Behavior of the Meat-Borne Bacterium Lactobacillus sakei during Its Transit through the Gastrointestinal Tracts of Axenic and Conventional Mice

A Lactobacillus sakei strain named FLEC01 was isolated from human feces and characterized genotypically. Comparison of the genetic features of this strain with those of both the meat-borne L. sakei strain 23K and another human isolate, LTH5590, showed that they belong to different but closely related clusters. The three L. sakei strains did not persist and only transited through the gastrointestinal tracts (GITs) of conventional C3H/HeN mice. In contrast, they all colonized the GITs of axenic mice and rapidly reached a population of 10⁹ CFU/g of feces, which remained stable until day 51. Five days after mice were fed, a first subpopulation, characterized by small colonies, appeared and reached 50% of the total L. sakei population in mice. Fifteen to 21 days after feeding, a second subpopulation, characterized by rough colonies, appeared. It coexisted with the two other populations until day 51, and its cell shapes were also affected, suggesting a dysfunction of the cell division or cell wall. No clear difference between the behaviors of the meat-borne strain and the two human isolates in both conventional and axenic mice was observed, suggesting that L. sakei is a food-borne bacterium rather than a commensal one and that its presence in human feces originates from diet. Previous observations of Escherichia coli strains suggest that the mouse GIT environment could induce mutations to increase their survival and colonization capacities. Here, we observed similar mutations concerning a food-grade gram-positive bacterium for the first time.Although initially characterized from rice wine (28), the lactic acid bacterium species Lactobacillus sakei is considered the main representative flora of meat products, representing the major population of many fermented meat products and of raw meat stored under vacuum-packaged conditions (10, 12, 13). L. sakei is naturally present in many fish and meat products that are traditionally processed without the use of starter cultures (33). In addition, when small-scale facilities producing traditional dry fermented sausage were searched, L. sakei was detected only in the meat matrix, suggesting that meat is contaminated by this species mainly during the early processing steps (certainly by hide or feces of the animals) and not later on or by contact with the environment or materials within the facilities (2).L. sakei shows high degrees of phenotypic and genomic diversity (11-13) that may explain the difficult detection and misidentification of it in the past. For instance, although the human gut microbiota has been intensively investigated by different microbial and molecular methods for many years, the presence of L. sakei in the feces of healthy humans was reported only recently (16, 17, 26, 39). The presence of the meat-borne species L. sakei in human feces, similar to that of several other lactobacilli, could be correlated to human diet, including raw and fermented meat (or fish), for millennia (37). Considering its relatively high concentration in human feces (10⁶ per g) that was previously reported (16), L. sakei was considered as one of the predominant food-associated Lactobacillus species present in human feces. Its natural reservoir and its origin prior to meat contamination are still not known. One can hypothesize that it belongs to the intestinal microbiota of animals used for meat production, although its presence has not yet been reported in mammals and has been reported only recently in the intestines of salmonids (5).Most of the available literature on L. sakei deals with its physiology in relation to preservation, fermentation, or spoilage of meat products (see references 10 and 13 and the references therein). Since its use as an ingredient or additive bioprotective culture, to ensure microbial safety of nonfermented meat products, has been proposed (8, 10), information on its behavior in the gastrointestinal tract (GIT) after ingestion of foodstuffs is required. The purpose of this study was thus to evaluate the ability of L. sakei to survive and transit in the GIT. Therefore, we compared two independent L. sakei strains isolated from human feces to the meat-associated L. sakei 23K model strain and analyzed their behaviors in the GITs of both conventional and axenic mice.     

Molecular Ecology of Listeria monocytogenes: Evidence for a Reservoir in Milking Equipment on a Dairy Farm

A longitudinal study aimed to detect Listeria monocytogenes on a New York State dairy farm was conducted between February 2004 and July 2007. Fecal samples were collected every 6 months from all lactating cows. Approximately 20 environmental samples were obtained every 3 months. Bulk tank milk samples and in-line milk filter samples were obtained weekly. Samples from milking equipment and the milking parlor environment were obtained in May 2007. Fifty-one of 715 fecal samples (7.1%) and 22 of 303 environmental samples (7.3%) were positive for L. monocytogenes. A total of 73 of 108 in-line milk filter samples (67.6%) and 34 of 172 bulk tank milk samples (19.7%) were positive for L. monocytogenes. Listeria monocytogenes was isolated from 6 of 40 (15%) sampling sites in the milking parlor and milking equipment. In-line milk filter samples had a greater proportion of L. monocytogenes than did bulk tank milk samples (P < 0.05) and samples from other sources (P < 0.05). The proportion of L. monocytogenes-positive samples was greater among bulk tank milk samples than among fecal or environmental samples (P < 0.05). Analysis of 60 isolates by pulsed-field gel electrophoresis (PFGE) yielded 23 PFGE types after digestion with AscI and ApaI endonucleases. Three PFGE types of L. monocytogenes were repeatedly found in longitudinally collected samples from bulk tank milk and in-line milk filters.Listeria monocytogenes can cause listeriosis in humans. This illness, despite being underreported, is an important public health concern in the United States (23) and worldwide. According to provisional incidence data provided by the Centers for Disease Control and Prevention (CDC), 762 cases of listeriosis were reported in the United States in 2007. In previous years (2003 to 2006), the number of reported annual listeriosis cases in the United States ranged between 696 and 896 cases per year (5).Exposure to food-borne L. monocytogenes may cause fever, muscle aches, and gastroenteritis (30), but does not usually cause septicemic illness in healthy nonpregnant individuals (7, 30). Elderly and immunocompromised people, however, are susceptible to listeriosis (22, 10), and they may develop more-severe symptoms (10). Listeriosis in pregnant women may cause abortion (22, 30) or neonatal death (22).Dairy products have been identified as the source of several human listeriosis outbreaks (4, 7, 10, 22). Listeria is ubiquitous on dairy farms (26), and it has been isolated from cows'' feces, feed (3, 26), and milk (21, 35). In ruminants, L. monocytogenes infections may be asymptomatic or clinical. Clinical cases typically present with encephalitis and uterine infections, often resulting in abortion (26, 39). Both clinically infected and healthy animals have been reported to excrete L. monocytogenes in their feces (20), which could eventually cause contamination of the bulk tank milk or milk-processing premises (39).On-farm epidemiologic research provides science-based information to improve farming and management practices. The Regional Dairy Quality Management Alliance (RDQMA) launched a combined United States Department of Agriculture (USDA)-RDQMA pilot project in January 2004 to scientifically validate intervention strategies in support of recommended best management practices among northeast dairy farms. The primary goal of the project was to track dynamics of infectious microorganisms on well-characterized dairy farms. Target species included Salmonella spp. (6, 36, 37), Mycobacterium avium subsp. paratuberculosis (13, 24), and L. monocytogenes.The objectives of this study were to describe the presence of L. monocytogenes on a dairy farm over time and to perform molecular subtyping by pulsed-field gel electrophoresis (PFGE) on L. monocytogenes isolates obtained from bulk tank milk, milk filters, milking equipment, feces, and the environmental samples to identify diversity among L. monocytogenes strains, persistence, and potential sources of bulk tank milk contamination.     

Competition of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed-Culture Biofilms

The majority of Listeria monocytogenes isolates recovered from foods and the environment are strains of serogroup 1/2, especially serotypes 1/2a and 1/2b. However, serotype 4b strains cause the majority of human listeriosis outbreaks. Our investigation of L. monocytogenes biofilms used a simulated food-processing system that consisted of repeated cycles of growth, sanitation treatment, and starvation to determine the competitive fitness of strains of serotypes 1/2a and 4b in pure and mixed-culture biofilms. Selective enumeration of strains of a certain serotype in mixed-culture biofilms on stainless steel coupons was accomplished by using serotype-specific quantitative PCR and propidium monoazide treatment to prevent amplification of extracellular DNA or DNA from dead cells. The results showed that the serotype 1/2a strains tested were generally more efficient at forming biofilms and predominated in the mixed-culture biofilms. The growth and survival of strains of one serotype were not inhibited by strains of the other serotype in mixed-culture biofilms. However, we found that a cocktail of serotype 4b strains survived and grew significantly better in mixed-culture biofilms containing a specific strain of serotype 1/2a (strain SK1387), with final cell densities averaging 0.5 log₁₀ CFU/cm² higher than without the serotype 1/2a strain. The methodology used in this study contributed to our understanding of how environmental stresses and microbial competition influence the survival and growth of L. monocytogenes in pure and mixed-culture biofilms.A prominent food-borne pathogen, Listeria monocytogenes can cause severe infections in humans, primarily in high-risk populations, though the disease (listeriosis) is relatively rare (11, 30, 43). Outbreaks of listeriosis have resulted from the contamination of a variety of foods by L. monocytogenes, especially meat and dairy products (27). L. monocytogenes is ubiquitous in the environment, able to grow at refrigeration temperature, and tolerant of the low pHs (3 to 4) typical of acidified foods (28, 32, 44). The capacity to produce biofilms confers protection against stresses common in the food-processing environment (13, 33).Biofilms are characterized by dense clusters of bacterial cells embedded in extracellular polymeric substances which are secreted by cells to aid in adhesion to surfaces and to other cells (4, 5). Strains of L. monocytogenes have been known to persist for years in food-processing environments, presumably in biofilms. Of the 13 known serotypes of L. monocytogenes, three (1/2a, 1/2b, and 4b) account for >95% of the isolates from human illness (21). Serotype 1/2a accounts for >50% of the L. monocytogenes isolates recovered from foods and the environment, while most major outbreaks of human listeriosis have been caused by serotype 4b strains (1, 3, 14, 15, 17, 22, 29, 31, 41, 47, 49,). No correlation between L. monocytogenes strain fitness and serotype has been identified (16, 19). Some studies have reported that strains repeatedly isolated from food and environmental samples (defined as persistent strains) had a higher adherence capacity than strains that were sporadically isolated (2, 36), while this phenomenon was not observed by others (7). Serotype 4b strains exhibited a higher capacity for biofilm formation than did serotype 1/2a strains (36), whereas this was not observed by Di Bonaventura and colleagues (6). It has been suggested that serotype 1/2a strains could be more robust than serotype 4b strains in biofilm formation under a variety of environmental conditions. Furthermore, strains of these serotypes differ in terms of the medium that promotes biofilm formation. Biofilm formation by serotype 4b strains was higher in full-strength tryptic soy broth than in diluted medium, whereas the opposite was observed with serotype 1/2a strains, which produced more biofilm in diluted medium (12).There is limited information on microbial competition between strains of different serotypes in biofilms or on how the environmental stresses present in food-processing environments may affect the biofilm formation and survival of L. monocytogenes of different serotypes. In food-processing plants, the environmental stresses encountered by bacteria are more complex and variable than most laboratory systems used for microbial ecology and biofilm studies. A simulated food-processing (SFP) system has been developed to address this issue (38). The SFP system incorporates several stresses that may affect bacteria in biofilms in the food-processing environment, including exposure to sanitizing agents, dehydration, and starvation. When biofilms were subjected to the SFP regimen over a period of several weeks, the cell numbers of L. monocytogenes strains in the biofilms initially were reduced and then increased as the culture adapted (38). The development of resistance to sanitizing agents was specific to the biofilm-associated cells and was not apparent in the detached cells (38). This suggested that extracellular polymeric substances present in the biofilm matrix were responsible for the resistance to sanitizing agents. It was subsequently found that real-time PCR, in combination with propidium monoazide (PMA) treatment of samples prior to DNA isolation, was an effective method for enumerating viable cells in biofilms (37).The objective of this study was to determine if strains of serotype 1/2a or 4b have a selective advantage under stress conditions. We investigated and compared the initial attachment and biofilm formation capabilities of L. monocytogenes strains of these two serotypes and analyzed the survival and growth of bacteria of each serotype in mixed-serotype biofilms in the SFP system by using PMA with quantitative PCR.     

Listeria monocytogenes Attachment to and Detachment from Stainless Steel Surfaces in a Simulated Dairy Processing Environment

The presence of pathogens in dairy products is often associated with contamination via bacteria attached to food-processing equipment, especially from areas where cleaning/sanitation is difficult. In this study, the attachment of Listeria monocytogenes on stainless steel (SS), followed by detachment and growth in foods, was evaluated under conditions simulating a dairy processing environment. Initially, SS coupons were immersed in milk, vanilla custard, and yogurt inoculated with the pathogen (10⁷ CFU/ml or CFU/g) and incubated at two temperatures (5 and 20°C) for 7 days. By the end of incubation, cells were mechanically detached from coupons and used to inoculate freshly pasteurized milk which was subsequently stored at 5°C for 20 days. The suspended cells in all three products in which SS coupons were immersed were also used to inoculate freshly pasteurized milk (5°C for 20 days). When SS coupons were immersed in milk, shorter lag phases were obtained for detached than for planktonically grown cells, regardless of the preincubation temperature (5 or 20°C). The opposite was observed when custard incubated at 20°C was used to prepare the two types of inocula. However, in this case, a significant increase in growth rate was also evident when the inoculum was derived from detached cells. In another parallel study, while L. monocytogenes was not detectable on SS coupons after 7 days of incubation (at 5°C) in inoculated yogurt, marked detachment and growth were observed when these coupons were subsequently transferred and incubated at 5°C in fresh milk or/and custard. Overall, the results obtained extend our knowledge on the risk related to contamination of dairy products with detached L. monocytogenes cells.Listeria monocytogenes is ubiquitous in nature due to its inherent ability to survive and grow under a wide range of adverse environmental conditions, such as refrigeration temperatures, high acidity and salinity, and reduced water activity (16). This microorganism is a major concern for the food industry, since it is the causal agent of listeriosis, a severe disease with high hospitalization and case-fatality rates (approximately 91% and 30%, respectively) (25). According to the European Centre for Disease Control and Prevention, listeriosis was the fifth most common zoonotic infection in Europe in 2006 (14), while it accounts for approximately 28% of the deaths resulting from food-borne illnesses in the United States (34).In the food industry, inadequately cleaned food-processing equipment (e.g., stainless steel [SS] surfaces) constitutes a potential source for L. monocytogenes, resulting in contamination of foods which come in contact with such equipment (36). Even though adherence to strict sanitation practices should minimize the risk of survivors on surfaces, existing evidence suggests that a considerable risk may occur in sites of processing plants which are not easily cleaned or sanitized, such as those that do not allow direct access of sanitation equipment for abrasion (e.g., edges, convex surfaces, etc.) (43, 45). Attachment to surfaces is believed to be important for the survival and persistence of this pathogen in such environments, with some strains able to remain on equipment surfaces for several years (32, 37). Thus, L. monocytogenes has been shown to adhere to and form biofilms on various food contact surfaces under laboratory conditions (3, 42, 44). Furthermore, attached L. monocytogenes cells are more difficult to mechanically remove from surfaces and are more resistant to sanitizers than their free-living counterparts (15, 40).Dairy products have been implicated in outbreaks of listeriosis (10, 31). However, most of the in vitro studies of the growth and survival of L. monocytogenes in such products have used strains previously cultivated planktonically (41). Although the results obtained in these studies are of great value, such studies have not taken into consideration that cells contaminating a product in a food-processing environment are usually attached to surfaces enclosed in biofilms. Limited information is available on the kinetic behavior of L. monocytogenes in dairy products inoculated with detached cells, although preincubation conditions have been shown to influence subsequent growth and survival of L. monocytogenes in foods (7, 13, 17, 18). Given the major physiological differences between attached and planktonic cells (15, 27, 48), an effect on subsequent growth might be possible.Considering the above, the main objective of the present study was to assess the influence of L. monocytogenes preincubation conditions with respect to mode of growth (either attached to SS or grown suspended in dairy products) on the subsequent growth of this pathogen in milk (at 5°C for 20 days). To prepare the two types of inocula, two different growth media (milk and vanilla custard) and temperatures (5 and 20°C) were studied. The unforced detachment of L. monocytogenes cells from SS coupons and growth in two dairy products (milk and custard) at 5°C for 20 days was also evaluated. In the latter case, previous attachment of cells to the coupons was done under especially adverse preincubation conditions (in yogurt at 5°C for 7 days).     

Effect of Octenidine Hydrochloride on Planktonic Cells and Biofilms of Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen capable of forming biofilms and persisting in food processing environments for extended periods of time, thereby potentially contaminating foods. The efficacy of octenidine hydrochloride (OH) for inactivating planktonic cells and preformed biofilms of L. monocytogenes was investigated at 37, 21, 8, and 4°C in the presence and absence of organic matter (rehydrated nonfat dry milk). OH rapidly killed planktonic cells and biofilms of L. monocytogenes at all four temperatures. Moreover, OH was equally effective in killing L. monocytogenes biofilms on polystyrene and stainless steel matrices in the presence and absence of organic matter. The results underscore OH''s ability to prevent establishment of L. monocytogenes biofilms by rapidly killing planktonic cells and to eliminate preformed biofilms, thus suggesting that it could be used as a disinfectant to prevent L. monocytogenes from persisting in food processing environments.Listeria monocytogenes is a major bacterial pathogen (2), accounting for approximately 28% of the deaths resulting from food-borne illnesses in the United States (22). It is widespread in nature and occurs in soil, vegetation, fecal matter, sewage, water, and animal feed (14). Because it is ubiquitous, L. monocytogenes is frequently isolated from foods and food processing environments (13, 23), thereby presenting a significant challenge to the food industry. Several studies have shown that L. monocytogenes is capable of adhering to food contact surfaces, such as glass, stainless steel, rubber, and polystyrene (6, 11, 28). Upon attachment to such surfaces, L. monocytogenes establishes biofilms and persists for long periods of time in the food processing environment (18, 30). This potentially poses a food safety hazard since biofilms are an important source of contamination of food products that come into contact with them. In addition, biofilms also protect the underlying bacteria from desiccation, antimicrobials, and sanitizing agents (7, 16). Thus, eradication of L. monocytogenes biofilms in processing plants is critical for improving food safety.When problems with persistent L. monocytogenes are encountered in food processing facilities, plant hygiene and sanitation are emphasized (31). This involves preventing the establishment of L. monocytogenes biofilms in the food processing environment and reducing contamination of product contact surfaces. A variety of cleaners and disinfectants, including quaternary ammonium compounds and hypochlorite, have been evaluated for this purpose (20). Although these compounds are approved by the Food and Drug Administration for use as disinfectants in processing plants, they are not effective in killing L. monocytogenes (24, 25), especially in the presence of soil or organic matter and at low temperatures. Therefore, there is a need for an effective disinfectant that can eliminate listerial biofilms in the presence of organic matter at a wide range of temperatures. Octenidine hydrochloride (OH) is a positively charged bispyridinamine that exhibits antimicrobial activity against plaque-producing organisms, such as Streptococcus mutans and Streptococcus sanguis (5). Toxicity studies with a variety of species have shown that OH is not absorbed through mucous membranes and the gastrointestinal tract, and there have been no reports of carcinogenicity, genotoxicity, or mutagenicity of this compound (17, 19, 29).The objective of this study was to investigate the efficacy of OH for inactivating planktonic cells and preformed biofilms of L. monocytogenes at 37, 21, 8, and 4°C in the presence and absence of organic matter on two matrices, polystyrene and stainless steel.     

Probabilistic Model for Listeria monocytogenes Growth during Distribution,Retail Storage,and Domestic Storage of Pasteurized Milk

A survey on the time-temperature conditions of pasteurized milk in Greece during transportation to retail, retail storage, and domestic storage and handling was performed. The data derived from the survey were described with appropriate probability distributions and introduced into a growth model of Listeria monocytogenes in pasteurized milk which was appropriately modified for taking into account strain variability. Based on the above components, a probabilistic model was applied to evaluate the growth of L. monocytogenes during the chill chain of pasteurized milk using a Monte Carlo simulation. The model predicted that, in 44.8% of the milk cartons released in the market, the pathogen will grow until the time of consumption. For these products the estimated mean total growth of L. monocytogenes during transportation, retail storage, and domestic storage was 0.93 log CFU, with 95th and 99th percentiles of 2.68 and 4.01 log CFU, respectively. Although based on EU regulation 2073/2005 pasteurized milk produced in Greece belongs to the category of products that do not allow the growth of L. monocytogenes due to a shelf life (defined by law) of 5 days, the above results show that this shelf life limit cannot prevent L. monocytogenes from growing under the current chill chain conditions. The predicted percentage of milk cartons—initially contaminated with 1 cell/1-liter carton—in which the pathogen exceeds the safety criterion of 100 cells/ml at the time of consumption was 0.14%. The probabilistic model was used for an importance analysis of the chill chain factors, using rank order correlation, while selected intervention and shelf life increase scenarios were evaluated. The results showed that simple interventions, such as excluding the door shelf from the domestic storage of pasteurized milk, can effectively reduce the growth of the pathogen. The door shelf was found to be the warmest position in domestic refrigerators, and it was most frequently used by the consumers for domestic storage of pasteurized milk. Furthermore, the model predicted that a combination of this intervention with a decrease of the mean temperature of domestic refrigerators by 2°C may allow an extension of pasteurized milk shelf life from 5 to 7 days without affecting the current consumer exposure to L. monocytogenes.L. monocytogenes is an important safety concern for the dairy industry. Several listeriosis outbreaks have been associated with the consumption of dairy products, including pasteurized milk (13, 22). An effective control of L. monocytogenes in pasteurized milk should be based on the selection of raw milk and the controls of the processing, packaging, distribution, and storage conditions. In general, the pathogen is effectively controlled during pasteurization. However, its presence in the finished product is possible as a result of postpasteurization contamination from sources in the plant environment. Considering that the levels of postpasteurization contamination are usually very low, the extent of L. monocytogenes growth during distribution, retail storage, and domestic storage is of major significance for the safety status of pasteurized milk at the time of consumption.The growth of L. monocytogenes during distribution and storage of pasteurized milk can be evaluated using the available predictive models. During the last decade, a large number of mathematical models for L. monocytogenes growth have been published (9, 11, 16, 19, 21, 24, 26, 31, 38), and some of them have been targeted to pasteurized milk (1, 40, 49). However, since the available data show that conditions that prevail during the chill chain vary significantly (8, 17, 23, 27, 28, 34, 44, 45, 48), the value of a deterministic application of these models as a tool in safety management of pasteurized milk would be limited.In recent years the need for taking into account the variabilities of the various factors in predictive microbiology has been increasingly recognized, leading to a more sophisticated modeling approach called probabilistic or stochastic modeling. The main characteristic of probabilistic modeling is the specific quantification of variabilities using probability distributions for the input data rather than point estimates. The importance of characterizing variability was stressed by Nauta (41), who illustrated the differences in decisions that could result if variability is ignored. Probabilistic modeling is being used with increasing frequency in the area of food safety. It has been extensively applied in quantitative microbial risk assessments (12, 18, 20), in quality and safety management systems (25, 30, 32), and recently for more specific topics, such as the evaluation of the effects of food processing (39) and the compliance of food products to safety criteria set by regulations (33).In the present study a probabilistic modeling approach was applied for evaluating the growth of Listeria monocytogenes in pasteurized milk from production to the time of consumption based on a Monte Carlo simulation. The objectives of the study were (i) to estimate the growth of the pathogen at the various stages of the chill chain, including transportation to retail, retail storage, and domestic storage, (ii) to analyze the importance of the chill chain factors, (iii) to evaluate the effects of selected intervention scenarios related to the improvement of the chill chain and handling conditions, and (iv) to evaluate the effect of a potential extension of milk shelf life on the growth of Listeria monocytogenes.     

Temperature-Dependent Requirement for Catalase in Aerobic Growth of Listeria monocytogenes F2365

Listeria monocytogenes is a Gram-positive, psychrotrophic, facultative intracellular food-borne pathogen responsible for severe illness (listeriosis). The bacteria can grow in a wide range of temperatures (1 to 45°C), and low-temperature growth contributes to the food safety hazards associated with contamination of ready-to-eat foods with this pathogen. To assess the impact of oxidative stress responses on the ability of L. monocytogenes to grow at low temperatures and to tolerate repeated freeze-thaw stress (cryotolerance), we generated and characterized a catalase-deficient mutant of L. monocytogenes F2365 harboring a mariner-based transposon insertion in the catalase gene (kat). When grown aerobically on blood-free solid medium, the kat mutant exhibited impaired growth, with the extent of impairment increasing with decreasing temperature, and no growth was detected at 4°C. Aerobic growth in liquid was impaired at 4°C, especially under aeration, but not at higher temperatures (10, 25, or 37°C). Genetic complementation of the mutant with the intact kat restored normal growth, confirming that inactivation of this gene was responsible for the growth impairment. In spite of the expected impact of oxidative stress responses on cryotolerance, cryotolerance of the kat mutant was not affected.Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen that has the ability to cause a severe disease (listeriosis) in humans and animals (13, 28, 30). L. monocytogenes is ubiquitously distributed in the environment and has the ability to grow over a wide range of temperatures (between 1 and 45°C) (13). Growth at low temperature has important implications for environmental persistence of the organism and for contamination of cold-stored, ready-to-eat foods, thus contributing to the food safety hazards associated with L. monocytogenes (19).L. monocytogenes is subjected to oxidative stress during both extracellular and intracellular growth and has evolved several responses to minimize the impact of reactive oxygen species (ROS). Catalase and superoxide dismutase (SOD) work synergistically in detoxification of ROS: superoxide anions are converted to H₂O₂ by SOD, with subsequent conversion of H₂O₂ into water and oxygen by catalase (22). Exposure to ROS may be especially acute during intracellular infection as well as under certain environmental conditions, such as those involved in repeated freezing and thawing (15, 16, 23, 29, 33).Previous studies revealed that the ability of L. monocytogenes to survive repeated freezing and thawing (cryotolerance) was markedly dependent on growth temperature, with bacteria grown at 37°C having significantly higher cryotolerance than those grown at either 4 or 25°C (1). However, mechanisms underlying Listeria''s cryotolerance have not been identified. Since oxidative damage is considered to take place during freezing and thawing, determinants such as catalase may be involved in cryotolerance.The catalase of L. monocytogenes has been investigated primarily in terms of its potential role in pathogenesis, with somewhat conflicting results. The isolation of catalase-negative strains from human listeriosis patients has led to the speculation that catalase is not required for human virulence (4, 8, 12, 31). On the other hand, under certain conditions (e.g., reduced serum levels), catalase-negative strains were impaired in their ability to survive in activated macrophages in comparison to catalase-positive strains (32). Furthermore, the catalase gene kat was among those for which expression was induced in infected cell cultures and in the spleens of mice infected with L. monocytogenes EGD-e, suggesting possible contributions to pathogenesis (5, 9).The potential role of catalase in environmental adaptations of L. monocytogenes such as growth at low temperature and cryotolerance was not addressed in these earlier investigations. In this study, we have characterized an isogenic mutant of L. monocytogenes F2365 to determine the involvement of catalase in growth at different temperatures, survival in selected foods, and cryotolerance of L. monocytogenes.     

Response of Listeria monocytogenes to Disinfection Stress at the Single-Cell and Population Levels as Monitored by Intracellular pH Measurements and Viable-Cell Counts

Listeria monocytogenes has a remarkable ability to survive and persist in food production environments. The purpose of the present study was to determine if cells in a population of L. monocytogenes differ in sensitivity to disinfection agents as this could be a factor explaining persistence of the bacterium. In situ analyses of Listeria monocytogenes single cells were performed during exposure to different concentrations of the disinfectant Incimaxx DES to study a possible population subdivision. Bacterial survival was quantified with plate counting and disinfection stress at the single-cell level by measuring intracellular pH (pH_i) over time by fluorescence ratio imaging microscopy. pH_i values were initially 7 to 7.5 and decreased in both attached and planktonic L. monocytogenes cells during exposure to sublethal and lethal concentrations of Incimaxx DES. The response of the bacterial population was homogenous; hence, subpopulations were not detected. However, pregrowth with NaCl protected the planktonic bacterial cells during disinfection with Incimaxx (0.0015%) since pH_i was higher (6 to 6.5) for the bacterial population pregrown with NaCl than for cells grown without NaCl (pH_i 5 to 5.5) (P < 0.05). The protective effect of NaCl was reflected by viable-cell counts at a higher concentration of Incimaxx (0.0031%), where the salt-grown population survived better than the population grown without NaCl (P < 0.05). NaCl protected attached cells through drying but not during disinfection. This study indicates that a population of L. monocytogenes cells, whether planktonic or attached, is homogenous with respect to sensitivity to an acidic disinfectant studied on the single-cell level. Hence a major subpopulation more tolerant to disinfectants, and hence more persistent, does not appear to be present.Listeria monocytogenes is a food-borne, human pathogen that has a remarkable ability to colonize food-processing environments (5, 16, 20, 21, 26, 29). Some L. monocytogenes strains can persist for years in food-processing plants (11, 14, 20, 27), and specific molecular subtypes can repeatedly be isolated from the processing environment (29) despite being very infrequent in the outdoor environment (9). This ability to persist has, hitherto, not been linked to any specific genetic or phenotypic trait.It has been suggested that persistent L. monocytogenes strains may be more tolerant or resistant to cleaning and especially disinfectants used in the food industry. Aase et al. (1) found increased tolerance to both benzalkonium chloride and ethidium bromide in L. monocytogenes isolates that had persisted for more than 4 years; however, other studies have not been able to link persistence and tolerance to disinfectants (6, 10, 11, 13). We recently compared disinfection sensitivities of persistent and presumed nonpersistent L. monocytogenes strains using viable-cell counts and did not find the latter group more sensitive to the two disinfectants Triquart SUPER and Incimaxx DES than persistent strains (13). However, we found that for all subtypes of L. monocytogenes, growth with NaCl increased the tolerance of planktonic L. monocytogenes cells to Incimaxx DES, whereas spot-inoculated, dried L. monocytogenes cells were not protected by NaCl against disinfection.There is no doubt that L. monocytogenes will be completely inactivated at the disinfectant concentrations recommended for use in the food industry; however, the efficiency of the disinfectant is very much influenced by the presence of organic material being inactivated by the presence of food debris. Hence, it is likely that the bacterial cell in a food production environment may be exposed to concentrations at a sublethal level. It is currently not known if treatment with a sublethal concentration of disinfectant affects the entire bacterial population or only attacks a fraction of the cell population, leaving another fraction of cells unaffected. In case of the latter, some bacterial cells may be able to survive the disinfection treatment. The potential presence of such tolerant subpopulations could, ultimately, ensure that the genome is propagated, leading to persistence.The presence of a more tolerant subpopulation can be determined on the single-cell level. Flow cytometry is a rapid method useable for measurements at the single-cell resolution (22); however, it cannot monitor the same single cells over time. Optical microscopy combined with microfluidic devices that allow measurement of growth of single cells is a useful technique (2), and in situ analyses of the physiological condition of single cells by the fluorescence ratio imaging microscopy (FRIM) technique represents another elegant approach (25). FRIM enables studies of dynamic changes with high sensitivity and on the single-cell level in important physiological parameters: e.g., intracellular pH (pH_i). Listeria maintains its pH_i within a narrow range of 7.6 to 8 at extracellular pH (pH_ex) values of 5.0 to 8.0 (4, 25) and at pH_ex 4.0 with the presence of glucose (23). It is believed that viable cells need to maintain a transmembrane pH gradient with their pH_i above the pH_ex, and failure to maintain pH_i homeostasis indicates that the bacterial cell is severely stressed and ultimately leads to loss of cell viability. FRIM has been used to determine the pH_i of L. monocytogenes after exposure to osmotic and acid stress (7, 23). Also, the dissipation of the pH gradient in L. monocytogenes after exposure to different bacteriocins has been determined with FRIM (4, 12). Hornbæk et al. (12) found that treatment with subinhibitory concentrations of leucocin and nisin gave rise to two subpopulations: one consisting of cells with a dissipated pH gradient (ΔpH) and the other consisting of cells that maintained ΔpH, which could indicate phenotypic heterogeneity.The aim of the present study was to investigate the physiological effects of the disinfectant Incimaxx DES at sublethal and lethal concentrations on single cells and the population level of a persistent L. monocytogenes strain to study a possible subdivision of sensitivity in the population. We also addressed the potential protective effect of NaCl against disinfection and compared sensitivities in a population of planktonic and attached bacteria. We applied the in situ technique FRIM and compared the pH_i measurements with the traditional viable-cell-count method.(Part of the results have been presented at a poster session at the 95th International Association for Food Protection annual meeting in Columbus, OH, 3 to 6 August 2008.)     

Influence of Stress on Single-Cell Lag Time and Growth Probability for Listeria monocytogenes in Half Fraser Broth

The impacts of 12 common food industry stresses on the single-cell growth probability and single-cell lag time distribution of Listeria monocytogenes were determined in half Fraser broth, the primary enrichment broth of the International Organization for Standardization detection method. First, it was determined that the ability of a cell to multiply in half Fraser broth is conditioned by its history (the probability for a cell to multiply can be decreased to 0.05), meaning that, depending on the stress in question, the risk of false-negative samples can be very high. Second, it was established that when cells are injured, the single-cell lag times increase in mean and in variability and that this increase represents a true risk of not reaching the detection threshold of the method in the enrichment broth. No relationship was observed between the impact on single-cell lag times and that on growth probabilities. These results emphasize the importance of taking into account the physiological state of the cells when evaluating the performance of methods to detect pathogens in food.Listeria monocytogenes has been involved in severe food-borne outbreaks with high mortality rates. This pathogen is widespread in many environments (16) and can be isolated from a large variety of foods which are the major routes of infection in humans. Ready-to-eat foods that can support the growth of L. monocytogenes may pose a major risk for public health, and the European Union legislation generally requires absence in 25 g at the production stage as a food safety criterion for this type of food (4).In food, L. monocytogenes is often affected by one or more stresses caused by a variety of processing treatments, including heating, freezing, and exposure to acids and to high osmotic pressures (15, 25, 29, 39). Recovering stressed L. monocytogenes from food is of great importance in food safety since sublethally injured bacteria may repair themselves under suitable conditions and regain or even increase their pathogenicity (19, 30).The injury of microbial cells has two major consequences for pathogen behavior in enrichment broths. First, injured cells become sensitive to selective components present in enrichment broths to which they normally show resistance (9, 10, 11, 42). Therefore, some cells of the stressed bacterial population do not initiate growth in enrichment broth, eventually resulting in an inefficient detection of pathogenic bacteria in food samples (50). This phenomenon can explain results obtained in several studies showing the effect of inoculum size on the growth limits of bacterial populations (26, 27, 37). Second, due to repair time, stressed cells show a longer lag phase than do healthy cells (5, 7, 37). This situation results in a true risk of not reaching the bacterial concentration necessary for the detection of the pathogen (in the range of 10² to 10⁴ CFU ml⁻¹) within the enrichment duration.The recent development of gene-based or immunologically based procedures, such as PCR, gene probes, and enzyme-linked immunosorbent assay, has facilitated the development of more-rapid methods which can identify positive samples in considerably shorter time periods. Nevertheless, these relatively rapid tests also require efficient enrichment steps to increase target organism numbers to detectable levels.At the moment of pathogen detection, low numbers of sublethally injured cells, as often encountered in naturally contaminated foods, show a wide distribution of lag-phase durations (45) and may not be able to multiply in broth containing selective components (11, 42). The challenge of the enrichment stage is to obtain appropriate enrichment conditions (2) which will favor pathogen resuscitation and limit the food microflora growth.In our study, we have focused on the primary enrichment phase of the International Organization for Standardization 11290-1 L. monocytogenes detection method (3), i.e., the half Fraser broth (1/2FB). The objectives were to investigate the impact of 12 different stresses on the single-cell growth probability and single-cell lag time of L. monocytogenes in 1/2FB. The intraspecific variability and the impact of food components and background microflora on single-cell growth probability were also studied.     

Survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG in the Human Gastrointestinal Tract with Daily Consumption of a Low-Fat Probiotic Spread

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. Therefore, probiotic strains should be able to survive passage through the human gastrointestinal tract. Human gastrointestinal tract survival of probiotics in a low-fat spread matrix has, however, never been tested. The objective of this randomized, double-blind, placebo-controlled human intervention study was to test the human gastrointestinal tract survival of Lactobacillus reuteri DSM 17938 and Lactobacillus rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. Forty-two healthy human volunteers were randomly assigned to one of three treatment groups provided with 20 g of placebo spread (n = 13), 20 g of spread with a target dose of 1 × 10⁹ CFU of L. reuteri DSM 17938 (n = 13), or 20 g of spread with a target dose of 5 × 10⁹ CFU of L. rhamnosus GG (n = 16) daily for 3 weeks. Fecal samples were obtained before and after the intervention period. A significant increase, compared to the baseline, in the recovery of viable probiotic lactobacilli in fecal samples was demonstrated after 3 weeks of daily consumption of the spread containing either L. reuteri DSM 17938 or L. rhamnosus GG by selective enumeration. In the placebo group, no increase was detected. The results of selective enumeration were supported by quantitative PCR, detecting a significant increase in DNA resulting from the probiotics after intervention. Overall, our results indicate for the first time that low-fat spread is a suitable carrier for these probiotic strains.The human intestinal microflora or microbiota constitutes a metabolically active microbial environment. This community is relatively stable in the guts of healthy individuals (20). Some of the microbial groups harbor species that are potentially harmful, whereas others, such as the bifidobacteria and lactobacilli, are regarded as beneficial (8). Specific members of the genera Lactobacillus and Bifidobacterium are being applied in functional foods as probiotics (25). Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (9). The current scientific consensus is that probiotics should be alive to exert their beneficial effect in the human gastrointestinal (GI) tract (6). Consequently, probiotics should remain alive in the product, such that the daily effective dose per serving is still present at the end of the shelf life (14). Food matrices, production processes, or product usages that involve heating can affect the viability of probiotics (24).Typically, those members selected for probiotic application are chosen for their resistance to passage through the upper GI tract and thus are able to transiently colonize the gut (25). Human GI tract survival of probiotics should lead to shedding of live cells in fecal samples. GI tract survival is, however, dependent on both the strain and the food matrix involved (27). Fecal recovery of several probiotic strains has been demonstrated in different food matrices, including fermented milk and yoghurt (10, 26, 29), fruit drinks (21), a cereal bar (22), supplements (13, 17, 27), and infant formula (29).For this study, we have selected two well-established probiotic strains to test the suitability of a low-fat spread as a probiotic carrier, namely, Lactobacillus reuteri DSM 17938 (BioGaia, Sweden) and Lactobacillus rhamnosus GG (ATCC 53103; Valio, Finland). L. reuteri DSM 17938 was derived from L. reuteri ATCC 55730 by curing of two plasmids harboring antibiotic resistance genes (23). A series of in vitro experiments confirmed the retention of the functional properties of the daughter strain, as no differences in colony morphology, fermentation patterns, production of reuterin, generation time, mucus-binding ability, or tolerance to bovine bile were found between L. reuteri ATCC 55730 and DSM 17938. The daughter strain is somewhat more resistant to low pH and grows to a higher density in vitro (23). Several studies have been published which provide data on the survival of L. reuteri ATCC 55730 in the human GI tract at doses of 4 × 10⁸ to 1 × 10¹⁰ CFU/day in freeze-dried matrices and chewable tablets (32-34). Furthermore, L. reuteri DSM 17938 was demonstrated to survive human GI tract passage in the same way as L. reuteri ATCC 55730 (23).L. rhamnosus GG has been isolated from a healthy human intestinal flora by Goldin et al. (10). L. rhamnosus GG is relatively resistant to acid and bile, adheres in vitro to epithelial cells, and can produce an antimicrobial substance (10, 15). A wide range of studies have been published which provide data on the survival of L. rhamnosus GG in the human GI tract (3, 4, 10, 18, 19, 27-30), as well as transient colonization of the intestinal microbiota in healthy adults in various formats, including freeze-dried powder, capsules, and tablets or via fermented milk drinks, yoghurt, or fruit juice. Saxelin et al. (28) evaluated the dose-response effect of orally administered L. rhamnosus GG in powder form on fecal colonization in healthy adults, which indicated that consumption of approximately 10¹⁰ to 10¹¹ CFU/day was required to reach detectable levels in fecal samples from volunteers. This was also the case when L. rhamnosus GG was administered in gelatin capsules (29). Additionally, Saxelin et al. (27) observed that milk, but possibly also other protective compounds in food, can improve the survival of L. rhamnosus GG. Fecal recovery of L. rhamnosus GG in milk-based products was shown at dose levels of around 2 × 10⁹ CFU/day.It is, however, not known whether probiotics can survive passage through the human GI tract after the consumption of a low-fat spread. The objective of this randomized, double-blind, placebo-controlled human intervention study was therefore to test the human GI tract survival of L. reuteri DSM 17938 and L. rhamnosus GG after daily consumption of a low-fat probiotic spread by using traditional culturing, as well as molecular methods. The primary outcome parameter of this study was a significant change from the baseline in the number of probiotic bacteria of the respective strains in fecal samples.     

Quantification of Shiga Toxin-Converting Bacteriophages in Wastewater and in Fecal Samples by Real-Time Quantitative PCR

Shiga toxin-converting bacteriophages (Stx phages) are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Stx phages are released from their bacterial hosts after lytic induction and remain free in the environment. Samples were analyzed for the presence of free Stx phages by an experimental approach based on the use of real-time quantitative PCR (qPCR), which enables stx to be detected in the DNA from the viral fraction of each sample. A total of 150 samples, including urban raw sewage samples, wastewater samples with fecal contamination from cattle, pigs, and poultry, and fecal samples from humans and diverse animals, were used in this study. Stx phages were detected in 70.0% of urban sewage samples (10 to 10³ gene copies [GC] per ml) and in 94.0% of animal wastewater samples of several origins (10 to 10¹⁰ GC per ml). Eighty-nine percent of cattle fecal samples were positive for Stx phages (10 to 10⁵ GC per g of sample), as were 31.8% of other fecal samples of various origins (10 to 10⁴ GC per g of sample). The stx₂ genes and stx₂ variants were detected in the viral fraction of some of the samples after sequencing of stx₂ fragments amplified by conventional PCR. The occurrence and abundance of Stx phages in the extraintestinal environment confirm the role of Stx phages as a reservoir of stx in the environment.Shiga toxin-producing Escherichia coli (STEC) is associated with diarrhea, hemorrhagic enterocolitis, and hemolytic-uremic syndrome in humans (46). Escherichia coli serotype O157:H7 is the main cause of these diseases, although other serotypes of E. coli and other enterobacteria species have been described (36). These E. coli serotypes produce at least two immunologically distinct Shiga toxins, called Stx1 and Stx2. In addition to these, several variations of these toxins have been reported in recent years, showing differences in virulence and distribution in the host populations examined (48, 51). Shiga toxin genes are carried by temperate bacteriophages (19, 35). Stx-encoding bacteriophages investigated to date consist of double-stranded DNA and have lambdoid genetic structures (19, 27, 32, 37, 47). The induction and regulation of these phages are directly involved in the production of toxin and, therefore, in the pathogenicity of the strains (8, 50). Stx phages are efficient vectors for the transfer of toxin genes, being able to convert nonpathogenic bacterial hosts into Stx-producing strains by transduction of stx, as has been demonstrated under various conditions (1, 4, 27, 28, 41, 49).Most of the reported outbreaks of STEC infections are associated with cattle products (10, 17), with the consumption of contaminated foods (10, 34), and with several waterborne infections (30). Stx phages are present within fecally contaminated aquatic environments (9, 28, 30, 32, 45). Moreover, a high percentage of STEC strains present in extraintestinal environments carry inducible Stx phages (14, 30).As individuals infected with STEC strains shed large quantities of Stx phages in feces, Stx phages should be prevalent in the environment, as are other viruses transmitted by the fecal-oral route (5, 11) or bacteriophages infecting bacteria present in the intestinal tract (16, 23). Moreover, those STEC strains isolated from food and animals carry inducible Stx phages (24, 27, 42). The virulence profiles of STEC strains isolated from food also suggest the presence of inducible Stx phages (10).Stx phages in sewage have been detected by nested PCR (28, 29, 31). However, to quantify them, the most probable number (MPN) method was applied, which allows only a rough estimate of the amount of Stx phages present in the sample. To assess the number of Stx phages accurately, real-time quantitative PCR (qPCR) technology is a useful tool. This technology is both sensitive and specific, and it gives accurate quantitative results (25). Comparison with a standard enables the number of copies of stx to be quantified, which can then be translated into the number of Stx phage particles.Little is known about the prevalence of phages carrying stx in fecal samples. The data available on the numbers of these phages in fecally contaminated water samples were only roughly estimated. The first step to evaluate the role of Stx phages in the environment as lateral gene transfer vectors is to know the extent of these viruses in the environment. The aim of this study is to report quantitative data on the abundance of Stx phages in urban sewage samples, in wastewater samples from cattle, pigs, and poultry, and in diverse fecal samples, calculated by means of a methodology based on qPCR.     

Temperature-Dependent Phage Resistance of Listeria monocytogenes Epidemic Clone II

Listeria monocytogenes epidemic clone II (ECII) has been responsible for two multistate outbreaks in the United States in 1998-1999 and in 2002, in which contaminated ready-to-eat meat products (hot dogs and turkey deli meats, respectively) were implicated. However, ecological adaptations of ECII strains in the food-processing plant environment remain unidentified. In this study, we found that broad-host-range phages, including phages isolated from the processing plant environment, produced plaques on ECII strains grown at 37°C but not when the bacteria were grown at lower temperatures (30°C or below). ECII strains grown at lower temperatures were resistant to phage regardless of the temperature during infection and subsequent incubation. In contrast, the phage susceptibility of all other tested strains of serotype 4b (including epidemic clone I) and of strains of other serotypes and Listeria species was independent of the growth temperature of the bacteria. This temperature-dependent phage susceptibility of ECII bacteria was consistently observed with all surveyed ECII strains from outbreaks or from processing plants, regardless of the presence or absence of cadmium resistance plasmids. Phages adsorbed similarly on ECII bacteria grown at 25°C and at 37°C, suggesting that resistance of ECII strains grown at 25°C was not due to failure of the phage to adsorb. Even though the underlying mechanisms remain to be elucidated, temperature-dependent phage resistance may represent an important ecological adaptation of L. monocytogenes ECII in processed, cold-stored foods and in the processing plant environment, where relatively low temperatures prevail.Listeria monocytogenes is responsible for an estimated 2,500 cases of serious food-borne illness (listeriosis) and 500 deaths annually in the United States. It affects primarily pregnant women, newborns, the elderly, and adults with weakened immune systems. L. monocytogenes is frequently found in the environment and can grow at low temperatures, thus representing a serious hazard for cold-stored, ready-to-eat foods (18, 31).Two multistate outbreaks of listeriosis in the United States, in 1998-1999 and in 2002, respectively, were caused by contaminated ready-to-eat meats (hot dogs and turkey deli meats, respectively) contaminated by serotype 4b strains that represented a novel clonal group, designated epidemic clone II (ECII) (3, 4). ECII strains have distinct genotypes as determined by pulsed-field gel electrophoresis and various other subtyping tools, and harbor unique genetic markers (6, 8, 11, 19, 34). The genome sequencing of one of the isolates (L. monocytogenes H7858) from the 1998-1999 outbreak revealed the presence of a plasmid of ca. 80 kb (pLM80), which harbored genes mediating resistance to the heavy metal cadmium as well as genes conferring resistance to the quaternary ammonium disinfectant benzalkonium chloride (10, 29).Listeria phages (listeriaphage) have long been used for subtyping purposes (33), and extensive research has focused on the genomic characterization (2, 24, 26, 35), transducing potential (14), and biotechnological applications of selected phages (25). In addition, applications of listeriaphage as biocontrol agents in foods and the processing plant environment have been investigated (12, 15, 22). However, limited information exists on phages from processing plant environments and on the impact of environmental conditions on susceptibility of L. monocytogenes strains representing the major epidemic-associated clonal groups to such phages. We have found that strains harboring ECII-specific genetic markers can indeed be recovered from the environment of turkey-processing plants (9). Furthermore, environmental samples from such processing plants yielded phages with broad host range, which were able to infect L. monocytogenes strains of various serotypes, and different Listeria species (20). In this study, we describe the impact of growth temperature on susceptibility of L. monocytogenes ECII strains to phages, including phages isolated from turkey-processing plant environmental samples.     

Genetic Features of Resident Biofilms Determine Attachment of Listeria monocytogenes

Planktonic Listeria monocytogenes cells in food-processing environments tend most frequently to adhere to solid surfaces. Under these conditions, they are likely to encounter resident biofilms rather than a raw solid surface. Although metabolic interactions between L. monocytogenes and resident microflora have been widely studied, little is known about the biofilm properties that influence the initial fixation of L. monocytogenes to the biofilm interface. To study these properties, we created a set of model resident Lactococcus lactis biofilms with various architectures, types of matrices, and individual cell surface properties. This was achieved using cell wall mutants that affect bacterial chain formation, exopolysaccharide (EPS) synthesis and surface hydrophobicity. The dynamics of the formation of these biofilm structures were analyzed in flow cell chambers using in situ time course confocal laser scanning microscopy imaging. All the L. lactis biofilms tested reduced the initial immobilization of L. monocytogenes compared to the glass substratum of the flow cell. Significant differences were seen in L. monocytogenes settlement as a function of the genetic background of resident lactococcal biofilm cells. In particular, biofilms of the L. lactis chain-forming mutant resulted in a marked increase in L. monocytogenes settlement, while biofilms of the EPS-secreting mutant efficiently prevented pathogen fixation. These results offer new insights into the role of resident biofilms in governing the settlement of pathogens on food chain surfaces and could be of relevance in the field of food safety controls.Listeria monocytogenes is a food pathogen that has been implicated in numerous food-borne disease outbreaks (5, 58). This organism is found not only in food products but also on surfaces in food-processing plants (18). It is well documented that L. monocytogenes is able to adhere and form persistent biofilms on a variety of solid materials, such as stainless steel, glass, or polymers (18, 48, 51, 52). However, in food-manufacturing plants (and particularly in fermented-food-processing environments), it is most likely that the first contact between a pathogen and a surface will concern a resident microbial biofilm covering the solid surface (10, 35, 46). In this context, such a resident biofilm may be regarded as a “conditioning film” that modifies the topographic and physicochemical characteristics of the surface and hence the adhesion capability of planktonic microorganisms coming into contact with this substratum (6).Once the pathogens are immobilized on the surface, interactions between the pathogens and their environment (physiological interactions with resident flora, nutrient availability, pH, water activity, temperature, and cleaning and disinfection procedures) govern the long-term settlement and persistence of the pathogens on the surface. Various studies have demonstrated the inhibition of L. monocytogenes development by natural “protective” biofilms (10, 66). Competition for nutrients has been demonstrated as a major mechanism underlying the inhibition of pathogen development (25, 27). The production of antimicrobial agents (bacteriocins, acids, and hydrogen peroxide) has also been reported as being of importance to such interactions (13, 20, 36). For example, Lactococcus lactis has been described as being exceptionally efficient in controlling the development of L. monocytogenes on food-processing surfaces by means of competitive exclusion (66) or bacteriocin production (35). It has been reported that treating a surface with a bacterial polysaccharide prevented the adhesion of different nosocomial pathogens (60). Furthermore, alginate-overexpressing Pseudomonas aeruginosa biofilms reduced the retention of Cryptosporidium parvum oocysts (54). Other recent studies have shown that the composition and quantity of specific exopolysaccharides (EPS) in Pseudomonas biofilms can inhibit the fixation of Escherichia coli or Erwinia chrysanthemi planktonic cells in porous media (37, 38).The present study investigated those properties of resident biofilms that could affect the settlement of L. monocytogenes. L. lactis was used as a model resident biofilm strain, as this is widely used in dairy fermentations and its cell wall properties have been the subject of considerable study (22, 23). Cell wall mutants of L. lactis MG1363 were used to create a set of model biofilms that differed in terms of their architecture, EPS synthesis, and cell surface hydrophobicity. These biofilms were used to evaluate the attachment of fluorescent inert polystyrene microbeads and of two reference strains of L. monocytogenes (LO28 and EGDe) using in situ confocal fluorescence imaging.     

Synergistic Effects of Sodium Chloride,Glucose, and Temperature on Biofilm Formation by Listeria monocytogenes Serotype 1/2a and 4b Strains

Biofilm formation by Listeria monocytogenes is generally associated with its persistence in the food-processing environment. Serotype 1/2a strains make up more than 50% of the total isolates recovered from food and the environment, while serotype 4b strains are most often associated with major outbreaks of human listeriosis. Using a microplate assay with crystal violet staining, we examined biofilm formation by 18 strains of each serotype in tryptic soy broth with varying concentrations of glucose (from 0.25% to 10.0%, wt/vol), sodium chloride (from 0.5% to 7.0%, wt/vol) and ethanol (from 1% to 5.0%, vol/vol), and at different temperatures (22.5°C, 30°C, and 37°C). A synergistic effect on biofilm formation was observed for glucose, sodium chloride, and temperature. The serotype 1/2a strains generally formed higher-density biofilms than the 4b strains under most conditions tested. Interestingly, most serotype 4b strains had a higher growth rate than the 1/2a strains, suggesting that the growth rate may not be directly related to the capacity for biofilm formation. Crystal violet was found to stain both bacterial cells and biofilm matrix material. The enhancement in biofilm formation by environmental factors was apparently due to the production of extracellular polymeric substances instead of the accumulation of viable biofilm cells.Listeria monocytogenes, a Gram-positive bacterium, is capable of causing severe food-borne infections in both humans and animals. The organism is ubiquitous in the environment and can grow in a wide variety of foods, including those stored at refrigeration temperatures. It is particularly difficult to eliminate this bacterium from ready-to-eat foods and food-processing equipment (19). The ability to form biofilms protects the bacterium from stresses in food-processing environments (13, 25). Among the 13 different serotypes described, serotypes 1/2a, 1/2b, and 4b are involved in the majority of human cases of listeriosis. Serotype 4b strains have accounted for most human outbreaks, whereas the majority of L. monocytogenes strains isolated from foods or food-processing plants belong to serotype 1/2a (19).Comparative studies to link the phenotypic attributes of L. monocytogenes strains to serotypes have obtained variable results. Buncic et al. (4) have shown that serotype 1/2a isolates were more resistant to antilisterial bacteriocins than serotype 4b strains at 4°C. They also found that 4b isolates exhibited greater resistance to heat treatments at 60°C and were easier to recover than 1/2a strains immediately following cold storage. Bruhn et al. (3) observed that 1/2a strains (lineage II) grew faster than 4b and 1/2b (lineage I) strains in commonly used enrichment broth media (University of Vermont media I and II). However, other studies have indicated that similar differences could not be linked to a serotype (14), and sequencing results have shown a syntenic relationship between strains of the two serotypes (27).Some L. monocytogenes strains have consistently been isolated from food-processing plants over many years (1, 28). Although several studies have been carried out to identify differences in cell adherence and biofilm formation among different serotypes, conflicting results were obtained. Lineage I isolates (including serotypes 4b, 1/2b, 3c, and 3b) were found to produce higher-density biofilms than lineage II isolates (including serotypes 1/2a, 1/2c, and 3a) (8, 28). However, this conclusion was not supported by other studies (1, 7, 18). For serotype 4b strains, the capacity to form biofilms was reduced when the nutrient level in a medium decreased, while serotype 1/2a strains were not similarly affected (11).It has been suggested that the formation of a biofilm is a stress response by bacterial cells (15, 16). Biofilm research under laboratory conditions may not reflect biofilm formation in the environment. To investigate the behavior of L. monocytogenes in biofilms, a simulated food-processing (SFP) system including several stresses was designed (30). The SFP system was used to study 1/2a and 4b strains in mixed-culture biofilms (31). Bacterial cells from a 1/2a cocktail predominated over 4b strains when exposed to the SFP system for 4 weeks, but no competitive inhibition was observed. Environmental factors, including temperature, sugar, salt, pH, and nutrients that are common in foods and food-processing environments, have been demonstrated to have impacts on L. monocytogenes adhesion and biofilm formation (25). The objectives of this study were to investigate and compare biofilm formation between L. monocytogenes serotype 1/2a strains and serotype 4b strains under a variety of environmental conditions, including different temperatures and varying concentrations of salt, sugar, and ethanol, and to examine the synergistic effects of these factors on biofilm formation by both serotypes.     

Differential Listeria monocytogenes Strain Survival and Growth in Katiki,a Traditional Greek Soft Cheese,at Different Storage Temperatures

Katiki Domokou is a traditional Greek cheese, which has received the Protected Designation of Origin recognition since 1994. Its microfloras have not been studied although its structure and composition may enable (or even favor) the survival and growth of several pathogens, including Listeria monocytogenes. The persistence of L. monocytogenes during storage at different temperatures has been the subject of many studies since temperature abuse of food products is often encountered. In the present study, five strains of L. monocytogenes were aseptically inoculated individually and as a cocktail in Katiki Domokou cheese, which was then stored at 5, 10, 15, and 20°C. Pulsed-field gel electrophoresis was used to monitor strain evolution or persistence during storage at different temperatures in the case of the cocktail inoculum. The results suggested that strain survival of L. monocytogenes was temperature dependent since different strains predominated at different temperatures. Such information is of great importance in risk assessment studies, which typically consider only the presence or absence of the pathogen.Listeria monocytogenes is a ubiquitous food-borne pathogen associated with outbreaks of listeriosis from consumption of various food commodities, especially dairy products, seafood, and meat (2, 26). The pathogen is of great health concern for the food industry because it is characterized by high mortality rates, amounting to 20 to 30% (14). Due to the severity of illness, especially for pregnant women, neonates, the elderly, and immunodeficient people, the level of the pathogen in food should remain low to ensure safe food products.The new regulation of the European Union (EU) for microbiological criteria for L. monocytogenes in foods has set maximum levels of 100 CFU g⁻¹ at the time of consumption for soft cheeses (8). In fact, the new EC 2073/2005 regulation in annex I lists the microbiological criteria for foodstuffs, which are classified into food safety criteria and process hygiene criteria. According to the new EU regulation, food safety criteria are those which “define the acceptability of a product or a batch of foodstuff applicable to products placed on the market” (8).Legislative amendments regarding the presence of L. monocytogenes in ready-to-eat (RTE) foods are of great importance. Indeed, for the first time RTE foods are legislatively distinguished according to the target population for which they are intended, i.e., whether they are intended for consumption (i) by infants, (ii) by people with special medical conditions (immunocompromised), or (iii) by other target human subpopulations. In the most recent amendment the RTE foods other than those intended for infants or for those with special medical needs are further subdivided into foods that are able to support the growth of L. monocytogenes and those that are not. Products with pH ≤ 5.0 and water activity of ≤0.94 and products with a shelf life of less than 5 days are automatically considered to belong to the category of RTE foods that are unable to support the growth of L. monocytogenes (8). The regulation also states that “other categories of products can also belong to this category, subject to scientific justification.” Last but not least, the food safety criteria for L. monocytogenes are adjusted according to the bacteria''s temporal stage in the food chain. Thus, for RTE foods that are able to support the growth of L. monocytogenes, the new regulation demands the absence of the pathogen (in 25 g) “before the food has left the immediate control of the food business operator, who has produced it” but allows up to 100 CFU g⁻¹ in “products placed on the market during their shelf life.” The 100-CFU g⁻¹ limit also applies throughout the shelf life of marketed RTE foods unable to support L. monocytogenes growth (8).The pH and the water activity of Katiki Domokou (Katiki), a spreadable RTE traditional Greek cheese, are within the limits mentioned in the regulation. This product, a white cheese with a creamy structure, was traditionally produced from goat milk or from a mixture of goat and sheep milk. It has been recognized as a Protected Designation of Origin product since 1994 (www.greekcheese.gr), and its consumption has readily increased in the last few years. The milk is initially pasteurized and cooled at 27 to 28°C. Coagulation is then conducted with or without the addition of rennet, and the mixture is left to stand at 20 to 22°C. The curd is pulped and placed in cloth sacks for draining, with high final moisture (ca. 75%) and low salt content (ca. 1%) and pH (4.3 to 4.5) while it is stored at 4 to 5°C.The quantitative estimation of kinetic parameters related to growth, survival, and death of L. monocytogenes has been described previously (2, 14, 20). The kinetic parameters of L. monocytogenes during storage at different temperatures have been the subject of many studies since temperature abuse of food products is often encountered (25, 28). However, strain characteristics or viability have not been taken into account (or have not been considered) as yet (20). This may explain the variability of findings in regard to different storage conditions (7, 17). Pulsed-field gel electrophoresis (PFGE) is a powerful subtyping tool, a gold standard for epidemiology, which provides repeatable results. It has the ability to generate profiles of a wide range of microorganisms and to discriminate strains with high fidelity (11, 19). PFGE has been used in several studies to type strains of epidemiological interest as well as to trace contaminants in the food chain (12, 13, 18).The purpose of the present study was to assess the survival of five strains of L. monocytogenes inoculated either individually or as a cocktail in Katiki cheese. The cheese was stored at 5, 10, 15, and 20°C over a period of 1 month. PFGE was used to monitor the strain(s) that might survive and/or grow at different temperatures in a complex ecosystem like Katiki. The strains used in the study to form the inoculum consisted of two type strains of serotype 4b and three isolates belonging to our laboratory collection that were isolated from soft cheese and the conveyor belt of RTE foods. The strains were chosen on the basis of their source of isolation since this could be crucial to the interpretation of the data. The population was monitored throughout storage with respect to its quantitative as well as its qualitative evolution.