Evaluation of selected mathematical models to predict the inactivation of Listeria innocua by pulsed electric fields

The inactivation of Listeria innocua ATCC 51742 by pulsed electric fields was investigated at 35, 40 and 45 kV/cm. Results indicate that at treatment times shorter than 37 μs at 40 and 45 kV/cm, and 49 μs at 35 kV/cm, there is a linear relationship between the logarithm of the survivor fraction and the treatment time. However, longer times result in an abrupt increase in the slope of the inactivation curve and in inactivation values greater than six logarithmic cycles. A model based on Weibull's survival function was used to describe microbial inactivation and then compared to a first-order kinetic model. Distribution parameters of Weibull's survival function and kinetic constant for the first-order kinetic model were calculated by fitting experimental data. Calculated mean times for microbial inactivation from Weibull's distribution were 11.55, 8.65 and 5.39 μs at 35, 40 and 45 kV/cm, respectively. The goodness-of-fit between experimental and predicted values was determined using an accuracy factor. The model based on the Weibull survival distribution provided better accuracy factors than first-order kinetics. The model based on Weibull's survival function seems promising for describing survival curves that exhibit concavity.     

Comparison of recovery methods for the enumeration of injured Listeria innocua cells under isothermal and non-isothermal treatments

This study compares the feature of different media with the combination of selective with non-selective media in a TAL method for recovery of Listeria innocua cells exposed to thermal treatments. Experiments were conducted in broth at constant temperature (52.5 and 65.0 °C) and pH (4.5 and 7.5) conditions, using NaCl or glycerol to adjust water activity to 0.95. Four different media were used in bacterial cell enumeration: (i) a non-selective medium – TSAYE, (ii) two selective media – TSAYE + 5%NaCl and Palcam Agar and (iii) TAL medium (consisting of a layer of Palcam Agar overlaid with one of TSAYE).     

Effect of cell immobilization on heat-induced sublethal injury of Escherichia coli, Salmonella Typhimurium and Listeria innocua

The occurrence of sublethally injured cells in foods poses major public health concerns and is an essential aspect when assessing the microbial response to food preservation strategies, yet there is limited research dealing with its specific implications for mild heating. All available studies so far have been performed in broths colonized by planktonic cells, although their susceptibility to lethal agents has often been reported to be markedly different to the stress tolerance of cell colonies developed in solid foods. In this work, the effect of planktonic and colony growth, as well as the influence of colony density on sublethal injury induced by mild heating of Escherichia coli, Salmonella Typhimurium and Listeria innocua were assessed in food model systems. Detection of injured survivors relied on their inability to form visible colonies on salt-based selective media, which do not affect the growth of healthy cells. Sublethal injury (SI) increased rapidly with shorter exposure times and afterwards, decreased progressively, suggesting a mechanism of cumulative damage triggering lethal instead of SI. Cell arrangement affected the degree of SI, higher values being generally found for gelified systems, although the effect of colony density depended on the target microorganism. This information is essential for optimizing the design of food safety assurance systems.     

Inactivation of Escherichia coli, Listeria innocua and Saccharomyces cerevisiae by UV-C light: Study of cell injury by flow cytometry

Flow cytometry (FCM) is a powerful tool for analyzing physiological characteristics of microorganisms on a single-cell basis and identifying heterogeneities within population. This work analyzed the UV-C induced damage on Escherichia coli ATCC 11229; Listeria innocua ATCC 33090 and Saccharomyces cerevisiae KE162 cells by applying flow cytometry technique. The UV-C doses, obtained by altering the exposure time and measured by the iodide-iodate chemical actinometer, ranged between 0 and 5 kJ/m². E. coli; L. innocua and S. cerevisiae populations were quantified by plate count technique. For flow cytometry studies, cells were labeled with fluorescein diacetate (FDA) for detecting membrane integrity and esterase activity, and with propidium iodide (PI) for monitoring membrane integrity. The results showed that mechanisms of cellular damage differed according to time of exposure to ultraviolet radiation and the organism tested. E. coli and S. cerevisiae sub-populations with PI increased within the first minutes of UV-C treatment, without much change afterwards. On the contrary, FCM was used to detect the inactivation of those L. innocua sub-populations of viable microorganisms (maintaining metabolic activity) which were non-culturable due to membrane rupture and thus not detectable by viable plate count technique.     

Pulsed Ligh inactivation of Listeria innocua on food packaging materials of different surface roughness and reflectivity

Inactivation of Listeria innocua on food packaging materials by Pulsed Light (PL) treatment was investigated. Coupons of low density polyethylene (LDPE), high density polyethylene (HDPE), polyethylene-laminated ultra-metalized polyethylene terephthalate (MET), polyethylene-coated paperboard (TR), and polyethylene-coated aluminum foil paperboard laminate (EP) were inoculated with L. innocua cells in stationary growth phase. Inoculated coupons (∼8 CFU/coupon) were treated with Pulsed Light fluence of up to 8.0 J/cm², and survivors were determined. Reductions up to 7.2 ± 0.29, 7.1 ± 0.06, 4.4 ± 0.85, 4.5 ± 1.32, and 3.5 ± 0.82 log CFU/coupon were obtained on LDPE, HDPE, MET, TR, and EP, respectively. Inactivation data were used to determine Weibull kinetic parameters and predict inactivation in a wide range of fluence. Increasing surface reflectivity and surface roughness appeared to induce lower inactivation. Minimal surface heating was observed for all materials except MET, on which significant heating occurred. These results demonstrate the potential of Pulsed Light as an effective method for decontaminating food packaging materials.     

Smearing of soft cheese with Enterococcus faecium WHE 81, a multi-bacteriocin producer,against Listeria monocytogenes

Enterococcus faecium WHE 81, a multi-bacteriocin producer, was tested for its antimicrobial activity on Listeria monocytogenes in Munster cheese, a red smear soft cheese. The naturally delayed and superficial contamination of this type of cheese allowed the use of E. faecium WHE 81 at the beginning of the ripening as a surface culture. A brine solution inoculated at 10⁵ CFU of E. faecium WHE 81 per mL was sprayed on the cheese surface during the first smearing operation. On day 7, smearing of cheese samples with a brine solution at 10² CFU of L. monocytogenes per mL yielded initial cell counts of approximately 50 CFU g⁻¹ of the pathogen on the cheese surface. Although, in some instances, L. monocytogenes could survive (<50 CFU g⁻¹) in the presence of E. faecium WHE 81, it was unable to initiate growth. In control samples however, L. monocytogenes counts often exceeded 10⁴ CFU g⁻¹. In other respects, E. faecium WHE 81, which naturally existed in Munster cheese, did not adversely impact on the ripening process.     

Use of natural antimicrobials to improve the control of Listeria monocytogenes in a cured cooked meat model system

Concern about nitrite in processed meats has increased consumer demand for natural products manufactured without nitrite or nitrate. Studies on commercial meat products labeled as "Uncured" and "No-Nitrite-or-Nitrate-Added" have shown less control of nitrite in these products and greater potential growth of bacterial pathogens. To improve the safety of the "naturally cured" meats, several natural ingredients were studied in a cured cooked meat model system (80:20 pork, 10% water, 2% salt, and 150 or 50 ppm ingoing sodium nitrite) that closely resembled commercial frankfurters to determine their inhibitory effect on Listeria monocytogenes. Results showed that cranberry powder at 1%, 2% and 3% resulted in 2-4 log cfu/g less growth of L. monocytogenes compared to the control with nitrite alone (P<0.05). Other natural compounds, such as cherry powder, lime powder and grape seed extract, also provided measureable inhibition to L. monocytogenes when combined with cranberry powder (P<0.05).     

Habituation to organic acid anions induces resistance to acid and bile in Listeria monocytogenes

We evaluated the intrinsic and inducible resistance of four human pathogenic strains of Listeria monocytogenes to acid and bile, factors associated with virulence. Cells were grown in media at pH 7.4, or in media at pH 6.0 containing 0 (HCl control) or 4.75 mM of different organic acids, harvested at stationary or mid log phase, and challenged for 1 h in acid or bile. Stationary phase cells were intrinsically more resistant to either challenge than log phase cells, and large differences between strains were evident among the latter. Compared to the HCl control, habituation to log phase with organic acids induced significant (p < 0.05) and meaningful (≥ 1 log) increases in acid resistance of three of four strains tested, and in bile resistance of two strains suggesting that exposure to organic acid anions may enhance virulence in L. monocytogenes.     

Foci of contamination of Listeria monocytogenes in different cheese processing plants

Listeria monocytogenes is a ubiquitous bacterium widely distributed in the environment that can cause a severe disease in humans when contaminated foods are ingested. Cheese has been implicated in sporadic cases and in outbreaks of listeriosis worldwide. Environmental contamination, in several occasions by persistent strains, has been considered an important source of finished product contamination. The objectives of this research were to (i) evaluate the presence of L. monocytogenes within the factory environments and cheeses of three processing plants, artisanal producer of raw ewe's milk cheeses (APC), small-scale industrial cheese producer (SSI) and industrial cheese producer (ICP) each producing a distinct style of cheese, all with history of contamination by L. monocytogenes (ii) and identify possible sources of contamination using different typing methods (arsenic and cadmium susceptibility, geno-serotyping, PFGE). The presence of markers specific for 3 epidemic clones (ECI–ECIII) of L. monocytogenes was also investigated. Samples were collected from raw milk (n = 179), whey (n = 3), cheese brining solution (n = 7), cheese brine sludge (n = 505), finished product (n = 3016), and environment (n = 2560) during, at least, a four-year period. Listeria monocytogenes was detected in environmental, raw milk and cheese samples, respectively, at 15.4%, 1.1% and 13.6% in APC; at 8.9%, 2.9% and 3.4% in SSI; and at 0%, 21.1% and 0.2% in ICP. Typing of isolates revealed that raw ewe's milk and the dairy plant environment are important sources of contamination, and that some strains persisted for at least four years in the environment. Although cheeses produced in the three plants investigated were never associated with any case or outbreak of listeriosis, some L. monocytogenes belonging to specific PFGE types that caused disease (including putative epidemic clone strains isolated from final products) were found in this study.     

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.     

Reduced growth of Listeria monocytogenes in two model cheese microcosms is not associated with individual microbial strains

Two model antilisterial microbial communities consisting of two yeasts, two Gram positive and two Gram negative bacteria, and originating from Livarot cheese smear were previously designed. They were used in the present study to analyse the impact of microbial population dynamics on growth of Listeria monocytogenes in cheese microcosm. Specific culture media and PCR primers were developed for simultaneous culture-dependent and real-time PCR quantification of strains belonging to Marinomonas sp., Paenibacillus sp., Staphylococcus equorum, Arthrobacter arilaitensis, Pseudomonas putida, Serratia liquefaciens, Candida natalensis, and Geotrichum candidum, in cheese microcosms. All strains were enumerated after 3, 5, 8 and 14 days at 15 °C. They established well at high counts in all cheese microcosms. Growth dynamics for all strains in presence of L. monocytogenes WSLC 1685 were compared to those of microbial communities obtained by omitting in turn one of the six members of the initial community. The growth of the microbial strains was neither markedly disturbed by Listeria presence nor by the removal of each strain in turn. Furthermore, these communities had a significant reducing effect on growth of L. monocytogenes independently of pH, as confirmed by mathematical modelling. A barrier effect was observed, that could be explained by specific competition for nutrients.     

Heat resistance of Listeria species to liquid whole egg ultrapasteurization treatment

The lethality of ultrapasteurization treatments (70 °C/1.5 min.) applied at constant temperature (isothermal condition) and at a constantly raising temperature of 2 °C/min (non-isothermal condition) in liquid whole egg (LWE) against two strains of Listeria monocytogenes (STCC 5672 and 4032) and one of Listeria innocua has been investigated. Isothermal survival curves up to 71 °C were obtained, which followed first-order inactivation kinetics. The obtained D_t values indicated that L. innocua was significantly (p < 0.05) more heat resistant than L. monocytogenes strains. Non-significant (p > 0.05) differences were observed among z values (12.4 ± 0.4 °C, 13.1 ± 0.4 °C and 12.2 ± 0.7 °C for L. innocua and L. monocytogenes 5672 and 4032, respectively). Based on obtained D_t and z values, isothermal ultrapasteurization treatment (70 °C/1.5 min.) would provide 3.5-, 5.0-, and 6.5-Log₁₀ cycles of L. innocua and L. monocytogenes 5672 and 4032, respectively. Non-isothermal heating lag phase increased the thermotolerance of Listeria species in LWE. The simulated industrial pasteurization treatment for LWE (heating-up phase from 25 to 70 °C followed by 1.5 min. at 70 °C) would attain 5-Log₁₀ reductions of L. monocytogenes 5672 and 4032, and 3.7-Log₁₀ reductions of L. innocua. Therefore, the safety level of industrial ultrapasteurization concerning L. monocytogenes could be lower than that estimated with data obtained under isothermal conditions.     

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.     

食源性单增李斯特菌与英诺克李斯特菌基因组特征比较

目的 通过全基因组测序对甘肃省市售食品中分离的单增李斯特菌和英诺克李斯特菌基因组特征进行比较分析。方法 收集2021—2022年甘肃省市售食品中分离的25株单增李斯特菌和7株英诺克李斯特菌作为研究对象,对菌株进行全基因组测序,分析其系统发育谱系、克隆复合群（CC）、序列型（ST）、毒力基因、抗性基因及泛基因组。结果 32株李斯特菌分属单增李斯特菌谱系Ⅰ和Ⅱ及英诺克李斯特菌3个群,单增李斯特菌分为10个亚群,英诺克李斯特菌分为5个亚群,与CC型保持一致,核心基因组多位点序列分型能将各谱系中不同CC型的菌株明显分开,谱系Ⅰ与英诺克李斯特菌的进化关系更近。25株单增李斯特菌均携带李斯特菌毒力岛LIPI-1和内化素基因,不携带LIPI-3,有2株ST87型菌株携带LIPI-4;7株英诺克李斯特菌均不携带LIPI-1和内化素基因,均携带LIPI-4,有5株菌携带LIPI-3。单增李斯特菌有16株携带SSI-1、3株携带SSI-2,7株英诺克李斯特菌均不携带SSI-1,有6株携带SSI-2。李斯特菌的泛基因组大小随着测序基因组数目的增加呈现线性增多,25株单增李斯特菌当菌株数量达到15后核心基因数目稳定在2 272个,占泛基因组基因数目的46.2%,25株单增李斯特菌和7株英诺克李斯特菌共同的核心基因1 487个,当菌株数量达到10后数目趋于稳定。结论 核心基因组多位点序列分型可将不同谱系不同克隆复合群的李斯特菌进行区分,英诺克李斯特菌与单增李斯特菌生化特性相似与其亲缘关系相近有关,致病性差异与英诺克李斯特菌缺失单增李斯特菌特有的毒力基因相关。     

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.     

A bacteriocin-like substance produced from Lactobacillus pentosus 39 is a natural antagonist for the control of Aeromonas hydrophila and Listeria monocytogenes in fresh salmon fillets

We studied the ability of Lactobacillus pentosus 39, a BLS (Bacteriocin-like substance)-producing strain, to control the growth of Aeromonas hydrophila ATCC 14715 and Listeria monocytogenes ATCC 19117 artificially added to fresh salmon fillets at refrigeration temperatures and under simulated cold-chain break conditions.At refrigeration temperatures, Lb. pentosus 39 protective culture and its putative bacteriocin significantly reduced A. hydrophila counts compared with the control (2.1 and 1.4 log CFU/g reductions, respectively). Similar behaviour was observed for L. monocytogenes (3.6 and 1.3 log CFU/g reductions, respectively).Under simulated cold-chain break conditions, an increase in temperature (30°C for 12h) produced an evident increase in the development of A. hydrophila, L. monocytogenes, but also of Lb. pentosus 39, with a consequent increase in BLS production. This condition resulted in a greater reduction of both pathogens compared with samples stored at 4°C throughout the experiment (2.8 log CFU/g reduction for A. hydrophila, 5.8 log CFU/g reduction for L. monocytogenes). In samples treated with the putative bacteriocin alone, a less marked decrease was observed.Our study demonstrates the capability of Lb. pentosus 39 to control the growth of psychrotrophic bacteria in an experimental seafood model system. A similar biopreservation technology could provide more prolonged shelf-life during storage of ready-to-eat seafood, ensuring safety, even under extreme conditions.     

Spray application of liquid smoke to reduce or eliminate Listeria monocytogenes surface inoculated on frankfurters

In a simulated post process contamination scenario liquid smoke was sprayed on the frankfurters after peeling, and then inoculated with Listeria monocytogenes (Lm). Samples that did not receive a liquid smoke spray remained at approximately 2 log cfu/cm² during the 48 h of storage while the levels on the liquid smoke treated frankfurters continued to decline until they were below detection level (1 cfu/100 cm²). A shelf-life study lasting 140 days indicated that liquid smoke suppressed the growth of Lm for up to 130 days. An application of 2 or 3 ml liquid smoke at packaging resulted in at least a 1 log reduction of Lm within 12 h post packaging.     

Effects of different sanitizing treatments on biofilms and attachment of Escherichia coli and Listeria monocytogenes on green leaf lettuce

The effects of ozone (2 mg/L), chlorine (100 mg/L) and organic acid (0.25 g/100 g citric acid plus 0.50 g/100 g ascorbic acid) treatments at 10 °C for 2 min on the removal of Escherichia coli and Listeria monocytogenes cells embedded inside biofilms on the surface of lettuce leaves were studied. None of the sanitizing treatments were found effective in removing the bacterial biofilms. Initiation of biofilms was observed after 24 h of incubation. Bacterial cells appeared as individual cells, rather than clusters after 6 h incubation, thus 99.9% reductions in both E. coli and L. monocytogenes counts were achieved with all the three treatments. However, after 48 h incubation, none of the treatments resulted in higher than 90% reduction in microbial counts. Biofilm formation was demonstrated for the 48 h incubated samples with SEM images.     

Antibacterial activity of oregano and thyme essential oils against Listeria monocytogenes and Escherichia coli O157:H7 in feta cheese packaged under modified atmosphere

The antibacterial activity of the essential oils (EO) of oregano and thyme added at doses of 0.1 or 0.2 and 0.1 ml/100 g, respectively, to feta cheese inoculated with Escherichia coli O157:H7 or Listeria monocytogenes was investigated during cheese storage under modified atmosphere packaging (MAP) of 50% CO₂ and 50% N₂ at 4 °C. Compositional analysis showed that the predominant phenols were carvacrol and thymol for both EO. In control feta inoculated with the pathogens and stored under MAP, results showed that E. coli O157:H7 and L. monocytogenes strains survived up to 32 and 28 days of storage. However, in feta cheese treated with oregano EO at the dose of 0.1 ml/100 g, E. coli O157:H7 or L. monocytogenes survived up to 22 and 18 days, respectively, whereas at the dose of 0.2 ml/100 g up to16 or 14 days, respectively. Feta cheese treated with thyme EO at 0.1 ml/100 g showed populations of E. coli O157:H7 or L. monocytogenes not significantly different (P > 0.05) than those of feta cheese treated with oregano at 0.1 ml/100 g. Although both essential oils exhibited equal antibacterial activity against both pathogens, the populations of L. monocytogenes decreased faster (P < 0.05) than those of E. coli O157:H7 during the refrigerated storage, indicating a stronger antibacterial activity of both essential oils against the former pathogen.