Non starter lactic acid bacteria biofilms: A means to control the growth of Listeria monocytogenes in soft cheese

The possibility to consider non starter lactic acid bacteria (NSLAB) biofilms as a means to control the growth of Listeria monocytogenes in soft cheeses was evaluated. In particular, the aptitude to form biofilm of four NSLAB strains (Lactobacillus plantarum DSM1055, Lactobacillus casei DSM20011, Lactobacillus curvatus DSM20019 and Lactobacillus paracasei DSM20207) was investigated. All tested strains were able to form biofilm on stainless steel and the highest quantities were produced when NSLAB were present simultaneously (about 6 Log CFU cm^?2). Then, experimental cheeses were made in presence of chips containing 7-days NSLAB biofilms and inoculated with L. monocytogenes (about 2 Log CFU g^?1). Results demonstrated that NSLAB biofilms can be considered a useful means to delay the growth of L. monocytogenes in soft cheeses: the maximum cell load attained at the stationary phase was about 6 Log CFU g^?1 in experimental cheeses against about 7 Log CFU g^?1 observed in control samples.     

Kinetics of Listeria monocytogenes cell reduction in processed cheese during storage

The present work utilized data from a series of challenge tests conducted on a processed cheese product (pH = 5.1, a_w = 0.93) aiming at elucidating whether the processed cheese can support or not the growth of Listeria monocytogenes when stored at 4, 12 or 22 °C (Angelidis, Boutsiouki, & Papageorgiou, 2010). In these challenge tests three L. monocytogenes strains (Scott A, CA and IS951) were used for inoculating samples of processed cheese at three different inoculation levels. Although the results of this previous work have shown that the product did not support the growth of L. monocytogenes at either storage temperature, there were noticeable differences in the inactivation rates of the different L. monocytogenes strains at the different storage temperatures. Therefore, in the present study, the data from the early experiments were used to describe the kinetics of L. monocytogenes cell reduction in processed cheese during storage. A Weibull-type model was initially employed to describe the reduction of L. monocytogenes viable cells as a function of storage time, while the Arrhenius relationship was used to express the effect of temperature on the rate of cell reduction. For each strain, cell reduction data at all temperatures and all inoculum levels were treated as a single data set for the simultaneous determination, through non-linear fitting, of the cell reduction kinetic parameters and their temperature dependence. The proposed approach gave satisfactory results, in terms of agreement between measured and predicted cell reduction, as far as the effect of initial inoculum level and storage time and temperature is concerned. Strain variability was noticeable at the lower temperatures tested, with Scott A being the most resistant among the L. monocytogenes strains examined.     

Cutting procedures might be responsible for Listeria monocytogenes contamination of foods: The case of Gorgonzola cheese

Commission Regulation (EC) No. 2073/2005 established, as a food safety criterion for tolerable levels of Listeria monocytogenes in ready-to-eat foods which do not support the growth of the pathogen or with shelf life below 5 days, a maximum of 100 cfu g⁻¹. Blue-veined cheeses are among these foods because their rinds can be contaminated, and the pathogen can be transferred to the paste during slicing. The aim of this research was to investigate whether cutting procedures could be responsible for cheese paste contamination. Considering that the Commission Regulation limit is allowed when the pathogen does not grow during the shelf life, we also wanted to verify whether, in the case of positive dragging, L. monocytogenes was able to grow on cut slices beyond the limit imposed, thereby becoming a risk for consumers during storage at 4 °C. Gorgonzola cheese was chosen for this investigation. The cutting simulation on artificially inoculated wheel rinds indicated that greater rind contamination corresponded to a higher percentage of contaminated paste samples. The growth of L. monocytogenes transferred to cut slices was variable relative to the physicochemical characteristics of the cheese, to the contamination level and to the time of storage. In particular, the sweet typology was able to support the growth of L. monocytogenes in the shelf life conditions considered and the quick overcoming of the limit imposed by food safety criteria would not ensure the safety for consumption.     

Heterologous production of pediocin for the control of Listeria monocytogenes in dairy foods

Pediocin is an antimicrobial peptide naturally produced by Pediococci with the potential to serve as a food-grade preservative for controlling Listeria contamination. The use of Pediococci in dairy products is limited due to their inability to ferment lactose, thus lactic acid bacteria (LAB) have been considered as potential hosts for the heterologous production of pediocin. In this study the four gene operon (papA-D) required for pediocin production was cloned on the nisin-inducible expression vector pMSP3535H3. The resulting vector, pRSNPed2, was electrotransformed into Streptococcus thermophilus, Lactococcus lactis ssp. lactis and Lactobacillus casei. Transformants containing the properly constructed vector were identified by PCR analysis and shown to inhibit the growth of Listeria monocytogenes Scott A. S. thermophilus transformants were all shown to constitutively express pediocin; however, in L. lactis and L. casei both constitutive and inducer-dependent expression was observed. In all cases nisin-induction resulted in optimal pediocin production. Transformants from each LAB host were also shown to inhibit the growth of L. monocytogenes NR30, a nisin-resistant variant of L. monocytogenes Scott A. These results suggest pediocin has the potential to serve as a hurdle component along with nisin for prevention of Listeria contamination of foods.     

Inhibition of Listeria monocytogenes by resident biofilms present on wooden shelves used for cheese ripening

The purpose of the present study was to characterize the development of Listeria monocytogenes on wooden shelves used for cheese ripening. The fate of two L. monocytogenes strains was analysed over time as a function of the presence of a native biofilm, the farmhouse origin of cheeses, and the wooden shelves properties. In presence of a native microbial flora on the shelves, deposited populations of L. monocytogenes remained stable or even decreased by up to 2 log₁₀(CFU/cm²) after 12 days of incubation at 15 °C in all tested conditions. By contrast, L. monocytogenes populations increased by up to 4 log₁₀(CFU/cm²) when the resident biofilm was thermally inactivated, suggesting a microbial origin of the observed inhibitory effect. All together, our results suggest that the biocontrol of pathogens multiplication on wooden shelves by resident biofilms should be considered for the microbiological safety of traditional ripened cheeses.     

Growth inhibition of Listeria monocytogenes by bacteriocin-producing Staphylococcus equorum SE3 in cheese models

The anti-listerial activity of Staphylococcus equorum SE3 isolated from cheese brine was tested in two different model cheese systems to ascertain its potential for use as a protective culture for smear cheese ripening. Co-cultivation of Listeria monocytogenes L129 and the antilisterial S. equorum SE3 was performed on “milk agar” or “modified milk agar” (model cheese surface systems). S. equorum inoculated at concentrations of 10⁶ cfu/cm² completely inhibited growth of L. monocytogenes inoculated at 10–500 cfu/cm² on modified milk agar within 24 h of incubation, in the negative controls L129 grew to >10⁷ cfu/cm². At a higher inoculation level, growth inhibition was still more than 7 log units after 24 h. L. monocytogenes strains of different serotypes were also inhibited. Co-cultivation of S. equorum SE3 with other smear bacteria or yeasts, however, showed no growth inhibition of these important ripening microorganisms. The antilisterial effect was not diminished on the modified milk agar when co-cultivation was performed with the added smear cheese microbiota. However, on milk agar with no adjuncts (“green cheese model”), only a slight (<1 log unit) growth inhibition of L. monocytogenes was observed. Addition of peptides or amino acids to milk agar could restore growth inhibition of listeriae at different levels.     

A multi-sampling approach to evaluate an infrared surface treatment for reducing Listeria monocytogenes contamination on whole Gorgonzola cheese rinds

The microbial ecology of Gorgonzola cheese rind is the focus of many studies because the surface can be contaminated by pathogenic microorganisms. Among food-borne pathogens, particular attention is focused on the behaviour of Listeria monocytogenes that is able to grow at refrigeration temperatures and it could also grow during ripening. The Consortium for the Protection of Gorgonzola Cheese declares the rind not edible but the pathogen may also be transferred during cutting and portioning. Therefore, the decontamination of rinds is important to increasing cheese safety. To achieve this goal, many different strategies have been proposed. In this study, the application of an infrared surface treatment to decontaminate cheese rinds is proposed. The presence of L. monocytogenes, which was artificially inoculated in cheese rinds together with cheese rind microflora, and the cheese rind microflora were monitored before and after the treatment of 32 samples of Gorgonzola cheese rinds.The infrared surface treatment provided good reduction of the rind microflora, and L. monocytogenes was particularly affected by this. The treatment, applied to cheeses at the end of ripening, does not interfere with the ripening process and offers the advantages of short time exposures and easy installation of the equipment in cheese plants. Moreover, this study demonstrated that the sampling method affects the detection of cheese rind microflora. In fact, a non-destructive sampling method, based on a sponge and often used for surface sampling but never before applied to ready to eat food sampling, was compared with a traditional but destructive method, based on rind scraping. Regarding L. monocytogenes, the sponge method allowed to estimate even only 5.71 ± 0.79 log cfu g⁻¹ of cells reduction after the treatment while the higher reduction when considering the rind scraping method was 4.06 ± 3.38 log cfu g⁻¹. The sponge method, combined with the classic scraping one, besides offering the great advantage of not being destructive, allowed to differentiate the effect that the treatment has on the microflora located on the surface from those in deeper layers.     

Evaluation of the Listeria monocytogenes inactivation during post-process storage of fermented sausages: A basis for the development of a decision support tool

In situ quantitative data on Listeria monocytogenes survival during storage of vacuum-packaged fermented sausages at various temperatures were collected from the literature to develop a generic predictive model regarding its fate at a specific storage temperature. The development of the tool was based on the z-concept. The time needed for 4D reduction of the pathogen was estimated and its influence by the temperature was further described by linear regression. A secondary model was developed for describing the effect of sausage water activity on the z-concept parameters at the reference temperature. The decision support tool was successfully validated against the studies not used during the development of the model. Based on the model predictions, a decision can be made about the required time of product storage before its distribution to achieve an additional pathogen inactivation. Such tools can be incorporated in a HACCP plan of a food-producing company to assure food safety.     

The presence,genetic diversity and behaviour of Listeria monocytogenes in blue-veined cheese rinds during the shelf life

Blue-veined cheeses may allow Listeria monocytogenes survival and multiplication due to the biochemical characteristics of the cheese and the growth characteristics of the pathogen. Because of the availability of a considerable number of samples, we wanted this study to take a large view of the microbiota and of the incidence and genetic diversity of L. monocytogenes in blue-veined cheese rinds. Moreover, we wanted to determine if the pathogen present on the rinds at the end of ripening represented a risk to the consumer if the cheese should exceed the domestic storage limit imposed by Commission Regulation (EC) No 2073/2005 for food safety criteria.The rind microflora showed a high level of heterogeneity, and the incidence of L. monocytogenes (never found in the paste samples) was 55%, with an increased presence and concentration associated with a longer ripening time. Amplified Fragment Length Polymorphism (AFLP) was used to characterise L. monocytogenes isolated from blue-veined cheese rinds and highlighted the heterogeneity among the strains, demonstrating its suitability for studying the biodiversity of the pathogen in this environment. L. monocytogenes was able to grow during the shelf life of the cheese and was influenced by the refrigeration temperatures and the physicochemical characteristics of the cheese.     

Evolution of Listeria monocytogenes populations during the ripening of naturally contaminated raw ewe’s milk cheese

The aim of this work was to study, in loco, the evolution of Listeria monocytogenes populations, during ripening (7, 42, 60 and 120 days) of naturally contaminated raw ewe’s milk cheese. Two batches of cheese consisting of 20 or 16 cheeses were obtained from two farmstead cheesemakers, respectively. A significant increase in numbers of L. monocytogenes was observed for both batches, from 7 to 42 days of ripening. These results suggest that this type of cheese has potential to support the survival of L. monocytogenes, while stressing the importance of cheese contamination in the dairies by resident strains.     

Listeria monocytogenes isolated from the smoked salmon industry: Growth potential under different environmental conditions

Forty isolates of Listeria monocytogenes were obtained from smoked salmon and production environment in a processing plant. Molecular characterisation evidenced the presence of biotypes corresponding to differences in technological conditions, particularly in salting.The growth behaviour of eight selected strains was evaluated in two media (BHI and Salmon Broth) at different temperatures and salt concentrations. In sub-optimal conditions, the physiological biodiversity of the strains was enhanced, especially in Salmon Broth, underlying the crucial importance of the medium in the evaluation of growth potential.     

Detection of Listeria in milk using non-targeted metabolic profiling of Listeria monocytogenes: A proof-of-concept application

Listeria is represented by ten known species, comprising pathogenic and non-pathogenic variants. Listeria monocytogenes is the type species and is primarily pathogenic to humans and causes serious illness. As a result, most countries have a zero tolerance towards the presence of Listeria in foods. Therefore, in order to  ensure food safety, robust techniques for detection are required. This paper describes a proof-of-concept application of a metabolomic technique for the rapid detection of Listeria, applied to nutrient media and a complex food sample (milk) inoculated with a pathogenic Listeria strain (L. monocytogenes). It was found that a profile of intracellular and extracellular metabolites associated with L. monocytogenes could be obtained using gas chromatography coupled to orthogonal acceleration time-of-flight mass spectrometry (GC-oaToFMS). Chemometric analysis showed that it is possible to differentiate between the uninoculated samples and samples inoculated with Listeria based on L. monocytogenes metabolic activity. This research demonstrates that metabolomics has the potential for rapidly identifying food contaminated with Listeria and could provide a means for enhancing monitoring programmes and ensuring food safety.     

Environmental sampling for Listeria monocytogenes control in food processing facilities reveals three contamination scenarios

Listeria monocytogenes enters the food processing facility via environment, or contaminated raw materials. To increase the understanding of L. monocytogenes environmental contamination in the meat and dairy food sector, six European scientific institutions sampled twelve food processing environments (FPEs) in a harmonized methodological approach. The selection of six previously assumed uncontaminated (UC) FPEs and six contaminated (C) FPEs was based on the L. monocytogenes occurrence information originating from the time prior to the current study. An aim of the study was to highlight, that FPEs regarded for years as uncontaminated, may also become L. monocytogenes contaminated and repeated environmental sampling could help to identify the potential sources of contamination.From a total of 2242 FPE samples, L. monocytogenes was present in 32% and 8.8% of meat and dairy processing environments, respectively. In the actual study, each FPE was contaminated with L. monocytogenes on at least one sampling occasion. Three contamination scenarios could be observed: (i) sporadic contamination in the interface of raw material reception and hygienic areas, (ii) hotspot contamination in the hygienic processing areas (iii), and widely disseminated contamination in the entirely FPE. These data demonstrate that L. monocytogenes are common colonizers of FPEs in the European processing facilities sampled and that a consistent cross-contamination risk exists. To avoid food contamination, a risk assessment approach should assign risk levels to critical control areas (CCAs) and identify those where cross-contamination should be essentially excluded.     

Influence of Listeria innocua on the growth of Listeria monocytogenes

The growth of Listeria monocytogenes and Listeria innocua strains was monitored during this study: (i) in TSB–YE media and (ii) in a food matrix (pasteurized milk) according to the ISO 11290-1 methodology. Different inocula concentrations and mixtures were tested. The response was shown to be strain dependent. In TSB–YE the inhibition of a L. monocytogenes strain was observed in just one of the three mixtures (L. monocytogenes_1340 with L. innocua_11288) showing a reduction of 1.37 log cfu/ml after 42.5 h and 1.85 log after 66.5 h of incubation. In pasteurized milk the inhibition of L. monocytogenes by L. innocua was always observed when L. innocua was present in higher concentrations than L. monocytogenes. The reverse was also observed but only in one mixture (cocktail of six L. monocytogenes with L. innocua_2030c) when the initial concentration of L. monocytogenes was 100 times higher than L. innocua suggesting the phenomenon of quorum sensing. Furthermore, inhibitory activity was not caused by bacteriocins, and no correlation between the growth rate and inhibition was demonstrated.     

Listeria monocytogenes in ready-to-eat foods in Italy: Prevalence of contamination at retail and characterisation of strains from meat products and cheese

In the framework of a European Union (EU) Coordinated Monitoring Programme, different types of ready-to eat (RTE) products, including soft and semi-soft cheese (n = 398) and cooked meat products (n = 403), were collected at retail in Italy and tested for detection and enumeration of Listeria monocytogenes. An Integrative Survey of 2696 samples, including soft and semi-soft cheese (n = 894) and cooked meat products (n = 1802) was carried out to have statistically representative results at the national level. Considering the results obtained both from the EU and the national Integrative Survey, prevalence of contamination of meat products was 1.66% (95% CL: 1.02–2.73%) at the arrival of the samples at the laboratory and 1.92% (95% CL: 1.31%–2.82%) at the end of shelf-life. Spalla cotta was the most frequently contaminated meat product. Prevalence of contamination in cheese was 2.13% (95% CL: 1.37%–3.3%) at the arrival at the laboratory and 1.01% (95% CL: 0.41%–2.55%) at the end of shelf-life. To get information about differences between cheese rinds and pastes, these two parts were separately analysed in the Integrative Survey samples. L. monocytogenes was detected in 4.02% (95% CL: 2.60%–6.19%) of cheese rinds (n = 473), whereas only the 0.34% (95% CL: 0.12%–0.98%) of cheese pastes (n = 894) were contaminated. This difference was statistically significant (χ² = 10.026, P < 0.05). Gorgonzola and Taleggio were the most frequently contaminated cheeses. Non-compliance with EU official criteria (100 CFU/g) was reported in 0.55% of meat products at arrival at the laboratory, in 0.46% at the end of shelf-life, and in 1.9% of cheese rinds. PFGE showed that cheese rinds could have been the origin of the few pastes' contamination, highlighting genetic similarity between isolates found in these two cheese components. Genetic subtyping also showed the presence of different pulsotypes, usually belonging to different clusters, in isolates from different food types. The presence of some pulsotypes predominantly prevalent over the others, in products from the same manufacturer, could suggest environmental strains as the main source of contamination.     

Occurrence of Listeria spp. in Brazilian fresh sausage and control of Listeria monocytogenes using bacteriophage P100

Since the 1980s, an increase in outbreaks of human listeriosis linked to contaminated food has been a concern of health authorities. Intensively manipulated foods, such as Brazilian fresh sausage, are frequently responsible for food-borne diseases. In this work the occurrence of Listeria monocytogenes and the efficacy of bacteriophage P100 (LISTEX?) to control the microorganism was evaluated in Brazilian fresh sausage. Eighty samples were analyzed, 40 each of swine and chicken Brazilian fresh sausage. Listeria spp. were isolated from 12 samples (15%), of which three (3.75%) were positive for L. monocytogenes. L. monocytogenes strains isolated belonged to serotype 1/2a. L. monocytogenes 1/2a was inoculated in Brazilian fresh sausage (2.1 × 10⁴ cfu/g) with the bacteriophage added thereafter (3.0 × 10⁷ pfu/g). Samples were analysed immediately (day zero) and then stored at 4 °C for 10 days. The bacteriophage P100 reduced L. monocytogenes counts by 2.5 log units at both 0 and 10 days compared to controls without bacteriophage. In spite of this, the populations of L. moncytogenes increased over the 10 day storage. Our data demonstrate that in one of the samples the use of the bacteriophage dropped the bacteria count below the level of direct detection. This study demonstrates a new alternative for pathogen control in the food industry, especially in the processes used to produce Brazilian fresh sausage.     

A review of minimal and defined media for growth of Listeria monocytogenes

Listeria monocytogenes is known to be an important foodborne pathogen and is the causative agent of one of the deadliest foodborne illnesses. The organism has a wide range of environmental conditions under which it will survive and grow, and often contaminates processing plants and retail environments in which ready-to-eat foods are manufactured, prepared and served. Although L. monocytogenes is not a fastidious organism and can grow in a variety of rich media, defined and minimal media are necessary to elucidate the minimal environmental nutrients that are required for the survival and growth of this organism. In addition, many of the virulence factors required for L. monocytogenes to invade and multiply in mammalian host cells are not produced in rich media and thus induction of these factors is best studied in a minimal medium. This review covers the historical development of minimal media for Listeria spp. and explores the various factors required for survival and growth of this organism. To the best of our knowledge, this is the first time that these media have been compared side by side. In order to better compare studies using different chemical substrates such as a different carbon source, all concentrations of components in each medium have been converted to molar concentrations.     

The international risk governance council framework and its application to Listeria monocytogenes in soft cheese made from unpasteurised milk

The International Risk Governance Council (IRGC) developed a Risk Governance Framework whose purpose is to help policy makers, regulators and risk managers both understand the concept of risk governance and apply it to their handling of risks. The Framework allows input from the scientific and public health perspective as well as the less-easily measurable side of freedom of choice. For food, the public wants many choices and less regulation, but at the same time safe products to consume. These ideals are at times in conflict. One example of this is the demand for soft cheese made from unpasteurised milk which may be contaminated with L. monocytogenes or other pathogens. Distinguishing between simple, complex, uncertain and ambiguous risks can help to design a risk management strategy. Public health advocates typically see management of soft cheese made from unpasteurised milk as a simple risk (protection of health), whereas for a small but vociferous proportion of the public sees it more of an uncertain or ambiguous one (choice trumps the slight risk of illness). The implications of five management options for the cheese are discussed. The new Codex Guidelines tend to put these cheeses into Option 1, a strict control on the presence of the pathogen, which will be ignored by the cheese aficionados. Other options are worth exploring to give some more choice but at low risk.     

Use of lactic acid bacteria (LAB) biofilms for the control of Listeria monocytogenes in a small-scale model

The antilisterial activity in biofilms developed in a small-scale model by two LAB (lactic acid bacteria) bacteriocin producers (Lactobacillus plantarum 35d, Enterococcus casseliflavus IM 416K1) and by two non-producers (L. plantarum 396/1, Enterococcus faecalis JH2-2) was evaluated against Listeria monocytogenes NCTC 10888. The LAB biofilms showed the capability to influence the survival and the multiplication of the pathogen with differences among the strains. L. plantarum 35d displayed the highest efficacy reducing L. monocytogenes by 5.4 log in the planktonic population and by 3.9 log in the adherent population at the end of the experiment (10 days). L. plantarum 396/1 reduced L. monocytogenes by 3.8 log in the adherent cells and by 4.9 log in the planktonic cells and this outcome could be attributed to the pH reduction.The E. casseliflavus IM 416K1 biofilm caused a L. monocytogenes reduction of 3.7 log in the adherent cells and of 4.8 log in the planktonic cells and the role of the bacteriocin production seemed to be predominant as the pH values did not significantly decrease. This hypothesis is confirmed by a slight capability to influence the L. monocytogenes survival by the non-bacteriocinogenic E. faecalis JH2-2. Studies performed with L. monocytogenes in co-culture with a Pseudomonas putida strain, revealed a reduction of the antilisterial activity only for the biofilms produced by lactobacilli.     

Antibacterial activity and mechanism of bifidocin A against Listeria monocytogenes

Bifidocin A, produced by Bifidobacterium animalis BB04, is a novel bacteriocin with antimicrobial activity against a wide range of gram-positive and gram-negative foodborne bacteria. The objective of this study was to investigate the antibacterial activity and mechanism of action of bifidocin A against Listeria monocytogenes, one of the most susceptible bacteria to this bacteriocin. The minimum inhibitory concentration (MIC) of bifidocin A for L. monocytogenes 35152 was 0.029 mg/mL. Time-kill assays showed that bifidocin A effectively inhibited the growth of L. monocytogenes in a time-and concentration-dependent manner. The mechanism of action of bifidocin A was studied by analyzing its effects at a MIC on the cell morphology, intracellular organization, membrane permeability, membrane integrity, and membrane proton motive force (PMF) of L. monocytogenes. Scanning and transmission electron microscopy analyses showed that bifidocin A induced alterations in the morphology and intracellular organization of L. monocytogenes cells. Confocal laser scanning microscopy images showed that L. monocytogenes cells treated with bifidocin A took up propidium iodide. Bifidocin A treatment also induced the leakage of K⁺ and inorganic phosphate, the hydrolysis and release of ATP, and a collapse of the transmembrane electrical potential and pH gradient in L. monocytogenes cells. These results suggested that bifidocin A exerted its anti-Listeria monocytogenes effect through the dissipation of the cytoplasmic membrane PMF, increased membrane permeability, cell membrane pore formation, destruction of membrane integrity, and ultimately complete disintegration of the cells.