Persistence Of Listeria Monocytogenes Research Articles

Persistence of Listeria monocytogenes in food commodities: foodborne pathogenesis, virulence factors, and implications for public health

Listeria monocytogenes is a ubiquitous pathogen found commonly in vast environment conditions and most commonly found in fresh-cut salads, raw fruits raw vegetables, sausages, cured meat, shellfish, refrigerated ready to eat food, soft cheeses, raw and under pasteurized milk. L. monocytogenes is a member of genus Listeria, along with five other members, such as L. innocua, L. ivanovii, L. seeligeri, L. grayi and L. murrayi. However, L. monocytogenes is the widely known species found in human cases, although a few cases with L. ivanovii have also reported. The infection caused by L. monocytogenes is known as listeriosis, and most affected populations are neonates, pregnant women, immune-suppressed and older people. Listeriosis is a severe condition with a higher level of hospitalization (94%) and around 16% fatality. Listeria can tolerate wide ranges of temperature, pH values, ions including Chlorides and Nitrites, different Oxygen levels. Therefore, L. monocytogenes is widely identified and survived easily on harsh environmental conditions. The ability of biofilm formation makes Listeria even harder to eliminate through food processing and associated surfaces. Although outbreaks are not very common, several large outbreaks have been documented in the literature. The extensive incubation period of this pathogen makes it challenging to identify the cause in most of the outbreaks, and most of the Listeria associated cases found to be sporadic. L. monocytogenes can cause substantial economic and public health impacts due to its effect on humans. Once contaminated, Listeria is hard to eliminate due to widespread presence in the environment, intrinsic physiological resistance, ability to adapt to external stresses, and the ability to grow at a wide range of temperatures. The objective of this review was to understand foodborne Listeria infections in humans and animals, the occurrence of infections and outbreaks, the growth and survival of L. monocytogenes in food-related industries, and risk reduction strategies.

Characterization of persistent Listeria monocytogenes strains from ten dry-cured ham processing facilities

The majority of cases of listeriosis are associated with the consumption of contaminated food. Some strains of Listeria monocytogenes can persist over months or years in meat processing plants increasing the risk of product contamination. The presence of L. monocytogenes was examined in 10 dry-cured ham processing facilities. A total of 1801 samples were collected from environment and equipment, during processing (1095) and after cleaning and disinfection (706). These samples were taken from non-food contact (736) and food contact (1065) surfaces. In addition, 204 samples from ham surfaces were also analysed. Prevalence varied from 6% to 34% among facilities, and was higher during processing than after cleaning and disinfection (24.8% vs 11.0%) and from non-food than from food contact surfaces (22.6% vs 17.4%). L. monocytogenes serotype 1/2a was predominant (53.9%), followed by 1/2c (26.0%) and 1/2b (15.3%) and less frequently 4b (4.8%). A total of 142 different pulsotypes were registered. Potential persistent L.monocytogenes strains were isolated in 9 out the 10 facilities, with no more than 6 pulsotypes in a given plant. Two pulsotypes were common in different installations, detected before and after cleaning and disinfection, highlighting the importance of monitoring the presence of this pathogen in dry-cured ham processing environments.

Control of Growth and Persistence of Listeria monocytogenes and β-Lactam-Resistant Escherichia coli by Thymol in Food Processing Settings

The main objective of this study was to evaluate the efficacy of thymol in controlling environmental contamination in food processing facilities. The effect of thymol was tested as an agent to prevent planktonic and bacterial biofilm growth of twenty-five Listeria monocytogenes isolates from a variety of foods and five Escherichia coli isolates from a farm. The E. coli isolates were positive for extended spectrum β-lactamase (ESBL) genes. All isolates and reference strains were susceptible to thymol at Minimum inhibitory concentration (MIC) values ranging from 250 to 800 μg/mL. An interesting activity of interference with biofilm formation of L. monocytogenes and E. coli was found for thymol at sub-MIC concentrations of 200, 100, 75, and 50 μg/mL. Anti-biofilm activity ranging from 59.71% to 66.90% against pre-formed 24-h-old L. monocytogenes biofilms at concentrations of 500 or 800 µg/mL, corresponding to 2× MIC, was determined against free-living forms of six isolates chosen as the best or moderate biofilm producers among the tested strains. The property of thymol to attack L. monocytogenes biofilm formation was also observed at a concentration of 100 µg/mL, corresponding to 1/4 MIC, by using a stainless-steel model to simulate the surfaces in food industries. This study gives information on the use of thymol in food processing setting.

Distribution and Persistence of Listeria monocytogenes in a Heavily Contaminated Poultry Processing Facility

We studied the colonization and distribution of Listeria monocytogenes in a heavily contaminated poultry processing plant over a 1-year period. A total of 180 nonfood contact surfaces, 70 food contact surfaces, 29 personnel, and 40 food samples were analyzed. L. monocytogenes isolates were subtyped by PCR serotyping, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. L. monocytogenes was detected in samples collected at every visit to the plant, and 43.8% (visit 4) to 65.6% (visit 7) of samples were positive, for an overall prevalence of 55.2%. The deboning area had the highest prevalence of positive samples (83.3%), and the processing area had the highest diversity of PFGE types. Ninety percent of the final products were positive for L. monocytogenes. Most of the isolates belonged to well-known persistent L. monocytogenes sequence types (ST9 and ST121). This study illustrates a well-established L. monocytogenes contamination problem in a poultry processing plant associated with a generalized failure of the food safety system as a whole. These findings reflect the potential for L. monocytogenes contamination when the food safety and quality management system is unsatisfactory, as described in the present study. It is essential to revise food safety and quality management systems to eliminate L. monocytogenes from food processing facilities, to control the entrance of sporadic sequence types, and to prevent L. monocytogenes spread within such facilities, especially in those premises with higher L. monocytogenes prevalence in the environment and final food products.

Detection and Pulsed-Field Gel Electrophoresis Typing of Listeria monocytogenes Isolates from Milk Vending Machines in Croatia

The aim of the present study was to evaluate the persistence of Listeria monocytogenes in raw milk from vending machines, based on culture and molecular detection of pathogen and Pulsed-Field Gel Electrophoresis typing. From December 2015 to January 2017, a total of 319 milk samples from 36 vending machines were examined for the presence and count of L. monocytogenes by reference methods ISO 11290:1 and ISO 11290:2. Molecular detection of pathogens was performed by loop-mediated isothermal DNA amplification (LAMP) coupled with bioluminescence (Molecular Detection Assay). L. monocytogenes was detected by MDA in 14 milk samples (4.38%) from four farms, compared to eight positive samples (2.5%) retrieved by a reference ISO method. Cultivable L. monocytogenes isolates were subjected to Pulsed-Field Gel Electrophoresis typing and pulsotypes were compared with those obtained during the previous survey in Croatia (2014‒2015). It was found that identical PFGE patterns of L. monocytogenes occur in milk samples of the same producer over a three-year period, indicating the persistence of pathogens in raw milk vending machines. The results obtained support the need for more effective control of milk in the entire food chain.

Distribution, adhesion, virulence and antibiotic resistance of persistent Listeria monocytogenes in a pig slaughterhouse in Brazil

Listeria monocytogenes is a relevant pathogen usually associated with meat and ready-to-eat products. This study aimed to assess the distribution, adhesion, virulence and antibiotic resistance of L. monocytogenes in a pork production chain. Environment, carcass and food samples (n = 894) were obtained from different steps of a pork production chain over a 6-month period (10 samplings), including from farms and the slaughterhouse (reception, slaughtering, processing, storage and end products). L. monocytogenes was detected in samples from the reception (lairage floor, 1/10), slaughtering (drains, 2/20) and cutting room stages (conveyor belts in the final packing stage – 11/20, knife – 1/40, and cutting boards – 1/20). Positive results for conveyor belts were recorded in seven consecutive samplings. L. monocytogenes isolates (n = 87) were characterized as belonging to serogroup IVb and presented positive PCR results for inlA, inlB, inlC, inlJ, hlyA, plcA, actA and iap. Isolates were selected according to the original samples (n = 31) and subjected to Pulsed Field Gel Electrophoresis (PFGE), demonstrating their high clonal identity (98.4–100%). According to PFGE results and their original samples, isolates were selected (n = 16) and subjected to phenotypic assay to assess their adhesion potential and tested for resistance against 15 antibiotics; all tested isolates presented weak adhesion potential and were resistant to ampicillin. The present study demonstrated the persistence of L. monocytogenes in the pork processing facility, indicating the potential risk for cross-contamination with a potential virulent and resistant clone.

Potential of oxygen and nitrogen reactive intermediates to disperse Listeria monocytogenes from biofilms.

Studying biofilm dispersal is important to prevent Listeria monocytogenes persistence in food processing plants and to avoid finished product contamination. Reactive oxygen and nitrogen intermediates (ROI and RNI, respectively) may trigger cell detachment from many bacterial species biofilms, but their roles in L. monocytogenes biofilms have not been fully investigated. This study reports on ROI and RNI quantification in Listeria monocytogenes biofilms formed on stainless steel and glass surfaces; bacterial culture and microscopy combined with fluorescent staining were employed. Nitric oxide (NO) donor and inhibitor putative effects on L. monocytogenes dispersal from biofilms were evaluated, and transcription of genes (prfA, lmo 0990, lmo 0807, and lmo1485) involved in ROI and RNI stress responses were quantified by real-time PCR (qPCR). Microscopy detected the reactive intermediates NO, peroxynitrite, H2O2, and superoxide in L. monocytogenes biofilms. Neither NO donor nor inhibitors interfered in L. monocytogenes growth and gene expression, except for lmo0990, which was downregulated. In conclusion, ROI and RNI did not exert dispersive effects on L. monocytogenes biofilms, indicating that this pathogen has a tight control for protection against oxidative and nitrosative stresses.

Preliminary results on prevalence and persistence of Listeria monocytogenes in different dairy and meat processing plants in Central Italy

Purpose: Listeria monocytogenes is a relevant food - borne pathogen able to cause severe and fatal infections in humans. Understanding correlations among different Listeria monocytogenes strains and evaluating Listeria monocytogenes isolates capacity to persist in processing plant environments is an essential tool for food surveillance. Most outbreaks of human listeriosis seem to be linked to strains able to persist in the environment and to replicate in foods. The aim of this study was to investigate prevalence and persistence of Listeria monocytogenes strains subtypes in different dairy and meat processing plants in the Abruzzo and Molise Region. Methods & Materials: Totally 14 food processing plants have been selected for the study (7 for dairy products – DP - and 7 for meat products - MP). Sampling was performed in 4 session from March to October 2017, from 30 to 35 environmental sampling have been sampled in all the division of the plants, both from food contact surfaces (FCS) and non food contact surfaces (NFCS). Sampling sites were selected as niches for Listeria monocytogenes presence. Samples were tested with FSIS MLG8-10:2017 method. Listeria monocytogenes strains were serotyped using the method described in the U.S. FDA Bacteriological Analytical Manual (Bennett and Weaver, 2001). PFGE subtyping was performed according to the PulseNet protocol (Pulsenet USA, 2013) and macrorestriction patterns were analyzed using the BioNumerics 7.6 (Applied Maths, Kortrijk, Belgium). Results: Results are based on 1772 environmental samples tested, 135 (7.6%) samples were found positive for Listeria monocytogenes (94 NFCS and 41 FCS) in 4 DP and 6 MP. PFGE showed indistinguishable persistent strains in 2 DP and 3 MP in all sampling sessions and in one MP in 3 sampling sessions. Conclusion: The persistence of Listeria monocytogenes underlines the ability to persist in food producing plants, in favourable niches inside the processing environment. To reduce Listeria monocytogenes prevalence, Good Manufacturing Practices and Good Hygiene Practices are needed. Analysis are still ongoing to model the contamination path, NGS on isolated strains will be carried out to confirm the persistence of the strains and to find the presence of gene devoted to resistance and persistence.

Modelling the fate and serogroup variability of persistent Listeria monocytogenes strains on grated cheese at different storage temperatures

Processed cheese from cow's milk is one of the most consumed dairy products worldwide. Since this product is defined as ready-to-eat, foodborne pathogens such as Listeria monocytogenes can represent a health concern for susceptible populations. In this study, the individual and combined kinetic behaviour of four L. monocytogenes serogroups (namely, 1/2a, 1/2b, 1/2c and 4b) persistently isolated from a Spanish cheesemaking factory was modelled on grated cheese at different isothermal conditions (4 and 12 °C) during a 120-days period. The serogroup variability was characterized over the storage time by the isolation and identification of the different serogroups in the cocktail containing the four strains. This processed cheese did not support the growth of L. monocytogenes during storage. Survival patterns described by the log-linear type model indicated a high variability of L. monocytogenes serotypes at the tested temperatures: L. monocytogenes serogroup 4b showed a more rapid decrease rate at 4 °C than at 12 °C, while the opposite trend was found for the rest of serogroups and the L. monocytogenes cocktail containing all the strains. Survival rate of L. monocytogenes serogroup 1/2c at 4 °C was 0.007 log CFU/d being the most resistant serotype while at 12 °C, serogroup 1/2a showed the lowest survival rate (0.011 log CFU/d), thus showing a prolonged survival at this temperature. This study highlights the potential implications of L. monocytogenes contamination in processed cheese and shows that serogroup variability should be considered when evaluating survival patterns in contaminated products. Finally, the predictive models developed here could be useful to assist food operators to set specific storage conditions and formulations to avoid L. monocytogenes growth and survival in grated cheeses.

Transcriptomic study on persistence and survival of Listeria monocytogenes following lethal treatment with nisin.

The aim of this study was to determine gene expression associated with the persistence of a Listeria monocytogenes stationary-phase population when facing lethal nisin treatment. RNA-Seq analysis was used for gene expression profiling of persister cells in nutrient-rich medium (persister TN) compared with untreated cells (non-persister). The results were confirmed using reverse transcription quantitative PCR (RT-qPCR). Functional genes associated with the persister population were identified in multiple systems, such as heat-shock-related stress response, cell wall synthesis, ATP-binding cassette (ABC) transport system, phosphotransferase system (PTS) and SOS/DNA repair. This study pointed to genetic regulation of persister cells exposed to lethal nisin concentrations and provides some insight into possible mechanisms of impeding bacterial persistence.

Prevalence and persistence of Listeria monocytogenes in premises and products of small food business operators in Northern Ireland

Listeriosis is a foodborne disease, with a high mortality rate, that predominantly affects the elderly. Under European Union legislation, EC 2073/2005, food business operators are encouraged to undertake sampling to ensure that the food processing environment, and required to ensure that food products, are free of Listeria monocytogenes. To determine the prevalence of L. monocytogenes in smaller food processing facilities in Northern Ireland, 24 companies submitted six processing environment swabs and two food samples every two months for eighteen months (July 2015 to November 2016) for L. monocytogenes examination. The prevalence of L. monocytogenes was 4.6% in food samples, and 6.3% in processing environment swabs. Over the duration of the study, 96 isolates of L. monocytogenes were obtained, one from each positive sample, except for two meat samples that had >100 cfu/g, where two isolates were obtained from each sample. No seasonality in occurrence of L. monocytogenes was seen for food isolates but significantly higher numbers of positive processing environment swabs were found in the warmer months of May, July and September (p = .007). Pulsed Field Gel Electrophoresis (PFGE) analysis revealed the presence of 27 pulsotypes; 9 pulsotypes were shared between different facilities and 9 were persistent. Based on a Combase predictive growth model, 77.5% (n = 130) of the foods tested were predicted to support the growth of L. monocytogenes. All of the isolates carried the pathogenicity genes inlA and actA and 71.4% carried qacH, which confers resistance to quaternary ammonium compounds which are frequently used in sanitizers. Whole genome sequencing of the isolates allowed multi-locus sequence typing to be undertaken. The data indicated that the sequence types identified included those with disease-causing ability, highlighting the disease-causing potential of the isolates.

Evaluation of Third-Party Deep Cleaning as a Listeria monocytogenes Control Strategy in Retail Delis

The objective of this study was to develop and assess the efficacy of an aggressive deep cleaning sanitation standard operating procedure (DC-SSOP) in nine retail delicatessens to reduce persistent Listeria monocytogenes environmental contamination. The DC-SSOP was developed from combined daily SSOPs recommended by the Food Marketing Institute and input from experts in Listeria control from food manufacturing and sanitation. The DC-SSOP was executed by a trained professional cleaning service during a single 12-h shutdown period. A modified protocol from the U.S. Food and Drug Administration Bacteriological Analytical Manual was used to detect L. monocytogenes in samples from 28 food and nonfood contact surfaces that were collected immediately before and after each cleaning and in samples collected monthly for 3 months. The DC-SSOP significantly reduced L. monocytogenes prevalence overall during the 3-month follow-up period and produced variable results for persistent L. monocytogenes isolates. Six delis with historically low to moderate L. monocytogenes prevalence had no significant changes in the number of samples positive for L. monocytogenes after deep cleaning. Deep cleaning in very high prevalence delis (20 to 30% prevalence) reduced L. monocytogenes by 25.6% (Padj < 0.0001, n = 294) overall during the follow-up period. Among delis with extremely high prevalence (>30%), positive samples from nonfood contact surfaces were reduced by 19.6% (Padj = 0.0002, n = 294) during the follow-up period. The inability of deep cleaning to completely eliminate persistent L. monocytogenes was likely due to the diverse infrastructures in each deli, which may require more individualized intervention strategies.

Listeria monocytogenes persistence and transfer to cantaloupes in the packing environment is affected by surface type and cleanliness

Investigation of the 2011 U.S. listeriosis outbreak associated with consumption of contaminated cantaloupes (Cucumis melo L) revealed that transfer of Listeria monocytogenes from equipment surfaces to melons in the packing facility was a potential route of contamination. The study presented here examined L. monocytogenes attachment and persistence on soiled materials and different types of equipment surfaces, and transfer to melons in a simulated-packing environment. Attachment involved the exposure of roller conveyors (polyvinyl chloride), conveyor belts (polyvinyl chloride, polyurethane, and nitrile rubber), and brush filaments (nylon and polyethylene) to ca. 7 log10 CFU/mL of a L. monocytogenes multi-strain inoculum for 10 and 60 min. L. monocytogenes showed similar (p > 0.05) attachment to all types of surfaces, and populations attached to surfaces following exposure for 10 min were not significantly different (p > 0.05) from that attached to surfaces after 60 min. Clean and soiled surfaces of nylon-brush filaments, conveyor belts, and foam pads were each inoculated with a L. monocytogenes multi-strain mixture (4.5 log10 CFU/mL) and subsequently stored at 25 °C, and L. monocytogenes populations on the surfaces were recovered for up to 15 days. L. monocytogenes populations on clean surfaces decreased from 4.5 log10 CFU/surface on day 0 to the detection limit by day 14; L. monocytogenes was still present on clean surfaces following enrichment on day 21. However, L. monocytogenes populations remained unchanged on soiled surfaces from day 0–14. Multivariate ANOVA revealed that soiled surfaces significantly (p < 0.05) promoted L. monocytogenes persistence compared to clean surfaces. Comparison of clean surfaces revealed that foam pads and brushes significantly (p < 0.05) promoted L. monocytogenes persistence compared to conveyor belt materials. Conveyor belts and foam pads were spot-inoculated (2.5 log10 CFU/surface), and 15 consecutive wet melons were manually rolled over each spot (n = 6 replicates), and melons were tested for the presence/absence of L. monocytogenes. Foam pads were found to contaminate significantly (p < 0.05) more melons than conveyor belts (polyvinyl chloride, polyurethane, and nitrile rubber). Soiled surfaces supported the survival of higher populations of L. monocytogenes, and surface type affected L. monocytogenes contamination transferred to specific melons.

Persistent Listeria monocytogenes strains isolated from mussel production facilities form more biofilm but are not linked to specific genetic markers

Contamination of mussels with the human pathogen Listeria monocytogenes occurs during processing in the factory, possibly from bacteria persisting in the factory's indoor and outdoor areas. In this study, a selection of persistent (n=8) and sporadic (n=8) L. monocytogenes isolates associated with mussel-processing premises in New Zealand were investigated for their phenotypic and genomic characteristics. To identify traits that favour or contribute to bacterial persistence, biofilm formation, heat resistance, motility and recovery from dry surfaces were compared between persistent and sporadic isolates. All isolates exhibited low biofilm formation at 20°C, however, at 30°C persistent isolates showed significantly higher biofilm formation after 48h using cell enumeration and near significant difference using the crystal violet assay. All 16 isolates were motile at 20°C and 30°C and motility was fractionally higher for sporadic isolates, but no significant difference was observed. We found persistent isolates tend to exhibit greater recovery after incubation on dry surfaces compared to sporadic isolates. Two of the three most heat-resistant isolates were persistent, while four of five isolates lacking heat resistance were sporadic isolates. Comparison of genome sequences of persistent and sporadic isolates showed that there was no overall clustering of persistent or sporadic isolates, and that differences in prophages and plasmids were not associated with persistence. Our results suggest a link between persistence and biofilm formation, which is most likely multifactorial, combining subtle phenotypic and genotypic differences between isolates.

A 3-year multi-food study of the presence and persistence of Listeria monocytogenes in 54 small food businesses in Ireland

The problem of assessing the occurrence of the food-borne pathogen Listeria monocytogenes in the food chain, and therefore the risk of exposure of the human population, is often challenging because of the limited scope of some studies. In this study the occurrence of L. monocytogenes in food from four major food groups, dairy products, meats, seafood and vegetables, and associated food processing environments in Ireland was studied over a three-year period. Fifty-four small food businesses participated in the study and sent both food and environmental samples every 2months between 2013 and 2015. L. monocytogenes was isolated using the ISO11290 standard method. Confirmation of L. monocytogenes and identification of serogroups were achieved using a multiplex PCR assay, and for some isolates serotype was determined using commercial antisera. Pulsed- field gel electrophoresis (PFGE) analysis was performed on all isolates allowing the relatedness of isolates from different food businesses to be compared nationwide. In total, 86 distinct pulsotypes were identified. The overall occurrence of L. monocytogenes in food samples was 4.2%, while in environmental samples it was 3.8%. In general, the occurrence of L. monocytogenes in food businesses decreased over the course of the study, presumably reflecting increased awareness and vigilance. The majority of the pulsotypes detected were unique to a particular food group (63/86), while only three pulsotypes were found in all four food groups investigated. The highest occurrence in food was found in the meat category (7.5%) while seafood had the lowest rate of occurrence (1.8%). Seventeen of the pulsotypes detected in the study were persistent, where persistence was defined as repeated isolation from a single facility with a minimum time interval of 6months. Using PFGE, 11 of the pulsotypes identified in this study were indistinguishable from those of 11 clinical isolates obtained from patients in Ireland over the last 4years, highlighting the fact that these pulsotypes are capable of causing disease. Overall, the study shows the diversity of L. monocytogenes strains in the Irish food chain and highlights the ability of many of these strains to persist in food processing environments. The finding that a significant proportion of these pulsotypes are also found in clinical settings highlights the need for continued vigilance by food producers, including frequent sampling and typing of isolates detected.

Persister cell formation of Listeria monocytogenes in response to natural antimicrobial agent nisin

The persistence of Listeria monocytogenes (L. monocytogenes) can be defined by its prolonged existence in food environments that poses a risk to food safety. The persistence of cells exposed to antibiotics and chemical sanitisers is well recorded however the persistence following treatment with natural antimicrobials like bacteriocins has not been determined. This study used two L. monocytogenes isolates, one from food and one from an animal origin, to obtain a persistent subpopulation following exposure to the natural antimicrobial nisin. Overnight cultures of L. monocytogenes were treated with 75 μg/ml of nisin over 24 h, and a biphasic killing pattern was observed for both strains. Persister cells became a population of cells showing tolerance to the high concentrations of nisin in our tests and proved not to be resistant with nisin re-exposure tests. XTT assays showed that the reduced level of metabolism maybe an important requirement for the L. monocytogenes persistence and the persister population which could survive at high concentration of nisin was probably related to less negative cell surface charges. The current data will be initial evidence to understand the mechanism of persistence through gene expression and in the future to give more reliable clues in developing strategies to control this food safety risk.

Diversity and persistence of Listeria monocytogenes within the Gorgonzola PDO production chain and comparison with clinical isolates from the same area

Listeria monocytogenes causes invasive syndromes with high fatality rates in specific population groups. Cheeses have been commonly implicated in outbreaks worldwide. Gorgonzola is a cheese only produced in Northwestern Italy (it is the third Italian cheese in terms of production and export) and L. monocytogenes is frequently isolated from the production chain. The aims of this study were to assess the distribution of L. monocytogenes Virulence Types (VTs) in isolates collected in Gorgonzola processing plants and to determine the presence of Epidemic Clones (ECs). Fifty-Six L. monocytogenes strains collected between 2004 and 2016 from cheese and environmental samples were subtyped with Multi-Virulence-Locus Sequence Typing (MVLST) and compared to previously typed strains. Most isolates (n=50) belonged to two new VTs (VT113 and VT114). The remaining isolates belonged to previously identified VTs: VT14-ECVIII (milk chocolate outbreak, 1994, USA) and VT80 (ricotta salata outbreak, 2012, USA). VT14, VT80 and VT113 were shared with isolates from apparently sporadic human cases in the same geographical area and temporal period (Piedmont and Lombardy, 2005–2016). The overall L. monocytogenes population appears to be homogeneous and may be characteristic of Gorgonzola production. Nevertheless, the detection in cheese and environmental samples of VTs observed in clinical isolates or outbreak related strains (VT80, VT14) contributed to better describe the current scenario and pointed out the need for increased surveillance.

Biofilm-producing ability of Listeria monocytogenes isolates from Brazilian cheese processing plants

The persistence of Listeria monocytogenes in food industry environments has been associated to the ability of specific isolates to produce biofilms. This study aimed to evaluate the biofilm production of 85 L. monocytogenes strains previously isolated from samples of cheese, brine and the environment of two cheese processing plants located in São Paulo, Brazil. The L. monocytogenes isolates belonged to serotypes 4b, 1/2b and 1/2c, yielded 30 different pulsotypes by pulsed-field gel electrophoresis (PFGE), and were submitted to biofilm-formation assays on polystyrene microplates and stainless steel coupons incubated statically at 35±0.5°C for 48h. All isolates from different sources showed ability to produce biofilms on polystyrene microplates, from which 21 (24.7%) also produced biofilms on stainless steel. Four isolates (4.7%) belonging to four different pulsotypes were classified as strong biofilms-producers on polystyrene microplates, while isolates belonging to four pulsotypes previously evaluated as persistent had weak or moderate ability to produce biofilms on polystyrene microplates. No relationship between the serotypes or pulsotypes and their biofilm-forming ability was observed. This study highlights the high variability in the biofilm production among L. monocytogenes strains collected from cheese and cheese-production environment, also indicating that strong biofilm-formation ability is not a key factor for persistence of specific isolates in cheese processing plants.

Prevalence and Persistence of Listeria monocytogenes in Ready-to-Eat Tilapia Sashimi Processing Plants

A 2-year study was performed at two ready-to-eat tilapia sashimi processing plants (A and B) to identify possible routes of contamination with Listeria monocytogenes during processing. Samples were collected from the aquaculture environments, transportation tanks, processing plants, and final products. Seventy-nine L. monocytogenes isolates were found in the processing environments and final products; 3.96% (50 of 1,264 samples) and 3.86% (29 of 752 samples) of the samples from plants A and B, respectively, were positive for L. monocytogenes . No L. monocytogenes was detected in the aquaculture environments or transportation tanks. The predominant L. monocytogenes serotypes were 1/2b (55.70%) and 4b (37.97%); serotypes 3b and 4e were detected at much lower percentages. At both plants, most processing sections were contaminated with L. monocytogenes before the start of processing, which indicated that the cleaning and sanitizing methods did not achieve adequate pathogen removal. Eleven seropulsotypes were revealed by pulsed-field gel electrophoresis and serotyping. Analysis of seropulsotype distribution revealed that the contamination was disseminated by the processing work; the same seropulsotypes were repeatedly found along the work flow line and in the final products. Specific seropulsotypes were persistently found during different sampling periods, which suggests that the sanitation procedures or equipment used at these plants were inadequate. Plant staff should improve the sanitation procedures and equipment to reduce the risk of L. monocytogenes cross-contamination and ensure the safety of ready-to-eat tilapia products.

Potential Impact of the Resistance to Quaternary Ammonium Disinfectants on the Persistence of Listeria monocytogenes in Food Processing Environments.

The persistence of certain strains of Listeria monocytogenes, even after the food processing environment has been cleaned and disinfected, suggests that this may be related to phenomena that reduce the concentration of the disinfectants to subinhibitory levels. This includes (i) the existence of environmental niches or reservoirs that are difficult for disinfectants to reach, (ii) microorganisms that form biofilms and create microenvironments in which adequate concentrations of disinfectants cannot be attained, and (iii) the acquisition of resistance mechanisms in L. monocytogenes, including those that lead to a reduction in the intracellular concentration of the disinfectants. The only available data with regard to the resistance of L. monocytogenes to disinfectants applied in food production environments refer to genotypic resistance to quaternary ammonium compounds (QACs). Although there are several well-characterized efflux pumps that confer resistance to QACs, it is a low-level resistance that does not generate resistance to QACs at the concentrations applied in the food industry. However, dilution in the environment and biodegradation result in QAC concentration gradients. As a result, the microorganisms are frequently exposed to subinhibitory concentrations of QACs. Therefore, the low-level resistance to QACs in L. monocytogenes may contribute to its environmental adaptation and persistence. In fact, in certain cases, the relationship between low-level resistance and the environmental persistence of L. monocytogenes in different food production chains has been previously established. The resistant strains would have survival advantages in these environments over sensitive strains, such as the ability to form biofilms in the presence of increased biocide concentrations.