acid bacteria (LAB) and nisin trapped in an alginate matrix, on Listeria monocytogenes growth on vacuum packed cold-smoked salmon. A film was formulated containing two LAB strains and nisin (100 IU/mL). LAB viability and bacteriocin like substance production (BLS) were assessed using the plate antagonism technique. To check the film antagonistic activity, pieces of salmon (4.0 × 4.0 cm²), inoculated with L. monocytogenes at a final concentration of 10⁴ CFU/cm², were covered with film containing both LAB strains plus nisin and stored at 4 °C. L. monocytogenes colonies on OXA agar were counted after 0, 7, 14, 21 and 28 days to evaluate pathogen inhibition. All treatments led to effective diffusion of the BLS that inhibited L. monocytogenes for 20 days after film preparation, with inhibition zones of 5.7 cm² for film coupons of 8 mm in diameter. After 28 days, salmon pieces covered with the film without inhibitors showed an increase of 2.4 log cycles in L. monocytogenes growth. In contrast, films with either LAB strain or a combination of both strains and nisin had a bacteriostatic effect on the pathogen over a period of 28 days, which exceeds the industrial standard shelf life for smoked salmon. The results demonstrate that these films inhibit L. monocytogenes growth on salmon during refrigerated storage.     

Microbial quality assessment and pathogen inactivation by electron beam and gamma irradiation of commercial seed sprouts

The microbiological quality of fresh sprouts and their seeds and the potential use of electron beam and gamma irradiation in inactivating inoculated pathogens in both samples were evaluated. High levels of aerobic bacteria and coliforms were enumerated in sprouts. Red radish, alfalfa and broccoli sprouts were positive for Listeria monocytogenes, while all seed samples were negative for pathogens. Red radish and broccoli sprouts and their seeds were inoculated with Escherichia coli O157:H7, Salmonella typhimurium, L. monocytogenes and Bacillus cereus and irradiated up to 3.0 kGy. The D₁₀ values of the inoculated pathogens were lower in both broccoli and red radish samples treated with gamma ray than with electron beam, while the D₁₀ values obtained in seeds were relatively higher compared with sprouts. This study demonstrated the poor microbiological quality of commercial sprout and the potential health risk it poses. Irradiation at appropriate doses is a promising approach for producing safe and pathogen-free sprouts for consumers.     

Effects of lactic acid and lauricidin on the survival of Listeria monocytogenes,Salmonella enteritidis and Escherichia coli O157:H7 in chicken breast stored at 4 °C

Lauricidin and lactic acid were evaluated for their effects on growth and survival of Listeria monocytogenes (L55), Salmonella enteritidis (S552) and Escherichia coli O157:H7 (E19) inoculated onto raw chicken breast. Fresh, raw chicken breasts were purchased immediately after slaughter and transported on ice to the laboratory within 20 min. Each chicken breast was decontaminated by briefly dipping in 70% ethanol and passed through a flame of a Bunsen burner and then allowed to cool. The decontaminated Chicken breast was dipped in TSB broth, at room temperature (25 °C) for 15 min, containing approximately log 9 CFU/ml of L. monocytogenes, S. enteritidis or E. coli O157:H7. Initial counts of L. monocytogenes, S. enteritidis or E. coli O157:H7 counts in chicken breast immediately after dipping in TSB broth were in the range of log 7–log 8 CFU/g. After inoculation, the chicken breasts were kept at room temperature for 20 min to allow attachment. Each inoculated chicken breast (25 °C) was dipped in 0 (control – sterile water), 0.5%, 1%, 1.5% or 2% of lauricidin (w/v) or lactic acid (v/v) for 10, 20 or 30 min and then individually placed in oxygen-permeable polyethylene bags. Breasts were subjected to microbiological analyses after treatment (day 0) and after storage for 2, 5, 7, 10 and 14 d at 4 °C. Initial counts of L. monocytogenes, S. enteritidis and E. coli O157:H7, in chicken breast treated with lauricidin decreased by 2.90, 1.31 and 2.27 log CFU/g, respectively. Lauricidin was more effective in reducing L. monocytogenes population than S. enteritidis and E. coli O157:H7 population. Dipping chicken breast in lauricidin for 30 min caused a significant reduction of L. monocytogenes, S. enteritidis and E. coli O157:H7 population compared to 10 and 20 min dipping. Initial L. monocytogenes, S. enteritidis and E. coli O157:H7 counts on chicken breast treated with lactic acid decreased by 1.97, 1.71 and 2.59 log CFU/g, respectively. Lactic acid caused a higher reduction in initial S. enteritidis and E. coli O157:H7 counts compared to lauricidin.     

Application of an optimized 18-h method involving one step culturing and single primer-based PCR assay for detection of Salmonella spp. in foods

A polymerase chain reaction (PCR) using 20-mer oligonucleotide single primer (named primer 3) randomly designed on the basis of Salmonella Typhimurium gatD gene encoding galactitol-1-phosphate dehydrogenase could produce the specific DNA product of approximate 770-bp in all 38 Salmonella strains used. No 770-bp DNA band was amplified from any DNA samples of 20 non-Salmonella bacteria. The DNA band was detected by 1% agarose gel electrophoresis and ethidium bromide staining. The sensitivity of the RAPD–PCR assay for detection of pure genomic DNA from S. Typhimurium ATCC 13311 and Salmonella Enteritidis DMST 15676 was as few as 0.01 ng. When simple boiling in TE buffer for 5 min was used for extraction both Salmonella spp. DNA, the detection limits were at least 230 and 320 cells, respectively. By using the RAPD–PCR assay following at least 14 h pre-enrichment in nutrient broth (NB), as few as 1 CFU of S. Typhimurium ATCC 13311 or S. Enteritidis DMST 15676 per 25 g of autoclaved chicken meat was detected. When the optimized 18-h method involving pre-enrichment in NB and DNA extraction by boiling protocol followed by RAPD–PCR using primer 3 was evaluated in comparison with the conventional method on 195 possibly naturally-contaminated food samples, 36 samples were found positive by both methods. In addition, the results of the developed RAPD–PCR-based assay proved to be identical to those by the conventional method. The optimized 18-h method was simple, rapid and sensitive, achieved the same detection limit as the conventional method and produced a zero level of false-negative results.     

Aqueous extracts of Hibiscus sabdariffa calyces as an antimicrobial rinse on hot dogs against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus

Contamination of ready-to-eat meat products by foodborne pathogens is a major concern in the food industry. Novel methods to control foodborne pathogens are made necessary by continuing outbreaks as well as the development of antibiotic-resistant pathogens. Hibiscus sabdariffa extracts could be useful as a natural source of antimicrobial rinse on ready-to-eat products to control pathogens. In this study, lyophilized Hibiscus flower extracts were examined for their antimicrobial activity as a rinse on all-beef hot dogs against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus (MRSA). Beef hot dogs were dip inoculated in overnight cultures of 1:1 mixtures of L. monocytogenes strains Scott A and 101 or MRSA strains ATCC 33591 and ATCC 33593 and were placed at 4 °C overnight to allow for bacterial attachment. Hot dogs were rinsed with extracts (120, 240 mg/mL) or water (control) for 5, 15, 30, or 60 min and then plated immediately (0 h; no storage) or stored at 4 °C overnight and plated at 24 h. Serial dilutions were plated in duplicate on both TSA and selection media, Modified Oxford (Listeria) or Baird Parker (MRSA), and the entire experiment was replicated 3 times. Higher extract concentrations, longer rinse times, and longer storage times were the most effective at inhibiting and/or killing L. monocytogenes and MRSA on hot dogs. L. monocytogenes was reduced to ca. 1.5 log CFU/g while MRSA was reduced to undetectable levels following rinsing of hot dogs with extracts at 240 mg/mL for 60 min and stored for 24 h. Both L. monocytogenes and MRSA were reduced ca. 2 log CFU/g following rinsing of hot dogs with extracts at 120 mg/mL for 60 min and stored for 24 h. This research demonstrates the effectiveness of Hibiscus extracts against L. monocytogenes and MRSA as an antimicrobial rinse on ready-to-eat meat products.     

Antibacterial effect of black seed oil on Listeria monocytogenes

Listeria monocytogenes is a major foodborne pathogen in the United States. Effective methods for reducing L. monocytogenes in foods would reduce the likelihood of foodborne outbreaks of listeriosis, and decrease economic losses to the food industry. Nigella sativa is a herbaceous plant, whose seeds (black seed) have been used as a spice and condiment in foods in the Middle East. The objective of this study was to determine the antibacterial effect of black seed oil on twenty strains of L. monocytogenes by disc diffusion method. A population of 7.0 log CFU of each strain of L. monocytogenes was inoculated on duplicate plates containing antibiotic medium one agar. The plates were allowed to dry at room temperature for 15 min. Three discs (6 mm diameter), each impregnated with 10 μl of black seed oil, vegetable oil (oil control), or gentamicin (positive control) were placed on each inoculated plate. The plates were incubated at 37 °C for 24 h, and were observed for zones of L. monocytogenes growth inhibition. Black seed oil exhibited a strong antibacterial activity against all the strains of L. monocytogenes, yielding a significantly (P<0.01) larger inhibition zone than that of gentamicin. The mean zones of inhibition produced by black seed oil and gentamicin were 31.50 ± 1.0 and 14.80 ± 0.50, respectively. The vegetable oil had no inhibitory effect on L. monocytogenes. Results indicate that black seed oil could potentially be used to inhibit L. monocytogenes, but appropriate applications in foods need to be validated.     

Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR

The wide application of nucleic acid amplification techniques and the increasing industrial interest toward rapid methods has led to the development and application of PCR based methods for the detection of microbial pathogens in food. In the present paper we describe the development of a multiplex PCR method for simultaneous detection of Salmonella enterica serovar Typhimurium, Listeria monocytogenes and Escherichia coli O157:H7 in a complex food matrix (liquid whole egg).Four different DNA extraction procedures were evaluated for their application on food and, among these, Chelex resin combined with a DNA purification step were found to better perform on the food system considered.A multiplex PCR system was developed, based on the evaluation and combination of published primer sets, and applied to the simultaneous detection of the target pathogens plus an internal amplification control, both in culture media and in a model food system.The overall system proposed, based on an overnight enrichment step followed by DNA isolation and multiplex PCR, was satisfactorily tested for its specificity and sensitivity and allowed the detection of the presence of bacterial DNA and the identification of the target pathogens down to 10 cells/25 g liquid whole egg.     

Detection of hazelnut (Corylus spp.) in processed foods using real-time PCR

Hazelnut seeds (Corylus spp.) are source of allergens and could cause severe adverse reactions in sensitized subjects, even if consumed in traces. This work presents two sensitive real-time PCR methods to quantify hazelnut in foods, one using the Sybr green dye and one based on hazelnut-specific Taqman probe designed on Cor a1.04 gene (specific amplicon: 82 bp). The sensibility and the robustness of the method were estimated analyzing spiked samples and some commercial hazelnut-containing creams. The lowest detection limit was 0.1 ng of genomic DNA. A qualitative specific PCR and a comparison of different DNA extraction protocols are also discussed.     

Lactic acid bacteria biodiversity in Italian marinated seafood salad and their interactions on the growth of Listeria monocytogenes

The aim of this research was to study the biodiversity of lactic acid bacteria (LAB) in marinated seafood salad (pH 5.0) and their interaction on the growth of Listeria monocytogenes. LAB were highly present in the samples considered in this study, reaching values of 8.0 log cfu/g at the end of product’s shelf-life. A high biodiversity in terms of LAB species and strains was detected by means of RAPD–PCR within the 171 bacterial isolates collected. Among them Lactobacillus curvatus, Lactobacillus sanfranciscensis and Enterococcus were present in all the salad batches considered. Three challenge tests against L. monocytogenes were carried out in order to assess the growth of this pathogen in the presence of dominant populations of LAB. L. monocytogenes tended to decrease in time, suggesting that a stable concentration of LAB inhibits the development of this pathogenic micro-organism.     

Prevalence of foodborne pathogens in open markets and supermarkets in Thailand

This study was conducted in Thailand (Bangkok and Pathum Thani provinces), from June 2006 to July 2007, in order to assess the prevalence of Listeria monocytogenes, Escherichia coli O157, Salmonella, Shigella and Vibrio parahaemolyticus in foods. Retail raw meats and seafood, including chicken (n = 109), pork (n = 80), beef (n = 108), shrimp (n = 43) and oysters (n = 48), from open markets and supermarkets were analyzed. Salmonella was found in 22 of 61 (36%) open market samples (48% of chicken, none of pork and beef, and 53% of shrimp) and in 12 of 75 (16%) samples from supermarkets (57%, 12%, 24%, 0% respectively). However, a small number of L. monocytogenes were isolated, where 6 of 217 (3%) were samples from open markets (6% of chicken and 3% of pork) and 17 of 171 (10%) were from supermarkets (3% of beef, 4% of chicken, and 32% of pork). In both markets, L. monocytogenes was not detected from shrimps, neither from oysters. E. coli O157, Shigella and tdh-positive V. parahaemolyticus were not isolated in this collection. Several Salmonella and L. monocytogenes isolates were multidrug-resistant. Both markets would need better assessment, since multidrug-resistant strains have been isolated and they may lead to therapeutic failure.     

Antimicrobial activity of clove (Syzgium aromaticum) oil in inhibiting Listeria monocytogenes on chicken frankfurters

The ability of Listeria monocytogenes to survive and grow at refrigeration temperature in some ready to eat (RTE) poultry products is a public health concern. The inhibitory effect of clove oil (1% and 2%, v/w) applied to the surface of RTE chicken frankfurters was determined on seven strains of L. monocytogenes inoculated at low (10²–10³ cfu/g) or high cell numbers (10⁴–10⁶ cfu/g), and stored at 5 °C for 2 weeks or at 15 °C for 1 week. All strains of L. monocytogenes survived and grew on control frankfurters at 5 °C and 15 °C but growth was inhibited under both storage conditions in the presence of either 1% or 2% clove oil. Depending on the sensory considerations, the addition of clove oil to frankfurters may be an effective strategy to control L. monocytogenes in chicken frankfurters.     

Use of lactic acid bacteria (LAB) biofilms for the control of Listeria monocytogenes in a small-scale model

The antilisterial activity in biofilms developed in a small-scale model by two LAB (lactic acid bacteria) bacteriocin producers (Lactobacillus plantarum 35d, Enterococcus casseliflavus IM 416K1) and by two non-producers (L. plantarum 396/1, Enterococcus faecalis JH2-2) was evaluated against Listeria monocytogenes NCTC 10888. The LAB biofilms showed the capability to influence the survival and the multiplication of the pathogen with differences among the strains. L. plantarum 35d displayed the highest efficacy reducing L. monocytogenes by 5.4 log in the planktonic population and by 3.9 log in the adherent population at the end of the experiment (10 days). L. plantarum 396/1 reduced L. monocytogenes by 3.8 log in the adherent cells and by 4.9 log in the planktonic cells and this outcome could be attributed to the pH reduction.The E. casseliflavus IM 416K1 biofilm caused a L. monocytogenes reduction of 3.7 log in the adherent cells and of 4.8 log in the planktonic cells and the role of the bacteriocin production seemed to be predominant as the pH values did not significantly decrease. This hypothesis is confirmed by a slight capability to influence the L. monocytogenes survival by the non-bacteriocinogenic E. faecalis JH2-2. Studies performed with L. monocytogenes in co-culture with a Pseudomonas putida strain, revealed a reduction of the antilisterial activity only for the biofilms produced by lactobacilli.     

Anti-listerial activity of ethanolic extracts of medicinal plants,Eremophila alternifolia and Eremophila duttonii,in food homogenates and milk

Listeria monocytogenes is a foodborne pathogen responsible for the disease listeriosis. Ethanolic extracts from two native Australian traditional medicinal plants, Eremophila alternifolia and Eremophila duttonii, have been found to inhibit the growth of L. monocytogenes. These plants were investigated for their ability to control the growth of L. monocytogenes in full cream milk and skim milk and in diluted homogenates of salami, pâté and brie cheese. Time-kill experiments indicated that the extracts were able to inhibit the growth of L. monocytogenes in food at 4 °C and 37 °C. However, components in the food appeared to inhibit the anti-listerial activity of the extracts, necessitating higher concentrations to control microbial growth relative to those used in laboratory media. Preliminary investigations suggested that the active components responsible for the antimicrobial activity of each extract are most likely to be terpenes or sterols. Our study suggests that natural products derived from medicinal plants have the potential to be used as food preservatives.     

Influence of food soiling matrix on cleaning and disinfection efficiency on surface attached Listeria monocytogenes

Cleaning and disinfection are essential steps in preventing contamination of foods with pathogenic and spoilage bacteria. The efficacy of cleaning and disinfection products differ depending on target bacteria and type of soiling. We evaluated the effectiveness of cleaning and disinfecting products against Listeria monocytogenes attached on food soiled inert surfaces in a laboratory model. The number of bacteria on surfaces before and after treatment was quantified using indirect conductometric measurements. L. monocytogenes attached to stainless steel surfaces in fish broth systems reached approx. 10⁴ CFU/cm². However, all cleaning and disinfection products were equally effective in this system since all bacteria were removed or killed. When the steel disks were immersed in a fish or meat emulsions, a level of approx. 10⁵–10⁶ L. monocytogenes per cm² was reached after 2–3 days at 20 °C. In this case, 2–3 log were removed or killed by alkaline cleaning products (MC103 or FC140). Direct use of a peracetic acid based disinfectant (Oxivit Active Plus) killed all bacteria when attached in salmon emulsions, whereas only 1–2 log were removed or killed in the meat emulsion system. Thus, the efficiency of cleaning and disinfection products against L. monocytogenes is strongly influenced by the food matrix.     

Listeria monocytogenes in raw salad vegetables sold at retail level in Malaysia

A range of commercially available vegetables (n = 306) that are consumed in the minimally processed state in Malaysia was examined for the presence of Listeria spp. and Listeria monocytogenes to provide information on the occurrence of such organisms in these vegetables. Analysis was carried out using the most probable number–polymerase chain reaction (MPN–PCR) method. It was found that Listeria spp. and L. monocytogenes could be detected in 33.3% and 22.5% of the vegetables respectively. L. monocytogenes was more frequently detected in Vigna unguiculata (Japanese parsley) at 31.3% and Oenanther stolonifera (yardlong bean) at 27.2%.