Listeria monocytogenes in food businesses: From persistence strategies to intervention/prevention strategies—A review

In 2023, Listeria monocytogenes persistence remains a problem in the food business. A profound understanding of how this pathogen persists may lead to better aimed intervention/prevention strategies. The lack of a uniform definition of persistence makes the comparison between studies complex. Harborage sites offer protection against adverse environmental conditions and form the ideal habitat for the formation of biofilms, one of the major persistence strategies. A retarded growth rate, disinfectant resistance/tolerance, desiccation resistance/tolerance, and protozoan protection complete the list of persistence strategies for Listeria monocytogenes and can occur on themselves or in combination with biofilms. Based on the discussed persistence strategies, intervention strategies are proposed. By enhancing the focus on four precaution principles (cleaning and disinfection, infrastructure/hygienic design, technical maintenance, and work methodology) as mentioned in Regulation (EC) No. 852/2004, the risk of persistence can be decreased. All of the intervention strategies result in obtaining and maintaining a good general hygiene status throughout the establishment at all levels ranging from separate equipment to the entire building.     

Biofilm in milking equipment on a dairy farm as a potential source of bulk tank milk contamination with Listeria monocytogenes

The objective of this study was to assess the presence of a Listeria monocytogenes-containing biofilm in milking equipment as a potential source of bulk tank milk contamination on a dairy farm where milk contamination had been previously documented. Samples were collected from milking equipment and milking parlor premises on 4 occasions and analyzed for the presence of L. monocytogenes. Pulsed-field gel electrophoresis (PFGE) typing was conducted on L. monocytogenes isolates from the milking equipment, parlor and storage room floors, bulk tank milk, and in-line milk filters. Pieces from milk meters and rubber liners were obtained to visually assess the presence of a biofilm using scanning electron microscopy. A total of 6 (15%), 4 (25%), and 1 (6%) samples were culture-positive for L. monocytogenes in the first, second, and third sample collection, respectively. Two samples were L. monocytogenes hly PCR-positive but were culture-negative in the fourth sample collection. Combined AscI and ApaI restriction analysis yielded 6 PFGE types for 15 L. monocytogenes isolates obtained from milking equipment, parlor, bulk tank milk, and milk filters. A predominant and persistent PFGE type (PFGE type T) was observed among these L. monocytogenes isolates (9/15 isolates). Scanning electron microscopy of samples from the bottom cover of 2 milk meters showed the presence of individual and clusters of bacteria, mainly associated with surface scratches. The presence of a bacterial biofilm was observed on the bottom covers of the 2 milk meters. Prevention of the establishment of biofilms in milking equipment is a crucial step in fulfilling the requirement of safe, high-quality milk.     

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.     

Microbial background flora in small-scale cheese production facilities does not inhibit growth and surface attachment of Listeria monocytogenes

The background microbiota of 5 Norwegian small-scale cheese production sites was examined and the effect of the isolated strains on the growth and survival of Listeria monocytogenes was investigated. Samples were taken from the air, food contact surfaces (storage surfaces, cheese molds, and brine) and noncontact surfaces (floor, drains, and doors) and all isolates were identified by sequencing and morphology (mold). A total of 1,314 isolates were identified and found to belong to 55 bacterial genera, 1 species of yeast, and 6 species of mold. Lactococcus spp. (all of which were Lactococcus lactis), Staphylococcus spp., Microbacterium spp., and Psychrobacter sp. were isolated from all 5 sites and Rhodococcus spp. and Chryseobacterium spp. from 4 sites. Thirty-two genera were only found in 1 out of 5 facilities each. Great variations were observed in the microbial background flora both between the 5 producers, and also within the various production sites. The greatest diversity of bacteria was found in drains and on rubber seals of doors. The flora on cheese storage shelves and in salt brines was less varied. A total of 62 bacterial isolates and 1 yeast isolate were tested for antilisterial activity in an overlay assay and a spot-on-lawn assay, but none showed significant inhibitory effects. Listeria monocytogenes was also co-cultured on ceramic tiles with bacteria dominating in the cheese production plants: Lactococcus lactis, Pseudomonas putida, Staphylococcus equorum, Rhodococcus spp., or Psychrobacter spp. None of the tested isolates altered the survival of L. monocytogenes on ceramic tiles. The conclusion of the study was that no common background flora exists in cheese production environments. None of the tested isolates inhibited the growth of L. monocytogenes. Hence, this study does not support the hypothesis that the natural background flora in cheese production environments inhibits the growth or survival of L. monocytogenes.     

Occurrence of Listeria Monocytogenes in a Serbian Salmon and Seafood Processing Line During 2013

The objective of this study was to examine the occurrence of L. monocytogenes in a selected fish and seafood processing line. Results showed that during 2013, 12.4%, 8.3% and 2.3% of fish, seafood salads and environmental swabs were positive for L. monocytogenes. All positive food samples showed a contamination level below 100 CFU/g. Environmental swabs from surface of slicing and trimming tables, slicing machines, fish filleting and trimming knives, belt glazer and working table were positive for L. monocytogenes. Therefore, strict attention must be paid to cleaning and disinfection to control the level of L. monocytogenes.     

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log₁₀ cfu/well) and that they contained 1-2 log₁₀ cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log₁₀ cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log₁₀ more L. plantarum than L. monocytogenes cells (total count, 9 log₁₀ cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log₁₀ cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log₁₀ cfu/well inactivation after exposure for 15 min to 100 μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log₁₀ cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log₁₀ cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.     

Listeria monocytogenes survival of UV-C radiation is enhanced by presence of sodium chloride, organic food material and by bacterial biofilm formation

The bactericidal effect on food processing surfaces of ceiling-mounted UV-C light (wavelength 254 nm) was determined in a fish smoke house after the routine cleaning and disinfection procedure. The total aerobic counts were reduced during UV-C light exposure (48 h) and the number of Listeria monocytogenes positive samples went from 30 (of 68) before exposure to 8 (of 68). We therefore in a laboratory model determined the L. monocytogenes reduction kinetics by UV-C light with the purpose of evaluating the influence of food production environmental variables, such as presence of NaCl, organic material and the time L. monocytogenes was allowed to adhere to steel before exposure. L. monocytogenes grown and attached in tryptone soy broth (TSB) with glucose were rapidly killed (after 2 min) by UV-C light. However, bacteria grown and adhered in TSB with glucose and 5% NaCl were more resistant and numbers declined with 4-5 log units during exposure of 8-10 min. Bacteria grown in juice prepared from cold-smoked salmon were protected and numbers were reduced with 2-3 log when UV-C light was used immediately after attachment whereas numbers did not change at all if bacteria had been allowed to form a biofilm for 7 days before exposure. It is not known if this enhanced survival is due to physiological changes in the attached bacterial cells, a physical protection of the cells in the food matrix or a combination. In conclusion, we demonstrate that UV-C light is a useful extra bacteriocidal step and that it, as all disinfecting procedures, is hampered by the presence of organic material.     

The microbiological safety of ready-to-eat specialty meats from markets and specialty food shops: A UK wide study with a focus on Salmonella and Listeria monocytogenes

From 2359 specialty meats (continental sausages, cured/fermented, dried meats) sampled from markets and specialty food shops, 98.9% of samples were of satisfactory or acceptable microbiological quality. However, 16 (0.7%) were unsatisfactory as a result of Escherichia coli, Staphylococcus aureus or Listeria spp. contamination (≥10² CFU/g), and nine (0.4%) were unacceptable due to presence of Salmonella spp. or Listeria monocytogenes (>10² CFU/g). Meats with unacceptable levels of L. monocytogenes were within shelf life (range: 8–143 days remaining). Nine different subtypes of L. monocytogenes were detected with sero/AFLP type 1/2c VII predominating (37%), although this subtype was not overrepresented in any particular meat type (P > 0.05). Ninety-six percent of continental sausages and cured/fermented products were stored at <8 °C at premises, including seven of the nine unacceptable samples. These nine meats were all pre-packed prior to supply to retail premises (OR = 0.1 P = 0.003) indicating that contamination with bacterial pathogens occurred earlier in the production chain. Most samples (72.7%, 8/11) with unsatisfactory levels of E. coli were sliced on request, suggesting cross-contamination at point of sale. This study highlights the importance of ensuring that products do not become contaminated before final packaging, that storage conditions are controlled, and that durability dates are an accurate indication of the shelf life of the product so as to minimise the potential for L. monocytogenes to be present at levels hazardous to health at the point of sale.     

Ecology and molecular typing of L. monocytogenes in a processing plant for cold-smoked salmon in the Republic of Ireland

A cold-smoked salmon factory was surveyed for a period of 1 year (2008–2009) for the presence of Listeria monocytogenes in the processing line, processing environment, personnel, raw materials and product (cold-smoked salmon). The purpose of the study was to determine whether genetically similar strains colonise different environmental niches in the processing factory and thereby determining the possible contamination source or pathways. The processing factory was divided into four zones (1–4) based on the proximity to the samples. The overall prevalence of L. monocytogenes was 24.54% (n = 444). The L. monocytogenes contamination pattern was identified by characterising 124 L. monocytogenes isolates (obtained from this survey) by Multiple Locus Variable number tandem repeats Analysis (MLVA). The isolates were divided into 8 MLVA types (Lm a, Lm b, Lm c, Lm d, Lm e, Lm f, Lm g and Lm i). The final product (cold-smoked salmon) was contaminated with two major types of L. monocytogenes; one type originating from the raw material (Lm a) and the other type colonising the production line (Lm c) in zone 1. This suggests that, in addition to the fish processing line, L. monocytogenes contaminated raw material can progress through the production chain and result in contamination of the final product. Each zone had one dominating strain type, thus leading to the hypothesis that specific L. monocytogenes strains may be better adapted to specific environmental niches in the processing factory. The results clearly indicated the problematic sites which were the raw material, cutting board, drains, floor, conveyer belt and slicer/skinner equipment. Although, these areas would be rigorously cleaned before the start of the production, there seems to be the existence of resistant L. monocytogenes strain types. In order to minimise the problem observed in this study, new cleaning and disinfection protocols should be considered.     

Survival of Listeria monocytogenes after cleaning and sanitation of wooden shelves used for cheese ripening

The effect of cleaning and heat disinfection processes of wooden shelves used for cheese ripening on the survival of Listeria monocytogenes was examined. The cut boards were inoculated with a suspension containing 5.5?×?10⁷ colony forming units (CFU)/ml of L.??monocytogenes. Survival of L.??monocytogenes was investigated in the wood shavings. During the 24-hour incubation period in a humidity chamber, Listeria counts increased by 0.8 log units on average (n?=?9), compared to results 1?h after incubation, indicating the absence of antimicrobial properties of the wood in use. Additionally, after incubation for 24?h at room temperature, the boards were cleaned by soaking them for 15?min in a solution of hot alkaline detergent followed by brushing and rinsing with warm water. Some of the cleaned boards were subsequently heat treated at 80°C for 5?min and at 65°C for 15?min, respectively. The cleaning procedure alone was not sufficient to render L.??monocytogenes from the upper 2?mm wood layer inactive. In the case of both temperature-time combinations for heat disinfection, however, L.??monocytogenes was not detectable. The present study shows that the use of wooden shelves does not affect the hygienic safety of cheeses if such shelves are in good repair and are thoroughly cleaned and sanitized by heat treatment. Therefore, there is no reason to replace wood employed in cheese ripening processes with other materials.     

Longitudinal monitoring of Listeria monocytogenes contamination patterns in a farmstead dairy processing facility

Contamination of dairy products with Listeria monocytogenes is a concern because multiple human listeriosis outbreaks have been linked to contaminated cheese and dairy products. Dairy production on farmstead operations may be a particular concern because L. monocytogenes is also an animal pathogen that can be shed by ruminants with and without clinical symptoms; physical proximity between production animal and dairy processing facilities may thus provide a higher risk for introduction of L. monocytogenes into the dairy production process. To better understand the risks of L. monocytogenes contamination associated with farmstead dairy production, samples from a farmstead dairy processing operation and the milking barn of the directly adjacent dairy sheep operation were tested for L. monocytogenes over a 3-yr period. Prevalence of L. monocytogenes for samples collected on the farm (n = 85) and the dairy production facility (n = 674) was 9.4 and 2.7%, respectively. Molecular subtyping using automated EcoRI ribotyping of L. monocytogenes isolates revealed that distinct subtypes were associated with the dairy production facility and the farm's milking parlor. Although a total of 5 and 4 different ribotypes were identified among isolates obtained from the dairy production facility and the milking parlor, respectively, only 1 ribotype (DUP-1030A) was isolated from both. Different ribotypes were predominant among isolates from the dairy production facility (ribotype DUP-1052A, representing 15 of 18 isolates) and the farm's milking parlor (ribotype DUP-1039A, representing 4 of 8 isolates); each of these ribotypes appeared to persist over time in the respective area. Our data support that i) in farmstead dairy processing facilities, L. monocytogenes present on the farm can largely be prevented from being introduced into the processing facility; and ii) L. monocytogenes can persist on farm and in processing areas, providing a potential high-risk source for contamination. Preventing cross contamination between dairy production and processing facilities and control of persistent L. monocytogenes are thus critical to assuring the microbial safety of farmstead dairy products.     

Prevalence and contamination patterns of Listeria monocytogenes in catfish processing environment and fresh fillets

Catfish skins, intestines, fresh fillets, processing surfaces at different production stages, chiller water and non-food contact surfaces were sampled for Listeria monocytogenes and other Listeria species. Among 315 samples, prevalence of L. monocytogenes, Listeria innocua and a group of Listeria seeligeri–Listeria welshimeri–Listeria ivanovii was 21.6, 13.0 and 29.5%, respectively. No Listeria grayi was detected in this survey. While no L. monocytogenes strains were isolated from catfish skins and intestines, the strains were found with a frequency of 76.7% in chilled fresh catfish fillets and 43.3% in unchilled fillets. L. monocytogenes and Listeria spp. were also detected in fish contact surfaces such as deheading machine, trimming board, chiller water, conveyor belts at different stages, and fillet weighing table. Among L. monocytogenes, 1/2b (47.0%), 3b (16.0%) and 4c (14%) were the predominant serotypes isolated, whereas 4b, 4e, 1/2c and 1/2a were detected at much lower frequencies. Genotype analyses of L. monocytogenes isolates using serotyping, pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed that chiller water represented an important contamination source of L. monocytogenes in the chilled catfish fillets of two processing facilities, whereas fillet weighing table significantly contributed to the catfish fillet contamination of the third facility. This study suggests that L. monocytogenes contamination in the processed catfish fillets originates from the processing environment, rather than directly from catfish. Results from this study can aid the catfish industry to develop a plant-specific proper cleaning and sanitation procedure for equipment and the processing environment designed to specifically target L. monocytogenes contamination.     

Influence of plasma characteristics on the efficacy of Cold Atmospheric Plasma (CAP) for inactivation of Listeria monocytogenes and Salmonella Typhimurium biofilms

The biofilm mode of growth protects bacterial cells from applied disinfection methods for abiotic (food) contact surfaces. Therefore, new inactivation technologies such as Cold Atmospheric Plasma (CAP) should be considered. However, the influence of different plasma characteristics on the CAP efficacy for biofilm inactivation requires further study. In this research, the influence of (i) the applied plasma configuration (Dielectric Barrier Discharge (DBD) and Surface Barrier Discharge (SBD)), (ii) the oxygen level of the gas flow (He + 0.0/0.5/1.0 (v/v) % O₂), and (iii) the plasma intensity (13.88, 17.88, and 21.88 V input voltage) on the CAP efficacy for inactivation of L. monocytogenes and S. Typhimurium biofilms was investigated. Depending on the applied plasma characteristics, log₁₀-reductions up to approximately 3.5 log(CFU/cm²) were obtained. Nevertheless, it could be concluded that the highest log-reductions were in general obtained while using the DBD electrode, 0.0 (v/v) % O₂, and an input voltage of 21.88 V.Industrial relevanceThis study demonstrated the potential application of CAP for inactivation of pathogenic biofilms developed on abiotic (food) contact surfaces. The effect of different plasma characteristics on the CAP inactivation efficacy was investigated and determined optimal conditions resulted in promising reductions of the biofilm-associated cells. By incorporating this novel technology in a complete cleaning and disinfection process, the risk of (cross) contamination of food products might extensively be reduced.     

Efficacy of bacteriocin-containing cell-free culture supernatants from lactic acid bacteria to control Listeria monocytogenes in food

Consumer demands have led to an increased interest in the use of natural antimicrobials for food protection. With the objective of developing novel products for enhancing the microbial safety of food, we have tested cell-free culture supernatants (CFS's) of eight antagonistic bacterial strains for their efficacy to inhibit Listeria monocytogenes in different food matrices. The antagonistic strains represented different members of the order Lactobacillales as well as one isolate of Staphylococcus sciuri and all showed strong inhibition of L. monocytogenes on agar plates. Cell-free supernatants were obtained after growing the bacteria in a yeast extract-glucose broth. In six of the CFS's, different class IIa bacteriocins, namely leucocin A, leucocin B, mundticin L, pediocin PA-1, sakacin A, and sakacin X, were identified as the major anti-listerial compounds. For the other two strains, the active substances could not be ascertained conclusively. The minimal effective concentration (MEC) of the individual CFS's to achieve a 2.3 log₁₀ reduction of L. monocytogenes was determined in culture broth, whole milk, and ground beef at 4 °C. While all bacteriocin-containing CFS's were effective in broth at concentrations from 52 to 205 AU/ml, significant higher concentrations were needed when applied in food. Best results were obtained using CFS's containing pediocin PA-1, that displayed only three- and ten-times higher MEC's in milk (307 AU/ml) and ground meat (1024 AU/g) compared to broth, respectively. A twenty-fold increase in the MEC (2048 AU/ml) was observed for a mundticin L-containing fermentate, and a CFS containing leucocin A and B was inactivated more than fifty-fold (> 1280 AU/ml) in both food matrices. Remarkably, the sakacin A and sakacin X containing CFS's displayed very selective inactivation rates, in which sakacin A was only effective in meat (512 AU/g), while sakacin X was only effective in milk (2048 AU/ml). In all cases, inhibition of L. monocytogenes was only transient and surviving or resistant bacteria started growing after prolonged storage. These results highlight the importance of careful testing the effectiveness of bacteriocins in the food systems for which they are intended to be applied against the selected target and non-target bacteria. Furthermore, the outgrowth of surviving or resistant bacterial populations points out that the tested bacteriocins are not suited to assure full inhibition of L. monocytogenes in a food product, if not applied in combination with additional preservative measures.     

Growth of Escherichia coli O157:H7 and Listeria monocytogenes with prior resistance to intense pulsed light and lactic acid

Previous study showed that repetitive mild decontamination treatments with intense light pulses (ILP) and lactic acid (LA) can induce increased resistance in surviving pathogenic cells. Research has evaluated the potential of increased resistance to enhance the persistence of resistant variants of Listeria monocytogenes and Escherichia coli O157:H7 under suboptimal growth conditions. Growth of resistant variants and parental strains was determined by optical density (OD) measurements in nutrient broths with different pH values and NaCl concentration, at low temperature. The real lag phase was calculated, and results indicated that intense light pulses (ILP) resistant variants needed longer time to initiate growth compared to their parental strains, for both L. monocytogenes and E. coli O157:H7 when incubated at 7 °C and 10 °C, respectively. These selected variants were of the similar resistance towards heat and low pH (no cross-tolerance). Nevertheless, lactic acid (LA) resistant variant of L. monocytogenes was cross-protected when exposed to low pH, but not when treated with heat.     

Growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham under refrigerated and temperature abuse conditions

This study examined the growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham at refrigerated and abuse temperatures. A five-strain mixture of L. monocytogenes and a native microflora, consisting of Brochothrix spp., isolated from cooked meat were inoculated alone (monocultured) or co-inoculated (co-cultured) onto cooked ham slices. The growth characteristics, lag phase duration (LPD, h), growth rate (GR, log₁₀ cfu/h), and maximum population density (MPD, log₁₀ cfu/g), of L. monocytogenes and the native microflora in vacuum-packed ham slices stored at 4, 6, 8, 10, and 12 °C for up to 5 weeks were determined. At 4-12 °C, the LPDs of co-cultured L. monocytogenes were not significantly different from those of monocultured L. monocytogenes in ham, indicating the LPDs of L. monocytogenes at 4-12 °C were not influenced by the presence of the native microflora. At 4-8 °C, the GRs of co-cultured L. monocytogenes (0.0114-0.0130 log₁₀ cfu/h) were statistically but marginally lower than those of monocultured L. monocytogenes (0.0132-0.0145 log₁₀ cfu/h), indicating the GRs of L. monocytogenes at 4-8 °C were reduced by the presence of the native microflora. The GRs of L. monocytogenes were reduced by 8-7% with the presence of the native microflora at 4-8 °C, whereas there was less influence of the native microflora on the GRs of L. monocytogenes at 10 and 12 °C. The MPDs of L. monocytogenes at 4-8 °C were also reduced by the presence of the native microflora. Data from this study provide additional information regarding the growth suppression of L. monocytogenes by the native microflora for assessing the survival and growth of L. monocytogenes in ready-to-eat meat products.     

Comparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy

Listeria monocytogenes ranks among the most frequent causes of death due to foodborne illness (20-30% case fatality rate).Discriminative subtyping methods are important to detect the relatedness of isolates and verify epidemiologic associations. AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. In this study, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance.A total of 103 unrelated L. monocytogenes strains isolated from different environmental and food sources were analyzed. Strains were isolated from samples obtained from food-production plants, supermarkets and small food markets in Piedmont, Italy.All methods clustered L. monocytogenes strains into two genetic lineages, Lineage I and II. The three methods were compared using the 82 isolates which were typeable with all techniques. The calculated pair-wise Pearson's correlation coefficients (r) showed close agreement between all three methods.Our findings suggest that the AFLP II method can be successfully used to subtype L. monocytogenes strains isolated from foods and food processing facilities.     

Kinetics of resuscitation and growth of L. monocytogenes as a tool to select appropriate enrichment conditions as a prior step to rapid detection methods

Rapid methods still rely on a prior (shortened) enrichment step before application. Quantitative information is a prerequisite for understanding the resuscitation kinetics of the growth during the enrichment step. In this study various basal and newly introduced selective enrichment broths were evaluated. First, growth parameters (λ, μ_max) of both healthy and sub-lethally injured cells were determined. Next, a selection of enrichment broths was compared for their capacity to support detection within 24 h of low numbers of Listeria monocytogenes in artificially and naturally contaminated food samples. Detection was performed either by phage protein-based capture (Listeria Capture kit, Profos, Regensburg, Germany) combined with plating on chromogenic medium or by fluorescence in situ hybridization (FISH) using the VIT-Listeria kit (Vermicon, Munich, Germany). Kinetics of resuscitation and growth of L. monocytogenes in various enrichment broths showed that for detection of low numbers of sub-lethally injured L. monocytogenes cells at least an overnight enrichment was needed. A selective enrichment broth was needed to enable proliferation of L. monocytogenes within the indigenous bacterial flora present in foods. However, combination of an appropriate enrichment condition with advanced detection techniques may enable a 24 h detection of L. monocytogenes.     

Potential of Escherichia coli O157:H7 to persist and form viable but non-culturable cells on a food-contact surface subjected to cycles of soiling and chemical treatment

Our aim was to assess the potential of Escherichia coli O157:H7 to persist in a processing environment. We studied E. coli behaviour under conditions modelling those of meat plants to establish one initial bacterial load that allows persistence and another that does not. Polyurethane coupons (3.5 cm²) were contaminated once with E. coli in meat exudate before being subjected daily to a cleaning product and a disinfectant, both at half the recommended in-use concentrations, and a further soiling with the exudate. This procedure aimed to model what occurs in harbourage sites. Because previous experiments showed that persistence could not be achieved at 15 °C (temperature of slaughter halls), we incubated the coupons at 20 °C. Viable cells were determined by ethidium monoazide-qPCR (EMA-qPCR). When the first chemical treatment (CT) was applied to 24-hour biofilms with 5.4 log CFU/cm², cells were no longer detectable after the first week. However, on 66-hour biofilms with 6.7 log CFU/cm², after initially decreasing, E. coli numbers reached 6.6 log CFU/cm² and 8.3 log viable cells/cm² on the 11th day. When E. coli was cultured with a Comamonas testosteroni previously shown to increase E. coli biofilm formation, and subjected to CT on alternate days, E. coli stabilized at 4.6 log CFU/cm² before the CT, from the 5th day of the experiment. The killing and detachment effects of the CT decreased over time and PCR quantification detected a resumption of growth after 2 days (CT on alternate days) or 3 days (daily CT). Intracellular pH (pHi) of individual cells was determined during an experiment in which the CT was applied on alternate days. The proportion of cells with no proton gradient towards the environment (pHi ≤ 5.4) increased after the CT as expected. But during the first week of the experiment only, a further increase in this proportion occurred 24 h after the CT, suggesting that some of the surviving viable but non-culturable cells finally died.This study shows that conditions leading to E. coli O157:H7 persistence are not likely to arise when good refrigeration and hygiene practices are applied, and highlights the usefulness of EMA or PMA-qPCR as a complement to CFU determination in studying bacterial survival after cleaning and disinfection.