Control of Listeria monocytogenes Biofilms in a Simulated Food-Processing Environment.

Biofilm-forming ability may vary significantly among different Listeria (L.) monocytogenes strains. This interstrain variation is also observed in L. monocytogenes biofilm resistance to antimicrobial compounds commonly used in the food-processing environment. The screening of a large set of L. monocytogenes strains with specific characteristics, such as serotype, MLST type, and other genetic characteristics under various environmental conditions, may lead to a better understanding of the mechanisms underlying the establishment of the pathogen on food contact surfaces. In this chapter, traditional methods for L. monocytogenes strains characterization with regard to biofilm formation and novel biofilm control methods will be described.

Field validation of predictive models for the growth of lactic acid bacteria in acidic cheese-based Greek appetizers.

A microbial model was developed for spoilage of two acidic Greek appetizers, namely, tyrosalata (TS) and tyrokafteri (TK), with pH values of 4.34 to 4.50 and 4.22 to 4.38, respectively. The specific spoilage organisms of these products were lactic acid bacteria (LAB), which dominated during storage, while yeasts, whenever present, remained at low levels (1 to 2 log CFU/g). Correlations of LAB populations with changes in pH and sensory characteristics indicated that the spoilage level of LAB ranged from 8.1 to 8.6 log CFU/g for both products. TK showed a relatively higher microbial stability than did TS. The growth of LAB was modeled with the Baranyi model, while their maximum specific growth rates were further modeled as a function of temperature with square-root model and Arrhenius equations for each appetizer. The validation of the model was performed under nonisothermal conditions in the laboratory and in a field validation trial with temperature logging during distribution of individual packages in the chill supply chain, including transportation from the plant to the distribution center, retail display, and household refrigerators. Models for both appetizers showed satisfactory agreement with data, with a slight tendency of overprediction of LAB in TS. The field validation process also confirmed the higher stability of TK over TS. The developed models may serve as a useful tool for monitoring the microbiological quality of such complex products and manage their distribution. Furthermore, depending on the seasonal variation of chill chain conditions, reassessment of shelf life may be performed.

Modelling the effect of osmotic adaptation and temperature on the non-thermal inactivation of Salmonella spp. on brioche-type products.

Salmonella spp. is known to survive in intermediate- and low-moisture foods. Bakery products such as cream-filled brioche (aw 0.82-0.84), depending mainly on the aw of the fillings and the baking they receive for food preservation, may support survival of the pathogen. The study aimed to model the inactivation of osmotically adapted and non-adapted Salmonella in cream-fillings (praline and biscuit) and cream-filled brioche at different storage temperatures. All matrices were inoculated with ca. 6.0 log CFU/g of osmotically adapted and non-adapted five-strain cocktail of Salmonella (Typhimurium, Agona, Reading, and Enteritidis) and stored aerobically in 120 mL screw-capped containers at 15, 20, and 30 °C. Adaptation of Salmonella was induced in cream-fillings (praline and biscuit) with aw adjusted to 0.88, by adding sterile water to each of the original fillings (aw 0.78-0.83) and incubating at 37 °C for 1 h. Survival of Salmonella was assessed at regular time intervals throughout storage using thin layer agar method to enhance the recovery of injured cells (n = 4). Inactivation curves were fitted best with the Weibull model using the freeware GInaFit tool and the estimated δ and ß values were used to calculate the time for 4D reduction-t4D. Results showed that inactivation of Salmonella increased with temperature, while osmotic adaptation enhanced its survival in a food matrix-related manner. Higher survival rates of adapted cells were observed in cream-fillings (t4D: 79.9 ±â€¯27.1 days on biscuit and 150.3 ±â€¯19.6 days on praline) compared to brioche (t4D: 61.3 ±â€¯0.9 days on biscuit and 52.5 ±â€¯4.6 days on praline) at 20 °C. Secondary (linear) modelling of t4D showed that the survival of Salmonella was affected by temperature and osmotic adaptation. Model simulation of pathogen inactivation in independent trials on cream-fillings agreed well with observed data. In conclusion, the present data could be used as a means to identify areas for improving the performance of existing models quantifying the survival of Salmonella in bakery-confectionary products with intermediate aw.

Effect of frozen storage, different thawing methods and cooking processes on the survival of Salmonella spp. and Escherichia coli O157:H7 in commercially shaped beef patties.

The effect of common handling practices (i.e., freezing, thawing and cooking) of beef patties on the survival of Salmonella spp. and Escherichia coli O157:H7, was evaluated. Inoculated ground beef was stored at -22 °C for 5 and 75 days. After thawing at 4 °C/16 h, 20 °C/12 h, in microwave/22-24 min, or without prior thawing, beef patties (90 g) were shaped and cooked in oven-broiler or in pan-grill to internal temperatures of 60 °C or 71 °C. Cooking in oven-broiler was more effective compared to pan-grill, especially when cooked to 71 °C. Defrosting methods did not affect significantly (P ≥ 0.05) the survival of the pathogens during subsequent cooking. Frozen storage for 75 days enhanced the survival of E. coli O157:H7, as the pathogen survived 3.1 logCFU/g when cooked in oven-broiler at 71 °C. Results may supplement the existing guidelines for the appropriate practices, associated with freezing, thawing and cooking of patties in households or catering services.

A 3-year hygiene and safety monitoring of a meat processing plant which uses raw materials of global origin.

A systematic approach in monitoring the hygiene of a meat processing plant using classical microbiological analyses combined with molecular characterization tools may assist in the safety of the final products. This study aimed: (i) to evaluate the total hygiene level and, (ii) to monitor and characterize the occurrence and spread of Salmonella spp. and Listeria monocytogenes in the environment and the final products of a meat industry that processes meat of global origin. In total, 2541 samples from the processing environment, the raw materials, and the final products were collected from a Greek meat industry in the period 2011-2013. All samples were subjected to enumeration of total viable counts (TVC), Escherichia coli (EC) and total coliforms (TCC) and the detection of Salmonella spp., while 709 of these samples were also analyzed for the presence L. monocytogenes. Pathogen isolates were serotyped and further characterized for their antibiotic resistance and subtyped by PFGE. Raw materials were identified as the primary source of contamination, while improper handling might have also favored the proliferation of the initial microbial load. The occurrence of Salmonella spp. and L. monocytogenes reached 5.5% and 26.9%, respectively. Various (apparent) cross-contamination or persistence trends were deduced based on PFGE analysis results. Salmonella isolates showed wide variation in their innate antibiotic resistance, contrary to L. monocytogenes ones, which were found susceptible to all antibiotics except for cefotaxime. The results emphasize the biodiversity of foodborne pathogens in a meat industry and may be used by meat processors to understand the spread of pathogens in the processing environment, as well as to assist the Food Business Operator (FBO) in establishing effective criteria for selection of raw materials and in improving meat safety and quality. This approach can limit the increase of microbial contamination during the processing steps observed in our study as well as the cross contamination of meat products.

Control of Listeria monocytogenes in the processing environment by understanding biofilm formation and resistance to sanitizers.

Listeria monocytogenes can colonize in the food processing environment and thus pose a greater risk of cross-contamination to food. One of the proposed mechanisms that facilitates such colonization is biofilm formation. As part of a biofilm, it is hypothesized that L. monocytogenes can survive sanitization procedures. In addition, biofilms are difficult to remove and may require additional physical and chemical mechanisms to reduce their presence and occurrence. The initial stage of biofilm formation is attachment to surfaces, and therefore it is important to be able to determine the ability of L. monocytogenes strains to attach to various inert surfaces. In this chapter, methods to study bacterial attachment to surfaces are described. Attachment is commonly induced by bringing planktonic cells into contact with plastic, glass, or stainless steel surfaces with or without food residues ("soil") in batch or continuous (e.g., with constant flow of nutrients) culture. Measurement of biofilm formed is carried out by detaching cells (with various mechanical methods) and measuring the viable counts or by measuring the total attached biomass. Resistance of biofilms to sanitizers is commonly carried out by exposure of the whole model surface bearing the attached cells to a solution of sanitizer, followed by measuring the survivors as described above.

A generic model for spoilage of acidic emulsified foods: combining physicochemical data, diversity and levels of specific spoilage organisms.

The spoilage pattern of three emulsified, vegetable-based spreads of low pH (3.90-4.15) adjusted with acetic acid was characterized by correlating the growth of spoilage flora with the organoleptic and physicochemical changes, as well as the changes in the species composition of the dominant microflora during storage under isothermal conditions. In a further step, a generic (hereafter called 'unified') model was developed to describe the maximum specific growth rate of the specific spoilage organisms (SSOs) in all acetic acid acidified products, including literature data and additional in-house data from similar products, as a function of the storage temperature, pH (3.61-4.25) and initial concentration of the undissociated acetic acid in each product. The predictions of the unified model were compared with those of product-specific models, with temperature as the sole predictor variable. Two independent batches of commercially prepared pepper- (PS), fava beans- (FS) and eggplant-based (ES) spreads were stored at 4, 7, 10, 12, 15, 18, 20 and 25°C. The growth of lactic acid bacteria (SSOs; LAB) was correlated with changes in pH, titratable acidity and organic acids concentration, as well as sensory characteristics, in order to define the shelf-life of the products. Isolates from each spread and storage temperature were grouped with SDS-PAGE and were identified with 16S rRNA, determining the association between spoilage and species diversity. Product-specific models were developed using the square root model, while a polynomial and the Ratkowsky model were used for the development of the unified model. Products with lower pH and/or higher acetic acid content showed higher microbial stability. Lactobacillus plantarum or Lactobacillus brevis dominated the LAB association in all three spreads, although their relative percentage at the beginning of storage varied significantly. These facultative or obligate hetero-fermentative bacteria increased lactic acid and, sporadically, acetic acid levels in the spreads. The developed models were validated under real chill chain conditions and showed very good agreement with the observed data in PS and FS. The spoilage perception patterns of the different products were similar and thus, the proposed unified model may provide accurate predictions for the spoilage of a wide variety of acetic acid-acidified spreads, regardless of differences in the formulation (e.g., raw materials) and the manufacturing procedure.

Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method.

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1.