Inactivation kinetics of pulsed electric field-resistant strains of Listeria monocytogenes and Staphylococcus aureus in media of different pH

A study of the effect of pulsed electric fields (PEF) on the inactivation of Listeria monocytogenes STCC 5672 and Staphylococcus aureus STCC 4459 in McIlvaine buffer covering a range from pH 3.5 to 7.0 was conducted. Mathematical models based on the Weibull distribution were developed to describe the influence of the electric field strength, treatment time and pH of the treatment medium on the lethality of both Gram positive pathogenic bacteria after PEF treatments. Both microorganisms were more sensitive to PEF in media of low pH, although the influence of the pH on the PEF resistance was more significant in S. aureus. In the best cases scenario, the highest inactivation levels achieved were 3.3 and 6.1 log₁₀ cycles for L. monocytogenes and S. aureus respectively in pH 3.5 after 500 μs of 35 kV/cm. Based on these results and those observed in literature, L. monocytogenes STCC 5672 at any pH investigated has been shown as one of the most PEF resistant microorganism. Therefore, this microorganism should be considered as a possible target microorganism to define process criterion for PEF pasteurization.     

Effects of high hydrostatic pressure and varying concentrations of sodium nitrite from traditional and vegetable-based sources on the growth of Listeria monocytogenes on ready-to-eat (RTE) sliced ham

The objective of this study was to determine the effect the source of added nitrite and high hydrostatic pressure (HHP) had on the growth of Listeria monocytogenes on ready-to-eat (RTE) sliced ham. Use of 600 MPa HHP for 3 min resulted in an immediate 3.9–4.3 log CFU/g reduction in L. monocytogenes numbers, while use of 400 MPa HHP (3 min) provided less than 1 log CFU/g reduction. With the 600 MPa HHP treatment, sliced ham with a conventional concentration of sodium nitrite (200 ppm) was not different in L. monocytogenes growth from use with 50 or 100 ppm of sodium nitrite in pre-converted celery powder. Instrumental color values as well as residual nitrite and residual nitrate concentrations for cured (sodium nitrite and nitrite from celery powder) and uncured ham formulations are discussed.     

Listeria monocytogenes and ready-to-eat seafood in Spain: Study of prevalence and temperatures at retail

The aim of this study was to obtain data from refrigerated ready-to-eat seafood products at retail in Spain (young eels, crabstick and smoked salmon), regarding prevalence and levels of Listeria monocytogenes, storage temperatures and the impact of transport conditions (type of bag) on the temperature of the product. The one-year surveillance period was carried out according to the EC Regulation No. 2073/2005, taking 5 units/batch and analyzing 250 samples following ISO 11290-1/A1 and ISO 11290-2/A methodologies. Low prevalence of L. monocytogenes was observed in surimi products, while 4.8% of smoked salmon samples were positive for Listeria with low levels (<10 cfu/g) and uneven pathogen distribution. A single company was responsible for 80% of the positive lots. All purchased products showed values higher than 4 °C at retail and an average increase of 2.5 °C or up to 6.2 °C was recorded when isothermal or plastic shopping bags were used for transport, respectively. To avoid noncompliance of the Food Safety Objective for L. monocytogenes in seafood RTE products more efforts from all stakeholders are needed, with special attention so as to improve control and maintenance of refrigerators at retail and to enhance consumer education regarding food safety practices.     

Survival and death of Listeria monocytogenes on cooked bacon at three storage temperatures

Survival of Listeria monocytogenes on cooked bacon cubes (a_w 0.910 ± 0.080), strips (a_w 0.726 ± 0.054), and bits (a_w 0.620 ± 0.038) was determined during a 25 week storage period at −20, 4.4, and 22 °C. Selective enrichment and subsequent enzyme-linked fluorescent antibody (ELFA) detection were used to asses survival on samples inoculated at ca. 1-log₁₀ CFU/g (LI). Samples inoculated at ca. 5.5-log₁₀ CFU/g (HI) were analyzed over time by direct plating on modified Oxford medium (MOX). The Baranyi model was fitted to the inactivation curves of HI samples using the DMFit program. At −20 °C, a decline of about 1-log₁₀ CFU/g occurred on all HI cooked bacon types by 14 weeks, although most LI samples remained positive by the ELFA detection method for 25 weeks. At 4.4 and 22 °C, some strips and bits LI samples were negative for the pathogen within 3 weeks, and >1.5 log₁₀ CFU/g reductions occurred on HI strips and bits by 8 weeks. Reductions on cubes at refrigeration and ambient temperature were ca. 0.5 log₁₀ CFU/g, and cubes remained positive on LI samples for 25 weeks. Rate parameter estimates indicated that the population declined fastest on strips and bits at 22 °C compared to all other product and temperature combinations. This study demonstrates that cooked bacon does not support the growth of L. monocytogenes and that the pathogen gradually dies off during storage.     

The effect of high hydrostatic pressure,sodium nitrite and salt concentration on the growth of Listeria monocytogenes on RTE ham and turkey

Growth of Listeria monocytogenes was evaluated for up to 182 days after inoculation on ready-to-eat (RTE) sliced ham and turkey breast formulated with sodium nitrite (0 or 200 ppm), sodium chloride (1.8% or 2.4%), and treated (no treatment or 600 MPa) with high hydrostatic pressure (HHP). HHP at 600 MPa for 3 min resulted in a 3.85–4.35 log CFU/g reduction in L. monocytogenes. With formulations at similar proximate analyses, one of the evaluation days (day 21) without HHP showed significantly greater growth of L. monocytogenes in ham than in turkey breast, but there were no significant differences on other evaluation days or with HHP. There were no differences in growth of L. monocytogenes due to sodium chloride level. Sodium nitrite provided a small, but significant inhibition of L. monocytogenes without HHP, but addition of sodium nitrite did not significantly affect growth of L. monocytogenes with use of HHP.     

Molecular subtyping and virulence gene analysis of Listeria monocytogenes isolates from food

A total of 67 Listeria monocytogenes isolates from 698 raw meat samples were characterized for molecular serogroup identification and antimicrobial susceptibility. Approximately one third (32.8%) of the isolates belonged to molecular serogroup 1/2a, 3a, followed by 1/2c, 3c (26.9%), 1/2b, 3b, 7 (22.4%), 4b, 4d, 4e (16.4%) and 4a, 4c (1.5%). Most of the L. monocytogenes isolates were susceptible to 14 antimicrobials tested but several were resistant to tetracycline, ciprofloxacin and nitrofurantoin. An additional 30 L. monocytogenes isolates from chicken and produce in our collection were also included to determine the presence of significant virulence markers. All 97 isolates carried inlC and inlJ except for a lineage III isolate 110-1. Most Listeriolysin S (LLS)-carrying isolates (11/12) belonged to lineage I, whereas the remaining one isolate belonged to lineage III. Five 4b, 4d, 4e isolates including two from turkey and three from produce belonged to Epidemic Clone I (ECI). Four molecular serogroup associated mutation types that lead to premature stop codons (PMSCs) in inlA were identified. PFGE and inlA sequence analysis results were concordant, and different virulence potential within 1/2a, 3a and 4b, 4d, 4e isolates were observed. The study revealed that a subset of isolates from meat and produce belonged to ECI, harbored inlC, inlJ and LLS, and produced full length InlA, suggesting that they be capable of causing human illness.     

Rhamnolipid and surfactin inhibit Listeria monocytogenes adhesion

The aim of this study was to assess the potential use of biosurfactants in inhibiting the Listeria monocytogenes strains adhesion to polystyrene surfaces. Surfactin and rhamnolipids were used. The adhesion profiles of 15 strains showed that most of these bacteria can be classified as moderate to strongly adherent. Both biosurfactants were able to reduce bacterial adhesion, and the effect was more pronounced against the strongly adherent strains. The most promising result was obtained for ATCC7644 strain, which showed an adhesion reduction of 84% for surfactin (30-h growth period). Rhamnolipids decreased the ATCC15313 adhesion by 82%. ATCC19112 adhesion was reduced by 53% for surfactin and purified rhamnolipid. Sodium dodecyl-sulphate was less effective than the biosurfactants, showing maximal adhesion reduction of 23% for 19112 and 7644. The purified rhamnolipid inhibited 100% of the growth of strongly adherent, suggesting that this surfactant can be exploited as a potential agent to control L. monocytogenes.     

Multiplex fiber optic biosensor for detection of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica from ready-to-eat meat samples

Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica are the most common foodborne bacterial pathogens and are responsible for many outbreaks. Therefore, multiplex detection of these three using a single assay platform is highly desirable. The objective was to develop and optimize a fiber optic sensor for simultaneous detection of these three from food. The streptavidin coated optical waveguides were immobilized with biotinylated polyclonal antibodies and exposed to the bacterial suspensions or enriched food samples for 2 h. Pathogens were detected after reacting with Alexa-Fluor 647-labeled monoclonal antibodies. Ready-to-eat beef, chicken and turkey meats were inoculated with each pathogen (∼100 cfu/25 g), enriched in SEL (Salmonella, E. coli, Listeria), a multipathogen selective enrichment broth for 18 h and tested with the biosensor. The biosensor was able to detect each pathogen, individually or in a mixture with very little cross-reactivity. The limit of detection for the sensor was ∼10³ cfu/ml for all three pathogens. Furthermore, the biosensor successfully detected each pathogen, grown in a mixture from enriched meat samples under 24 h. The pathogen presence was further verified by PCR and immunofluorescence assay. The multiplex fiber optic sensor shows promise for detection of the three pathogens if present in the same sample eliminating the use of multiple single pathogen detection platforms.     

Evaluation of atmospheric pressure plasma to improve the safety of sliced cheese and ham inoculated by 3-strain cocktail Listeria monocytogenes

The objective of this study was to evaluate the efficacy of atmospheric pressure plasma (APP), which is capable of operating at atmospheric pressure in air, in sliced cheese and ham inoculated by 3-strain cocktail of Listeria monocytogenes (ATCC 19114, 19115, and 19111, LMC). The process parameters considered were input power (75, 100, 125, and 150 W) and plasma exposure time (60, 90, and 120 s). Microbial log reduction increased with increases of input power and plasma exposure time. After 120 s APP treatments at 75, 100, and 125 W, the viable cells of LMC were reduced by 1.70, 2.78, and 5.82 log in sliced cheese, respectively. More than 8 log reductions can be achieved in 120 s at 150 W. In contrast, reductions after 120 s ranged from 0.25 to 1.73 log CFU/g in sliced ham. Calculated D values, the exposure time required to inactivate 90% of a population, from the survival curves of 75, 100, 125, and 150 W of APP treatments were 71.43, 62.50, 19.65, and 17.27 s for LMC in sliced cheese, respectively, and those in sliced ham were 476.19, 87.72, 70.92, and 63.69 s. No viable cells were detected at 125 and 150 W of APP treatment in sliced cheese, irrespective of plasma exposure time, after 1 week at a detection limit of 10¹ CFU/g. These results indicate that the inactivation effects of APP on L. monocytogenes are strongly dependent on the type of food.     

Effect of nanovesicle-encapsulated nisin on growth of Listeria monocytogenes in milk

Commercial nisin was encapsulated in nanovesicles (mean diameter 140 nm) prepared from partially purified soy lecithin. Nisin-loaded liposomes and unencapsulated (free) nisin were initially tested in BHI medium and skim milk inoculated with Listeria monocytogenes and incubated for 48 h at 30 °C. At such abuse temperature conditions, free nisin showed better inhibitory than the liposomal counterparts. Subsequently, the effect of encapsulated or free nisin was evaluated in combination with refrigeration (7 ± 1 °C) in both whole (3.25% fat) and skim (0% fat) milk for up to 14 day. A decrease of 3–4 log cycles in L. monocytogenes counts was observed for free and encapsulated nisin at 0.5 mg/ml concentration. Liposome encapsulation of antimicrobial peptides may be important to overcome stability issues and interaction with food components. The utilization of nanovesicle-encapsulated nisin in combination with low temperatures appeared to be effective to control L. monocytogenes in milk, emphasizing the importance of hurdle technology to assure food safety.     

The probability of growth of Listeria monocytogenes in cooked salmon and tryptic soy broth as affected by salt,smoke compound,and storage temperature

The objectives of this study were to examine and model the probability of growth of Listeria monocytogenes in cooked salmon containing salt and smoke (phenol) compound and stored at various temperatures. A growth probability model was developed, and the model was compared to a model developed from tryptic soy broth (TSB) to assess the possibility of using TSB as a substitute for salmon. A 6-strain mixture of L. monocytogenes was inoculated into minced cooked salmon and TSB containing 0–10% NaCl and 0–34 ppm phenol to levels of 10^2–3 cfu/g, and the samples were vacuum-packed and stored at 0-–25 °C for up to 42 days. A total 32 treatments, each with 16 samples, selected by central composite designs were tested. A logistic regression was used to model the probability of growth of L. monocytogenes as a function of concentrations of salt and phenol, and storage temperature. Resulted models showed that the probabilities of growth of L. monocytogenes in both salmon and TSB decreased when the salt and/or phenol concentrations increased, and at lower storage temperatures. In general, the growth probabilities of L. monocytogenes were affected more profoundly by salt and storage temperature than by phenol. The growth probabilities of L. monocytogenes estimated by the TSB model were higher than those by the salmon model at the same salt/phenol concentrations and storage temperatures. The growth probabilities predicted by the salmon and TSB models were comparable at higher storage temperatures, indicating the potential use of TSB as a model system to substitute salmon in studying the growth behavior of L. monocytogenes may only be suitable when the temperatures of interest are in higher storage temperatures (e.g., >12 °C). The model for salmon demonstrated the effects of salt, phenol, and storage temperature and their interactions on the growth probabilities of L. monocytogenes, and may be used to determine the growth probability of L. monocytogenes in smoked seafood.     

Reduction of Listeria monocytogenes on frankfurters treated with lactic acid solutions of various temperatures

United States regulations require ready-to-eat meat and poultry processors to control Listeria monocytogenes using interventions which may include antimicrobials that reduce post-processing contamination by at least 1 log-cycle; if the treatment achieves ≥2 log reductions, the plant is subject to less frequent microbial testing. Lactic acid (LA) may be useful as a post-lethality intervention and its antimicrobial properties may increase with temperature of application. The aim of this study was to evaluate the effect of LA solution concentration and temperature on L. monocytogenes counts of inoculated frankfurters and to identify parameters (concentration, temperature, and time) that achieve 1 and 2 log-unit immediate reductions. Frankfurters were surface-inoculated with a 10-strain mixture of L. monocytogenes (4.4 ± 0.1 log CFU/cm²) and then immersed in distilled water or LA solutions (0–3%) of 4, 25, 40, or 55 °C for 0–120 s. A regression equation for L. monocytogenes reduction included significant (P < 0.05) effects by the terms of concentration, time, temperature, and the interaction of concentration and temperature; other tested parameters (other interactions, quadratic and cubic terms), within the experimental range examined, did not affect (P ≥ 0.05) the extent of reduction. Results indicated that the effectiveness of LA against L. monocytogenes, in addition to concentration, increased with solution temperature (in the range of 0.6–2.8 log CFU/cm²). The developed equation may allow processors to vary conditions of treatment with LA to achieve a 1 or 2 log-unit reduction of the pathogen and comply with United States regulations.     

Sensory,physical and chemical characteristics of cooked ham manufactured from rapidly chilled and earlier deboned M. semimembranosus

Effects of rapid chilling of carcasses (at − 31 °C in the first 3 h of chilling, and then at 2–4 °C) and earlier deboning (8 h post-mortem), compared to rapid (till 24 h post-mortem) and conventional chilling (at 2–4 °C, till 24 h post-mortem), on quality characteristics of pork M. semimebranosus and cooked ham were investigated. Quality measurements included pH value, colour (CIEL*a*b* values) and total aerobic count of M. semimebranosus, as well as sensory (colour, juiciness, texture, and flavour), physical (pH value, colour – CIEL*a*b* values and texture – Warner–Bratzler shear and penetration forces) and chemical (protein, total fat, and moisture content) characteristics of cooked ham. The cooked ham was manufactured from pieces of M. semimebranosus with ultimate lightness (CIEL* value) lower than 50. Rapid chilling and earlier deboning significantly increased quantity of M. semimebranosus desirable for cooked ham manufacturing. Earlier start of pork fabrication did not affect important quality characteristics of cooked ham.     

Practical relevance of methodologies for detecting and tracing of Listeria monocytogenes in ready-to-eat foods and manufacture environments - A review

The multifaceted properties of Listeria monocytogenes allow the microorganism to grow and multiply in various food matrices even under adverse conditions. Therefore methods are needed to detect and trace this pathogen along the entire food supply network. Analytical methods have to fulfill several needs and also should meet the requirements of governmental, scientific and industrial parties. Among these demands, the level of detection based on genus and/or species or even strain specific information is of high practical significance to the food manufacturer. Hence, the release of sufficiently resolved information should be integrated into risk analysis and elucidation of contamination routes. This review aims at providing a current overview of methods for detecting, isolating and subtyping L. monocytogenes in various matrices, taking into account recent studies indicating the different drawbacks and advantages of commonly applied methods.     

Growth kinetics of Listeria monocytogenes and spoilage microorganisms in fresh-cut cantaloupe

The main objective of this study was to investigate the growth kinetics of Listeria monocytogenes and background microorganisms in fresh-cut cantaloupe. Fresh-cut cantaloupe samples, inoculated with three main serotypes (1/2a, 1/2b, and 4b) of L. monocytogenes, were incubated at different temperatures, ranging from 4 to 43 °C, to develop kinetic growth models. During storage studies, the population of both background microorganisms and L. monocytogenes began to increase almost immediately, with little or no lag phase for most growth curves. All growth curves, except for two growth curves of L. monocytogenes 1/2a at 4 °C, developed to full curves (containing exponential and stationary phases), and can be described by a 3-parameter logistic model. There was no significant difference (P = 0.28) in the growth behaviors and the specific growth rates of three different serotypes of L. monocytogenes inoculated to fresh-cut cantaloupe. The effect of temperature on the growth of L. monocytogenes and spoilage microorganisms was evaluated using three secondary models. For L. monocytogenes, the minimum and maximum growth temperatures were estimated by both the Ratkowsky square-root and Cardinal parameter models, and the optimum temperature and the optimum specific growth rate by the Cardinal parameter model. An Arrhenius-type model provided more accurate estimation of the specific growth rate of L. monocytogenes at temperatures <4 °C. The kinetic models developed in this study can be used by regulatory agencies and food processors for conducting risk assessment of L. monocytogenes in fresh-cut cantaloupe, and for estimating the shelf-life of fresh-cut products.     

Short communication: Biofilm production characterization of mecA and mecC methicillin-resistant Staphylococcus aureus isolated from bovine milk in Great Britain

Staphylococcus aureus is an important cause of contagious intramammary infection in dairy cattle, and the ability to produce biofilm is considered to be an important virulence property in the pathogenesis of mastitis. The aim of this study was to characterize the biofilm formation capacity of methicillin-resistant Staph. aureus (MRSA), encoding mecA or mecC, isolated from bulk tank milk in Great Britain. For this purpose, 20 MRSA isolates were grown on microtiter plates to determine the biofilm production. Moreover, the spa-typing and the presence of the intercellular adhesion genes icaA and icaD were analyzed by PCR. All MRSA isolates tested belonged to 9 spa-types and were PCR-positive for the ica genes; 10 of them (50%) produced biofilm in the microtiter plate assay. This is also the first demonstration of biofilm production by mecC MRSA.     

Fat content increases the lethality of ultra-high-pressure homogenization on Listeria monocytogenes in milk

Listeria monocytogenes CCUG 15526 was inoculated at a concentration of approximately 7.0 log₁₀ cfu/mL in milk samples with 0.3, 3.6, 10, and 15% fat contents. Milk samples with 0.3 and 3.6% fat content were also inoculated with a lower load of approximately 3.0 log₁₀ cfu/mL. Inoculated milk samples were subjected to a single cycle of ultra-high-pressure homogenization (UHPH) treatment at 200, 300, and 400 MPa. Microbiological analyses were performed 2 h after the UHPH treatments and after 5, 8, and 15 d of storage at 4°C. Maximum lethality values were observed in samples treated at 400 MPa with 15 and 10% fat (7.95 and 7.46 log₁₀ cfu/mL), respectively. However, in skimmed and 3.6% fat milk samples, complete inactivation was not achieved and, during the subsequent 15 d of storage at 4°C, L. monocytogenes was able to recover and replicate until achieving initial counts. In milk samples with 10 and 15% fat, L. monocytogenes recovered to the level of initial counts only in the milk samples treated at 200 MPa but not in the milk samples treated at 300 and 400 MPa. When the load of L. monocytogenes was approximately 3.0 log₁₀ cfu/mL in milk samples with 0.3 and 3.6% fat, complete inactivation was not achieved and L. monocytogenes was able to recover and grow during the subsequent cold storage. Fat content increased the maximum temperature reached during UHPH treatment; this could have contributed to the lethal effect achieved, but the amount of fat of the milk had a stronger effect than the temperature on obtaining a higher death rate of L. monocytogenes.     

Bactericidal activity of lauric arginate in milk and Queso Fresco cheese against Listeria monocytogenes cold growth

Lauric arginate (LAE) at concentrations of 200 ppm and 800 ppm was evaluated for its effectiveness in reducing cold growth of Listeria monocytogenes in whole milk, skim milk, and Queso Fresco cheese (QFC) at 4°C for 15 to 28 d. Use of 200 ppm of LAE reduced 4 log cfu/mL of L. monocytogenes to a nondetectable level within 30 min at 4°C in tryptic soy broth. In contrast, when 4 log cfu/mL of L. monocytogenes was inoculated in whole milk or skim milk, the reduction of L. monocytogenes was approximately 1 log cfu/mL after 24 h with 200 ppm of LAE. When 800 ppm of LAE was added to whole or skim milk, the initial 4 log cfu/mL of L. monocytogenes was nondetectable following 24 h, and no growth of L. monocytogenes was observed for 15 d at 4°C. With surface treatment of 200 or 800 ppm of LAE on vacuum-packaged QFC, the reductions of L. monocytogenes within 24 h at 4°C were 1.2 and 3.0 log cfu/g, respectively. In addition, the overall growth of L. monocytogenes in QFC was decreased by 0.3 to 2.6 and by 2.3 to 5.0 log cfu/g with 200 and 800 ppm of LAE, respectively, compared with untreated controls over 28 d at 4°C. Sensory tests revealed that consumers could not determine a difference between QFC samples that were treated with 0 and 200 ppm of LAE, the FDA-approved level of LAE use in foods. In addition, no differences existed between treatments with respect to flavor, texture, and overall acceptability of the QFC. Lauric arginate shows promise for potential use in QFC because it exerts initial bactericidal activity against L. monocytogenes at 4°C without affecting sensory quality.     

Diversity of culturable microorganisms and occurrence of Listeria monocytogenes and Salmonella spp. in Tuber aestivum and Tuber melanosporum ascocarps

The aim of this study was to investigate the total mesophilic microorganisms, Pseudomonas genus, Enterobacteriaceae family, mold and yeast counts and the presence of Listeria monocytogenes and Salmonella spp on Tuber aestivum and Tuber melanosporum ascocarps. The results confirmed that the major percentage of the microorganisms, approximately 9.0 log ufc/g, were present in the peridium, the glebas of healthy truffles being practically free of microorganisms. The predominant microbial group was the Pseudomonas averaging 8.3 and 8.4 log cfu/g on T. aestivum and T. melanosporum whole ascocarps, respectively. The Enterobacteriaceae also achieved high populations, especially in T. aestivum truffles, with 6.3 log cfu/g. Molds and yeasts never exceeded 5.0 log cfu/g. The characterization of the isolates revealed that the fluorescens pseudomonads were the most prevalent. Raoultella terrigena and Enterobacter intermedius were the dominant Enterobacteriaceae. The identification of the yeast isolates revealed five species: Debaryomyces hansenii, Issatchenkia scutulata, Rhodotorula aurantiaca, Saccharomyces dairensis and Trichosporon beigelii subspecies A and B. The mold genera detected in both species of truffles were Aspergillus, Cladosporium, Penicillium and Fusarium, Trichoderma being present only in T. aestivum. L. monocytogenes was found in 10% of the samples of T. aestivum analysed but Salmonella spp. was not detected. Knowledge of the microbial population in terms of possible food borne and pathogen microorganisms is very useful for establishing successful disinfection and storage methods to prolong the shelf-life of ascocarps of T. aestivum and T. melanosporum.