Potential of a nisin-containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meats

A bacterially produced cellulose film containing nisin was developed and used in a proof-of-concept study to control Listeria monocytogenes and total aerobic bacteria on the surface of vacuum-packaged frankfurters. Bacterial cellulose pellicles were produced by Gluconacetobacter xylinus K3 in Corn Steep Liquor-Mannitol Medium and were subsequently purified before nisin was incorporated into them. Investigations into the effect of nisin concentrations and contact times on incorporation of nisin into cellulose films showed that the lowest nisin concentration and shortest time needed for production of an effective antimicrobial cellulose film were 625IUml(-1) and 6h, respectively. The active cellulose films produced under these conditions did not, however, significantly reduce L. monocytogenes populations on frankfurters (P>0.05) during refrigerated storage for 14 days as compared to the controls. Films produced using a higher concentration of nisin (2500IUml(-1)) with the same exposure time (6h) resulted in a significant (P<0.05) decrease in L. monocytogenes counts on frankfurters of approximately 2logCFUg(-1) after 14 days of storage as compared to the control. Both the above-mentioned films showed a similar effectiveness in reducing total aerobic bacterial populations as measured by total aerobic plate counts on frankfurters. For both films, total aerobic bacterial levels were significantly (P>0.05) reduced by approximately 3.3logCFUg(-1) after 14 days of storage as compared to control samples. Bacterial cellulose films were demonstrated in this study to have potential applicability as antimicrobial packaging films or inserts for processed meat products.     

Strain dependent expression of stress response and virulence genes of Listeria monocytogenes in meat juices as determined by microarray

A subgenomic array, encompassing 54 probes targeting genes responsible for virulence, adhesion and stress response in Listeria monocytogenes, was used in order to study their expression in food systems. RNA extracted from L. monocytogenes inoculated in BHI and in situ (i.e. in minced meat and fermented sausage juices) and incubated at 4 °C, was hybridized on the array and the results obtained were compared in order to understand the effect that the food juice has on the expression. Three different strains of L. monocytogenes were tested, in order to determine the effect of the strain provenience. As determined by cluster analysis, each strain behaved in a different way when inoculated in food juices. The goal was to respond to acidic and osmotic stresses encountered in the food, particularly in the fermented sausage juice. No differences in the expression profile between the three strains were observed, when they were inoculated in BHI. On the other hand, in the meat and sausage juices, the iap, gadC and gadE genes, together with different internalin encoding genes, were significantly differentially expressed in the three strains.     

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.     

Molecular Subtyping and Tracking of Listeria monocytogenes in Latin-Style Fresh-Cheese Processing Plants

Latin-style fresh cheeses, which have been linked to at least 2 human listeriosis outbreaks in the United States, are considered to be high-risk foods for Listeria monocytogenes contamination. We evaluated L. monocytogenes contamination patterns in 3 Latin-style fresh-cheese processing plants to gain a better understanding of L. monocytogenes contamination sources in the manufacture of these cheeses. Over a 6-mo period, 246 environmental samples were collected and analyzed for L. monocytogenes using both the Food and Drug Administration (FDA) method and the Biosynth L. monocytogenes detection system (LMDS). Finished cheese samples from the same plants (n = 111) were also analyzed by the FDA method, which was modified to include L. monocytogenes plating medium (LMPM) and the L. monocytogenes confirmatory plating medium (LMCM) used in the LMDS method. Listeria monocytogenes was detected in 6.3% of cheese and 11.0% of environmental samples. Crates, drains, and floor samples showed the highest contamination rates, with 55.6, 30.0, and 20.6% L. monocytogenes positive samples, respectively. Finished products and food contact surfaces were positive in only one plant. The FDA method showed a higher sensitivity than the LMDS method for detection of L. monocytogenes from environmental samples. The addition of LMPM and LMCM media did not further enhance the performance of the FDA method for L. monocytogenes detection from finished products. Molecular subtyping (PCR-based allelic analysis of the virulence genes actA and hly and automated ribotyping) was used to track contamination patterns. Ribotype DUP-1044A, which had previously been linked to a 1998 multistate human listeriosis outbreak in the United States, was the most commonly identified subtype (20/36 isolates) and was isolated from 2 plants. This ribotype was persistent and widespread in one factory, where it was also responsible for the contamination of finished products. We hypothesize that this ribotype may represent a clonal group with a specific ability to persist in food processing environments. While previous listeriosis outbreaks were linked to Latin-style fresh cheeses made from unpasteurized milk, the presence of this organism in pasteurized cheese products illustrates that persistent environmental contamination also represents an important source of finished product contamination.     

Detection and characterization of Listeria monocytogenes in Sao Jorge (Portugal) cheese production

Listeria monocytogenes is a foodborne pathogen that can cause serious invasive disease in humans. Because human listeriosis cases have previously been linked to consumption of contaminated cheese, control of this pathogen throughout the cheese production chain is of particular concern. To understand the potential for L. monocytogenes transmission via São Jorge cheese, a Portuguese artisanal cheese variety that bears a Protected Denomination of Origin classification, 357 raw milk, curd, natural whey starter, and cheese samples representative of the production chain of this cheese were collected over one year and tested for the presence of L. monocytogenes and selected physicochemical parameters. Although neither L. monocytogenes nor other Listeria spp. were detected in whey, curd, or cheese samples, 2 of the 105 raw milk samples analyzed were positive for L. monocytogenes. These 2 raw milk isolates represented a ribotype that has previously been linked to multiple human listeriosis outbreaks and cases elsewhere, indicating the potential of these isolates to cause human listeriosis. On average, physicochemical parameters of São Jorge cheese ripened for 4 mo presented values that likely minimize the risk of L. monocytogenes outgrowth during ripening and storage (mean pH = 5.48; mean moisture = 37.79%; mean NaCl concentration = 4.73%). However, some cheese samples evaluated in this study were characterized by physicochemical parameters that may allow growth and survival of L. monocytogenes. Even though our results indicate that raw milk used for São Jorge cheese manufacture as well as finished products is rarely contaminated with L. monocytogenes, continued efforts to control the presence of this pathogen in the São Jorge cheese production chain are urged and are critical to ensure the safety of this product.     

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.     

Enumeration and growth of naturally occurring Listeria spp. in unpackaged ham

A total of 301 unpackaged retail ham samples were tested for the presence and number of Listeria spp. after 7 days at 5 degrees C to simulate domestic storage. Thirteen samples (4.3%) contained Listeria monocytogenes, with the highest count being 1.6 x 10(3)cfu g(-1). Thirteen samples contained other Listeria spp. Genotyping showed that only one L. monocytogenes isolate from the 14 tested was of a type previously identified in New Zealand human cases. Listeria-contaminated batches were incubated at 5 degrees C over approximately 3 weeks to assess the growth rate of natural contaminants. None contained L. monocytogenes, but growth occurred in one sample containing Listeria welshimeri and four containing Listeria innocua. Growth was usually slow at 0.002-0.004 log h(-1). In one sample, L. innocua grew at 0.02 log h(-1) although the maximum number reached was only 4.0-5.0 x 10(3)cfu g(-1). In five other samples little growth, if any, occurred. Growth of naturally occurring Listeria spp. at 5 degrees C was therefore generally slower than predicted by the Pathogen Modelling Programme (PMP) or did not occur.     

The inhibitory effect of natural microflora of food on growth of Listeria monocytogenes in enrichment broths

The aims of this study were to (i) compare the inhibitory effects of the natural microflora of different foods on the growth of Listeria monocytogenes during enrichment in selective and non-selective broths; (ii) to isolate and identify components of the microflora of the most inhibitory food; and (iii) to determine which of these components was most inhibitory to growth of L. monocytogenes in co-culture studies. Growth of an antibiotic-resistant marker strain of L. monocytogenes was examined during enrichment of a range of different foods in Tryptone Soya Broth (TSB), Half Fraser Broth (HFB) and Oxoid Novel Enrichment (ONE) Broth. Inhibition of L. monocytogenes was greatest in the presence of minced beef, salami and soft cheese and least with prepared fresh salad and chicken pâté. For any particular food the numbers of L. monocytogenes present after 24 h enrichment in different broths increased in the order: TSB, HFB and ONE Broth. Numbers of L. monocytogenes recovered after enrichment in TSB were inversely related to the initial aerobic plate count (APC) in the food but with only a moderate coefficient of determination (R²) of 0.51 implying that microbial numbers and the composition of the microflora both influenced the degree of inhibition of L. monocytogenes. In HFB and ONE Broth the relationship between APC and final L. monocytogenes counts was weaker. The microflora of TSB after 24 h enrichment of minced beef consisted of lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas spp., Enterobacteriaceae, and enterococci. In co-culture studies of L. monocytogenes with different components of the microflora in TSB, the lactic acid bacteria were the most inhibitory followed by the Enterobacteriaceae. The least inhibitory organisms were Pseudomonas sp., enterococci and B. thermosphacta. In HFB and ONE Broth the growth of Gram-negative organisms was inhibited but lactic acid bacteria still reached high numbers after 24 h. A more detailed study of the growth of low numbers of L. monocytogenes during enrichment of minced beef in TSB revealed that growth of L. monocytogenes ceased at a cell concentration of about 10² cfu/ml when lactic acid bacteria entered stationary phase. However in ONE Broth growth of lactic acid bacteria was slower than in TSB with a longer lag time allowing L. monocytogenes to achieve much higher numbers before lactic acid bacteria reached stationary phase. This work has identified the relative inhibitory effects of different components of a natural food microflora and shown that the ability of low numbers of L. monocytogenes to achieve high cell concentrations is highly dependent on the extent to which enrichment media are able to inhibit or delay growth of the more effective competitors.     

Prevalence, characterization and sources of Listeria monocytogenes in blue crab (Callinectus sapidus) meat and blue crab processing plants

Seven blue crab processing plants were sampled to determine the prevalence and sources of Listeria spp. and Listeria monocytogenes for two years (2006–2007). A total of 488 raw crabs, 624 cooked crab meat (crab meat) and 624 environmental samples were tested by standard methods. Presumptive Listeria spp. were isolated from 19.5% of raw crabs, 10.8% of crab meat, and 69.5% of environmental samples. L. monocytogenes was isolated from 4.5% of raw crabs, 0.2% of crab meat, and 2.1% of environmental samples. Ninety-seven percent of the isolates were resistant to at least one of the ten antibiotics tested. Eight different serotypes were found among 76 L. monocytogenes isolates tested with the most common being 4b, 1/2b and 1/2a. Automated EcoRI ribotyping differentiated 11 ribotypes among the 106 L. monocytogenes isolates. Based on ribotyping analysis, the distribution of the ribotypes in each processing plant had a unique contamination pattern. A total of 92 ApaI and 88 AscI pulsotypes among the 106 L. monocytogenes isolates were found and distinct pulsotypes were observed in raw crab, crab meat and environmental samples. Ribotypes and serotypes recovered from crab processing plants included subtypes that have been associated with listeriosis cases in other food outbreaks. Our findings suggest that molecular methods may provide critical information about sources of L. monocytogenes in crab processing plants and will augment efforts to improve food safety control strategies such as targeting specific sources of contamination and use of aggressive detergents prior to sanitizing.     

Effect of preinoculation growth media and fat levels on thermal inactivation of a serotype 4b strain of Listeria monocytogenes in frankfurter slurries

Preinoculation growth conditions and fat levels were evaluated for effects on the heat resistance of Listeria monocytogenes strain MFS 102 in formulated frankfurter slurries and on frankfurter surfaces. Comparison of linear inactivation rates (D-values) for cells heated in frankfurter slurry showed that growth conditions were significant (P<0.05) factors affecting subsequent thermal resistance. The average D(60 degrees C)-values for the five preinoculation growth media tested from most resistant to least heat resistant were: tryptic soy broth with 0.6% yeast extract (TSBYE) (2.2 min) and 8.5% fat slurry (2.2 min), followed by 23% fat slurry (1.7 min) and 11% fat slurry (1.7 min), and then TSYBE with quaternary ammonium compounds added (TSBYE+Q) (1 min). The fat level in the frankfurter heating media also had a significant (P<0.05) effect on the thermal death rate of L. monocytogenes. Cells heated in 8.5% fat slurry had a significantly higher (P<0.05) D(60 degrees C)-value (2.2 min) than those heated in 11% fat (1.0 min) and 23% fat slurry (0.9 min). Growth media (TSBYE, 8.5% fat slurry, and TSBYE+Q), and fat level (15% and 20%), however, were not significant factors (P>0.05) affecting thermal inactivation rates on frankfurter surfaces. Heat inactivation rates were consistently higher on frankfurter surfaces compared to similar treatments done in frankfurter slurry. On frankfurter surfaces, a 2.3- to 5.1-log(10) reduction was achieved after 15 min depending on frankfurter surface type. The time necessary to achieve a 3-log(10) reduction using post-processing pasteurization of frankfurters in a hot water-bath at 60 degrees C almost doubled for cells grown in TSBYE and heated in 23% fat frankfurter slurry (19.6 min) versus cells grown and heated in 8.5% fat frankfurter slurry (10.8 min).     

Role of quantitative risk assessment and food safety objectives in managing Listeria monocytogenes on ready-to-eat meats

Listeria monocytogenes may be found on ready-to-eat (RTE) meats, posing a public health risk. To minimize the public health impact, an appropriate level of protection (ALOP) can be established for a population with respect to L. monocytogenes, and ideally should be based on a scientific assessment of the risk, as well as societal and economic factors. Food safety systems can be based on meeting the ALOP. Food safety objectives (FSO) provide a link between the ALOP and performance objectives that are established to control a foodborne hazard. An FSO can be used as a risk management tool for L. monocytogenes in RTE meats, as the FSO establishes the stringency of the measures being used to control the hazard, by specifying the frequency and/or cell number of the pathogen in the food that should not be exceeded at the time of consumption. Typically, this requires setting performance objectives or performance criteria at an earlier point in the food chain, to ensure that the product will meet the FSO. Establishing an FSO requires an assessment of the risk of the hazard to the population of interest. Risk management strategies such as use of HACCP systems and Good Manufacturing Practices can then be used to ensure that the FSO is met.     

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).     

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.     

Occurrence and characterization of Listeria spp. in ready-to-eat retail foods from Vancouver, British Columbia

The occurrence of Listeria spp. and Listeria monocytogenes in retail RTE meat and fish products in Vancouver, British Columbia (B.C.) was investigated. To assess potential consumer health risk, recovered L. monocytogenes isolates were subjected to genotypic and phenotypic characterization. Conventional methods were used to recover Listeria spp. from deli meat (n = 40) and fish (n = 40) samples collected from 17 stores. Listeria spp. were recovered only from fish samples (20%); 5% harboured Listeria innocua, 5% had L. monocytogenes and 10% contained Listeria welshimeri. L. monocytogenes isolates serotyped as 1/2a and 1/2b, possessed dissimilar PFGE patterns, and had full-length InlA. Three 1/2a clonal isolates encoded the 50 kb genomic island, LGI1. Antimicrobial resistance (AMR) profiling showed all Listeria spp. possessed resistance to cefoxitin and nalidixic acid. L. monocytogenes were resistant to clindamycin, two were resistant to streptomycin, and one to amikacin. Reduced susceptibility to ciprofloxacin was seen in all L. monocytogenes, L. innocua and three L. welshimeri isolates. Reduced susceptibility to amikacin and chloramphenicol was also observed in one L. monocytogenes and three L. welshimeri isolates, respectively. Recovery of L. monocytogenes in fish samples possessing AMR, full-length InlA, LGI1, and serotypes frequently associated with listeriosis suggest B.C. consumers are exposed to high-risk strains.     

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.     

Validation of a stochastic modelling approach for Listeria monocytogenes growth in refrigerated foods

A stochastic modelling approach was developed to describe the distribution of Listeria monocytogenes contamination in foods throughout their shelf life. This model was designed to include the main sources of variability leading to a scattering of natural contaminations observed in food portions: the variability of the initial contamination, the variability of the biological parameters such as cardinal values and growth parameters, the variability of individual cell behaviours, the variability of pH and water activity of food as well as portion size, and the variability of storage temperatures. Simulated distributions of contamination were compared to observed distributions obtained on 5 day-old and 11 day-old cheese curd surfaces artificially contaminated with between 10 and 80 stressed cells and stored at 14 °C, to a distribution observed in cold smoked salmon artificially contaminated with approximately 13 stressed cells and stored at 8 °C, and to contaminations observed in naturally contaminated batches of smoked salmon processed by 10 manufacturers and stored for 10 days a 4 °C and then for 20 days at 8 °C. The variability of simulated contaminations was close to that observed for artificially and naturally contaminated foods leading to simulated statistical distributions properly describing the observed distributions. This model seems relevant to take into consideration the natural variability of processes governing the microbial behaviour in foods and is an effective approach to assess, for instance, the probability to exceed a critical threshold during the storage of foods like the limit of 100 CFU/g in the case of L. monocytogenes.     

Persistent Listeria monocytogenes subtypes isolated from a smoked fish processing facility included both phage susceptible and resistant isolates

Contamination of Ready-To-Eat foods with Listeria monocytogenes can typically be traced back to post-processing contamination from environmental sources; contamination is often linked to subtypes that persist in food associated environments. Although phage-based biocontrol strategies have been proposed for controlling this pathogen, information on the efficacy of phage treatment against diverse L. monocytogenes subtypes from food associated environments is still limited. We identified subtypes that were repeatedly found (“persistent”) in a smoked fish processing facility by using EcoRI ribotyping data for isolates obtained in 1998–2009. PFGE analysis of 141 isolates (9 ribotypes) supported persistence for up to 11 years. Characterization of selected isolates, representing persistent subtypes, against a panel of 28 listeriaphages showed a wide range of likelihood of phage susceptibility, ranging from 4.6% (for 7 ribotype DUP-1043A isolates) to 95.4% (for 7 ribotype DUP-1044A isolates). In challenge studies with 10⁵ and 10⁶ CFU/ml L. monocytogenes, using phage cocktails and a commercial phage product at different phage-host ratios, one isolate (ribotype DUP-1043A) was not affected by any treatment. A reduction in L. monocytogenes counts of up to 4 log units was observed, after 8 h of treatment, in isolates of two ribotypes, but subsequent re-growth occurred. Survivor isolates obtained after 24 h of treatment showed decreased susceptibility to individual phages included in the phage cocktail, suggesting rapid emergence of resistant subtypes.     

Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese

Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 °C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 °C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese.     

Modeling the growth rate and lag time of different strains of Salmonella enterica and Listeria monocytogenes in ready-to-eat lettuce

The growth parameters (growth rate, μ and lag time, λ) of three different strains each of Salmonella enterica and Listeria monocytogenes in minimally processed lettuce (MPL) and their changes as a function of temperature were modeled. MPL were packed under modified atmosphere (5% O₂, 15% CO₂ and 80% N₂), stored at 7–30 °C and samples collected at different time intervals were enumerated for S. enterica and L. monocytogenes. Growth curves and equations describing the relationship between μ and λ as a function of temperature were constructed using the DMFit Excel add-in and through linear regression, respectively. The predicted growth parameters for the pathogens observed in this study were compared to ComBase, Pathogen modeling program (PMP) and data from the literature. High R² values (0.97 and 0.93) were observed for average growth curves of different strains of pathogens grown on MPL. Secondary models of μ and λ for both pathogens followed a linear trend with high R² values (>0.90). Root mean square error (RMSE) showed that the models obtained are accurate and suitable for modeling the growth of S. enterica and L. monocytogenes in MP lettuce. The current study provides growth models for these foodborne pathogens that can be used in microbial risk assessment.