Safety assessment of smoked fish related to Listeria monocytogenes prevalence using risk management metrics

One of the fundamental objectives of European food law is the protection of human health. In this framework, the administration has to ensure that there are control measures from “farm to fork”, which maintain product safety in each stage of the food chain. With this in mind, the objective of this paper was to assess the level of safety of smoked fish in relation to Listeria monocytogenes in the early stages of the chain. This was carried out by evaluating the results obtained by the official control of the Valencian region related to the level of implementation of pre-requisites and HACCP. The prevalence of this organism in the industry and the retail stage was also measured. In order to discern whether these values were within the international consumer protection objectives a practical case focusing on smoked salmon was studied. The results showed that the management system in the industry is effective. However, there is a real increase in the prevalence in the samples taken in the supermarket. The ALOP values estimated for smoked salmon indicated that the level of safety achieved is good in a very high percentage of cases, though governments and the different agents in the food chain must continue working to improve and attain new safety goals.     

Listeria monocytogenes prevalence, contamination levels and strains characterization throughout the Parma ham processing chain

In order to estimate prevalence, levels and patterns of Listeria monocytogenes contamination, a total of 774 swine carcasses were traced along the Parma ham production chain. Analyses were conducted on isolates originated from the same carcass, collected at different stages during processing, resulting in 0.2% (faeces at intestine removal from carcasses), 3.0% (swabbing of carcasses), 12.5% (fresh hams) and 2.0% prevalence (dry-cured hams). The highest contamination levels of L. monocytogenes were reached in fresh hams after cutting and were followed by a marked decrease during the subsequent processing stages. All the 132 isolates were characterized by serotyping and Pulsed-Field Gel Electrophoresis (PFGE). Transfer of L. monocytogenes between different stages of the processing chain was not reported, whereas processing itself has proved to be an important cause of contamination. The sole isolate of fecal origin belonged to a pulsotype that was uncommon to any of those recovered in carcasses, fresh hams and dry-cured hams, indicating that contaminations from farms does not significantly affect Parma ham production. For the majority of the strains isolated from the same production plants, PFGE profiles were highly similar. In several cases, the same pulsotypes were recurrently detected, over time, in carcasses and fresh ham samples sharing the same processing environment. A_w levels were also measured, showing that drying of the ham surface was able to induce a considerable decrease of the contamination levels, although unable to ultimately remove L. monocytogenes.     

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.     

Inhibition of Listeria monocytogenes by pomegranate (Punica granatum) peel extract in meat paté at different temperatures

Natural antimicrobials are being more and more considered as alternative approach for controlling growth of microorganisms in food. The objective of this study was to evaluate the pomegranate extract’s (PE) potential to be used as a natural preservative in ready to eat meats. Listeria monocytogenes was the main target. In a preliminary assessment with the disk diffusion method PE showed inhibitory effect against all five tested species, in the following order of increasing sensitivity: L. monocytogenes, Bacillus subtilis, Bacillus cereus, Escherichia coli and Staphylococcus aureus.No viable cells of L. monocytogenes were detected after incubation in BHI broth in presence of 7.5% v/v of the liquid PE (or 24.7 mg dry PE/ml). This concentration was considered as the Minimal Bactericidal Concentration (MBC) of the tested PE. Two pure components commonly found in PE, namely gallic and ellagic acids were also tested in BHI broth, however they did not show considerable inhibition of L. monocytogenes. PE in a concentration equal to the measured MBC was tested against L. monocytogenes in meat paté at different temperatures. At 4 °C during 46 days the extract inhibited the growth in meat paté by 4.1 log CFU/g compared to the control, which had reached log 9.2 CFU/g already on the 18th day. Inhibition was less pronounced at higher temperatures. The results indicate that the PE has a potential to be used as a natural preservative in meat products.     

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.     

Use of epifluorescence microscopy to assess the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces

One source of foodborne listeriosis is related to a virulent strain established in the food-processing environment. We used direct epifluorescence microscopy (DEM) to evaluate the effectiveness of phage P100 in controlling Listeria monocytogenes biofilms on stainless steel surfaces, in wet conditions at room temperature. Biofilms that were allowed to develop for 72 h were subsequently treated with different concentrations of phage (5, 6, 7, and 8 log PFU/ml). L. monocytogenes was monitored up to 48 h, using both DEM and cultivation method. When L. monocytogenes was monitored using a cultivation method, the first significant reduction was observed after phage treatment with 7 and 8 log PFU/ml at 8 h. Subsequently, these treatments achieved undetectable levels of pathogen at 48 h, with a mean reduction of 5.29 log CFU/cm². Conversely, when samples were evaluated using DEM in treatments with 6, 7, and 8 log PFU/ml, although disaggregation of biofilms could be observed after 8 h, viable cells were still present up to 48 h (maximal reduction 1.5 log units). The phage titer remained stable or increased up to 2.59 log units during the study period. In conclusion, phage P100 may provide an adjuvant measure to control L. monocytogenes biofilm on stainless steel surfaces. However, phage treatment must be used in combination with other hygienization measures to increase efficacy. In this study, DEM was a good tool to quickly and accurately assess the real effect of phage P100 on L. monocytogenes biofilms.     

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.     

Prevalence of Listeria spp. in ready to eat foods (RTE) from Algiers (Algeria)

The aim of this study was to establish the occurrence of Listeria spp., especially Listeria monocytogenes in ready to eat RTE food marketed in Algiers (Algeria).A total of 227 samples were collected from different producers and retailers.All samples were analyzed using a conventional cultivation method AFNOR V08-055.Out of 227 samples tested, 21 (9.3%) tested positive for Listeria spp. among them, 6 (2.6%) tested positive for L. monocytogenes. L. innocua was the most common Listeria species found being detected in 11 samples (4.8%), although both Listeria ivanovii and Listeria welshimeri were detected in 3 (1.3%) and 1 (0.4%) food samples respectively.The study of the antimicrobial sensitivity of Listeria monocytogenes strains showed no resistance.The study has enabled us to detect these contaminants in a wide range of RTE foods, to suggest that contamination likely occurs after heat treatment, and to assess the danger represented by this category of food for populations at risk.     

Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR

The increase of foodborne outbreaks associated with fresh vegetables has highlighted the importance of developing rapid and specific methods for the detection and quantification of foodborne pathogens. In this sense, real-time PCR (qPCR) fulfills these requirements although it may detect dead cells. Recently, a potential strategy to specifically detect viable cells has been proposed relying on the use of DNA binding molecules as sample pretreatment previous to the qPCR. In this study propidium monoazide (PMA) and reagent D, combined with qPCR, were evaluated for the detection and quantification of viable Escherichia coli O157:H7, Salmonella and Listeria monocytogenes. Initially, the optimal concentration of both reagents was determined for discrimination between viable and dead bacteria in cell suspensions. Although both reagents showed similar reductions for the three pathogens, reagent D was toxic to L. monocytogenes and Salmonella and therefore only PMA was used to evaluate the applicability of this technique on food samples. A final concentration of 50 μM PMA was assayed in artificially inoculated spinach and mixed salad. PMA-qPCR signal was negative for all dead cell concentrations tested except for mixed salad inoculated with L. monocytogenes at the highest concentration. These results demonstrate that PMA-qPCR is a suitable technique for the detection and quantification of viable pathogens in fresh-cut vegetables at the levels normally found in vegetable samples.     

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.     

Inactivation of Listeria innocua in brined white cheese by a combination of nisin and heat

The objective of this study was to investigate the effect of nisin alone and in combination with heat (63 °C/5 min) on the inactivation of Listeria innocua in white cheese. Nisin was added at different concentrations (500, 1000, and 1500 IU ml⁻¹) to pasteurized milk before curd formation. The curd was soaked for 24 h in 10% solution of brine containing ca 10⁶ CFU ml⁻¹ of a cocktail mixture of three strains of L. innocua. Part of the nisin treated samples were heat treated at 63 °C/5 min. Total mesophilic count (TMC), L. innocua survivors and changes in the pH of white cheese were monitored each 2 d for a period of 12 d of storage at 4 or 10 °C. Nisin at 500 IU ml⁻¹ did not diminish TMC in white cheese compared to the control. The combination of heat and nisin (1000 or 1500 IU ml⁻¹) exhibited a bacteriostatic effect on TMC throughout the storage period at 4 or 10 °C. Nisin at 500 IU ml⁻¹ had a marginal inhibitory activity against L. innocua. However, nisin at 1000 and 1500 IU ml⁻¹l resulted in a more than 2 log₁₀ reduction in L. innocua count and the effect was more prominent at 10 °C. In comparison, the combination of nisin (1000 or1500 IU ml⁻¹) and heat treatment exhibited a synergistic inhibitory activity against L. innocua, where a complete elimination of the organism was accrued after 6 and 8 d of storage at 10 and 4 °C. Therefore, nisin and heat combination could be used as a prudent hurdle to preclude the growth of Listeria in white cheese, especially under the condition of abused refrigeration conditions.     

Seasonal prevalence of Vibrio species in retail shrimps with an emphasis on Vibrio parahaemolyticus

Seasonal prevalence of Vibrio species in shrimp samples from retail outlets in the South-western part of Iran was studied. A total of 300 samples were analyzed (75 samples in each season). Special attention was paid to the prevalence of total and pathogenic Vibrio parahaemolyticus. All the TCBS isolates were first identified to the genus level with PCR and then identified to the species level using a battery of biochemical reactions and tests. To investigate the pathogenicity of the isolated V. parahaemolyticus, multiplex PCR (tl, tdh and trh genes) was performed. Vibrios were detected during the whole investigation period, depending on the sampling season. They were detected in 18.6% of the winter samples, 64% of the spring samples, 70.6% of the summer samples and 41.3% of the autumn samples. Vibrio calviensis and Vibrio alginolyticus were dominant in samples of different seasons, with the average prevalence of 18.6% and 17.6%, respectively. V. parahaemolyticus was found in 4.0% of the winter samples, 13.3% of the spring samples, 18.6% of the summer samples and 8% of the autumn samples. During the period of this study, two tdh-positive strains were isolated, while no trh-positive V. parahaemolyticus strain was detected in samples of different seasons.     

Antibiotic resistance in Campylobacter jejuni and Campylobacter coli isolated from food in Poland

This study presents the results of investigations on the susceptibility of Campylobacter spp. strains isolated from chicken meat and giblets to fluorochinolones (ciprofloxacin), macrolides (erythromycin), tetracyclines (tetracycline) and aminoglycosides (gentamicin) andV an analysis of the molecular mechanisms of resistance to the selected antibiotics.Between January 2008 and December 2009 a total of 218 samples of chicken meat and giblets from retail trade in Poland were examined. Campylobacter bacteria were found in 143 samples, that is in 65.6% of the total number embraced by the study. Campylobacter coli was the most ubiquitous - its presence was determined in 108 samples out of 143 (75.5%), whereas Campylobacter jejuni was found in 35 of the contaminated samples (24.5%).The results obtained point to the high percentage (97.9%) of Campylobacter isolates resistant to ciprofloxacin. 92 strains (64.3%) were resistant to tetracycline, 14 (9.1%) to erythromycin and only 9 (6.3%) to gentamicin. Moreover, ten out of the 143 resistant Campylobacter strains (7.0%) were found to be resistant to at least three unrelated antibiotics.     

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.     

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.     

Anti-listeria activity of Pediococcus pentosaceus BCC 3772 and application as starter culture for Nham, a traditional fermented pork sausage

Lactic acid bacteria forming bacteriocin active against Listeria monocytogenes were screened for potential use in controlling growth of L. monocytogenes in Nham, a Thai traditional fermented pork sausage. Based on the spot-on-lawn assay, Pediococcus pentosaceus BCC 3772 was found to produce the highest anti-listeria activity. Through a series of chromatographic techniques, Edman N-terminal sequencing and mass spectrometry, this anti-listeria compound showed a complete homology to pediocin PA-1/AcH. Inoculation of P. pentosaceus BCC 3772 in Nham caused a significant decrease of 3.2 log in population of spiked L. monocytogenes within 18-24 h of fermentation, in comparison with the initial count with no significant changes in sensory properties and consumer acceptability on the overall characteristics of the final fermented Nham products. These results indicate that the P. pentosaceus BCC 3772 should be useful as a functional starter culture for control of L. monocytogenes without compromising the unique quality of Nham.     

Removal of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 biofilms on stainless steel using scallop shell powder

Biofilms on steel surfaces containing Listeria monocytogenes, Staphylococcus aureus and Escherichia coli O157:H7 continue to threaten dairy and meat processors. In this study, the ability of scallop shell powder (SSP) to remove biofilms formed by these three pathogens on stainless steel plates was examined. Whey powder solution (WPS) and bench wash water (BWW) provided by dairy and meat factories, respectively, were inoculated with L. monocytogenes, S. aureus or E. coli O157:H7 (9 log₁₀ CFU/ml). Stainless steel plates (10 cm²) were placed in the inoculated fluids and incubated at 20 °C at 48 h to form biofilms. After drying and washing in sterile water, the plates were treated with 0.0, 0.25, or 0.50% (w/v) SSP slurries for 1, 5, or 10 min and then quantitatively examined for the three pathogens. Both 0.25 and 0.50% SSP reduced L. monocytogenes on the plates by 4 log CFU/cm² with a 1 min exposure to 0.50% SSP decreasing S. aureus by 5 logs CFU/cm². After 1 min in 0.25 and 0.50% SSP, E. coli O157:H7 populations in WPS and BWW biofilms decreased 4 and 6 log CFU/cm² and 3 and 5 log CFU/cm², respectively. Increasing the concentration of SSP led to significantly increased efficacy against the tested pathogens (P < 0.05). In conclusion, this study showed that SSP slurries could significantly reduce the numbers of L. monocytogenes, S. aureus and E. coli O157:H7 in biofilms on stainless steel surfaces.     

Presence of Fusarium emerging mycotoxins in tiger-nuts commercialized in Spain

Tiger-nuts are tubers from the plant Cyperus esculentus which are commercialized in some regions of West and Central Africa, USA and Spain. These tubers have a high microbial charge such as bacteria, moulds and yeasts which could remain until the finished product. In this study we examined 47 samples of tiger-nut tubers purchased from Spanish supermarkets for contamination with the emerging Fusarium mycotoxins: Enniatins ENs (EN A, EN A₁, EN B and EN B₁), beauvericin (BEA) and fusaproliferin (FUS). The extraction of the samples was carried out with methanol using an Ultraturrax homogenizer. Mycotoxins were analyzed with a liquid chromatography (LC) coupled to a diode array detector (DAD).The percentage of contaminate samples with one or more emerging mycotoxins was 21.3%. EN A₁ result the most common EN found with the highest prevalence of 17% with levels ranging from 32.3 to 4400 μg/g. EN B₁ and BEA were present simultaneously in 5 samples, whereas the maximum contamination observed of the EN B₁ was of 346 μg/g. The lowest incidence were observed for the ENs A and B evidenced only in one sample of the total studied. Only one emerging mycotoxin, fusaproliferin (FUS), has not been detected in any sample studied.The present work is the first one ever drafted on the presence of the emerging Fusarium mycotoxins in Spanish tiger-nuts.