Influence of food soiling matrix on cleaning and disinfection efficiency on surface attached Listeria monocytogenes

Cleaning and disinfection are essential steps in preventing contamination of foods with pathogenic and spoilage bacteria. The efficacy of cleaning and disinfection products differ depending on target bacteria and type of soiling. We evaluated the effectiveness of cleaning and disinfecting products against Listeria monocytogenes attached on food soiled inert surfaces in a laboratory model. The number of bacteria on surfaces before and after treatment was quantified using indirect conductometric measurements. L. monocytogenes attached to stainless steel surfaces in fish broth systems reached approx. 10⁴ CFU/cm². However, all cleaning and disinfection products were equally effective in this system since all bacteria were removed or killed. When the steel disks were immersed in a fish or meat emulsions, a level of approx. 10⁵–10⁶ L. monocytogenes per cm² was reached after 2–3 days at 20 °C. In this case, 2–3 log were removed or killed by alkaline cleaning products (MC103 or FC140). Direct use of a peracetic acid based disinfectant (Oxivit Active Plus) killed all bacteria when attached in salmon emulsions, whereas only 1–2 log were removed or killed in the meat emulsion system. Thus, the efficiency of cleaning and disinfection products against L. monocytogenes is strongly influenced by the food matrix.     

Disinfectant action of Cymbopogon sp. essential oils in different phases of biofilm formation by Listeria monocytogenes on stainless steel surface

Disinfectant solutions based on the essential oils of Cymbopogon citratus (D.C.) Stapf. and Cymbopogon nardus (L.) Rendle, applied alone or in combination, and a control disinfectant solution were tested in two phases (3 and 240 h) of biofilm formation by Listeria monocytogenes ATCC 19117 on AISI 304 (#4) stainless steel surface. Disinfectant solutions based on essential oils have effectively reduced the number of surface-adhered cells, especially after 60 min of contact. The disinfectant solutions based on a combination of essential oils were capable of reducing 100% (5.64 Log CFU cm⁻²) the number of surface-adhered cells after 60 min of contact, at 240 h of biofilm formation. Essential oils of C. citratus and C. nardus, alone or in combination, are new alternatives for disinfection of industrial stainless steel surfaces contaminated by L. monocytogenes.     

Production of biofilm by Listeria monocytogenes in different materials and temperatures

Listeria monocytogenes, considered as one of the most important foodborne pathogens, is easily found on surfaces, particularly in the form of a biofilm. Biofilms are aggregates of cells that facilitate the persistence of these pathogens in food processing environments conferring resistance to the processes of cleaning and may cause contamination of food during processing, thus, representing a danger to public health. Little is known about the dynamics of the formation and regulation of biofilm production in L. monocytogenes, but several authors reported that the luxS gene may be a precursor in this process. In addition, the product of the inlA gene is responsible for facilitating the entry of the microorganism into epithelial cells that express the receptor E-cadherin, also participates in surface attachment. Thus, 32 strains of L. monocytogenes isolated from different foods (milk and vegetables) and from food processing environments were analyzed for the presence of these genes and their ability to form biofilms on three different surfaces often used in the food industry and retail (polystyrene, glass and stainless steel) at different temperatures (4, 20 and 30 °C). All strains had the ilnA gene and 25 out of 32 strains (78.1%) were positive for the presence of the luxS gene, but all strains produced biofilm in at least one of the temperatures and materials tested. This suggests that genes in addition to luxS may participate in this process, but were not the decisive factors for biofilm formation. The bacteria adhered better to hydrophilic surfaces (stainless steel and glass) than to hydrophobic ones (polystyrene), since at 20 °C for 24 h, 30 (93.8%) and 26 (81.3%) produced biofilm in stainless steel and glass, respectively, and just 2 (6.2%) in polystyrene. The incubation time seemed to be an important factor in the process of biofilm formation, mainly at 35 °C for 48 h, because the results showed a decrease from 30 (93.8%) to 20 (62.5%) and from 27 (84.4%) to 12 (37.5%), on stainless steel and glass, respectively, although this was not significant (p = 0.3847). We conclude that L. monocytogenes is capable of forming biofilm on different surfaces independent of temperature, but the surface composition may be important factor for a faster development of biofilm.     

Inhibitory effects of chitosan in combination with antibiotics on Listeria monocytogenes biofilm

Microorganisms on living or inert surfaces usually form biofilms which makes microbes highly resistant to antibiotics and immune clearance. Herein we investigated the synergistic effect of several antibiotics with chitosan on eradication biofilms built by Listeria monocytogenes, a causative organism of the serious foodborne illness in a wide variety of mammalian species including human and food animals. Our data demonstrated that chitosan combined with the aminoglycoside antibiotic such as amikacin, but not clindamycin, vancomycin and erythromycin was more effective in inhibition or disruption of L. monocytogenes biofilms than the antibiotic alone did. To elicit an optimal anti-biofilm and bactericidal efficiency, chitosan with specialized molecular size (∼13 kDa) and high N-deacetylation degree (∼88%) was required in this combination. Further evidence showed that this strategy was effective in removing biofilms built by multiple L. monocytogenes strains and other Listeria species. Thus, our data highlighted that chitosan/amikacin combination might be useful for the treatment of Listeria biofilms and help prevent the spread of antibiotic resistance through improving antibiotic effectiveness.     

Lactobacillus sakei 1 and its bacteriocin influence adhesion of Listeria monocytogenes on stainless steel surface

Listeria monocytogenes is of particular concern for the food industry due to its psychrotolerant and ubiquitous nature. In this work, the ability of L. monocytogenes culturable cells to adhere to stainless steel coupons was studied in co-culture with the bacteriocin-producing food isolate Lactobacillus sakei 1 as well as in the presence of the cell-free neutralized supernatant of L. sakei 1 (CFSN-S1) containing sakacin 1. Results were compared with counts obtained using a non bacteriocin-producing strain (L. sakei ATCC 15521) and its bacteriocin free supernatant (CFSN-SA). Culturable adherent L. monocytogenes and lactobacilli cells were enumerated respectively on PALCAM and MRS agars at 3-h intervals for up to 12 h and after 24 and 48 h of incubation. Bacteriocin activity was evaluated by critical dilution method. After 6 h of incubation, the number of adhered L. monocytogenes cells in pure culture increased from 3.8 to 5.3 log CFU/cm² (48h). Co-culture with L. sakei 1 decreased the number of adhered L. monocytogenes cells (P < 0.001) during all sampling times with counts lower than 3.0 log CFU/cm². The CFNS-S1 also led to a significant and similar reduction in culturable adhered L. monocytogenes counts for up to 24 h of incubation, however after 48 h of incubation, re-growth of L. monocytogenes number of adhered cells was observed, likely due to lack of competition for nutrients. L. sakei ATCC 15521 or its supernatant (CFNS-SA) did not reduce the number of adhered L. monocytogenes cells on stainless steel surface and from 6 h of incubation, listerial counts were between 4.3 and 4.5 log CFU/cm². These results indicate that L. sakei 1 and its bacteriocin sakacin 1 may be useful to inhibit early stages of L. monocytogenes adherence to abiotic surface.     

Antibacterial effect of black seed oil on Listeria monocytogenes

Listeria monocytogenes is a major foodborne pathogen in the United States. Effective methods for reducing L. monocytogenes in foods would reduce the likelihood of foodborne outbreaks of listeriosis, and decrease economic losses to the food industry. Nigella sativa is a herbaceous plant, whose seeds (black seed) have been used as a spice and condiment in foods in the Middle East. The objective of this study was to determine the antibacterial effect of black seed oil on twenty strains of L. monocytogenes by disc diffusion method. A population of 7.0 log CFU of each strain of L. monocytogenes was inoculated on duplicate plates containing antibiotic medium one agar. The plates were allowed to dry at room temperature for 15 min. Three discs (6 mm diameter), each impregnated with 10 μl of black seed oil, vegetable oil (oil control), or gentamicin (positive control) were placed on each inoculated plate. The plates were incubated at 37 °C for 24 h, and were observed for zones of L. monocytogenes growth inhibition. Black seed oil exhibited a strong antibacterial activity against all the strains of L. monocytogenes, yielding a significantly (P<0.01) larger inhibition zone than that of gentamicin. The mean zones of inhibition produced by black seed oil and gentamicin were 31.50 ± 1.0 and 14.80 ± 0.50, respectively. The vegetable oil had no inhibitory effect on L. monocytogenes. Results indicate that black seed oil could potentially be used to inhibit L. monocytogenes, but appropriate applications in foods need to be validated.     

Ralstonia insidiosa serves as bridges in biofilm formation by foodborne pathogens Listeria monocytogenes,Salmonella enterica,and Enterohemorrhagic Escherichia coli

Biofilm formation on abiotic surfaces in fresh produce processing facilities may play a role in foodborne outbreaks by providing protective microniches for pathogenic bacteria. Our previous study showed that a strain of Ralstonia insidiosa isolated from a fresh produce processing plant could enhance the incorporation of Escherichia coli O15:H7 in biofilms under various environmental conditions. These results raised the concern that R. insidiosa might have the ability to incorporate other foodborne pathogens and promote their survival and growth in biofilms. To test this hypothesis, 6 strains of Shiga toxin producing E. coli, 2 strains of Salmonella, and 6 strains of Listeria monocytogenes were examined for dual-species biofilm formation with R. insidiosa. A significant increase in biomass formation was observed in 7 of the 14 R. insidiosa-pathogen combinations, while significantly enhanced incorporation of pathogenic cells into biofilms was seen in 12 of the 14 R. insidiosa-pathogen combinations. The synergistic interactions between R. insidiosa and the tested foodborne pathogens seemed dependent on intimate cellular contact between the two strains. Overall, this study showed that R. insidiosa could enhance the incorporation of biofilms of different types of foodborne pathogenic bacteria and should be considered a bridging bacterium for biofilm formation in various food processing environments.     

Inactivation and induction of sublethal injury of Listeria monocytogenes in biofilm treated with various sanitizers

This study determined the effects of different sanitizers (one phenolic-based, one chlorine-based, two QACs-based and one levulinic acid and SDS-based) on Listeria monocytogenes biofilm. The induction of the sub-lethal injury state and the biofilm formation characteristics as a result of exposure to sanitizers were also evaluated. The results revealed that QACs-based and phenolic-based sanitizers most effectively reduced L. monocytogenes, resulting in a reduction of 3.7–6.9 log CFU/ml and 4.9–8.2 log CFU/ml after a 60-min treatment for 37°C- and 15°C-grown biofilms, respectively. An enhanced level of sanitizer resistance was observed in biofilms when they were multiply exposed to QACs-based and phenolic-based sanitizers, with a reduction of 0.7–3.5 log CFU/ml and 1.6–>9.3 log CFU/ml for 37°C- and 15°C-grown biofilms, respectively. As biofilm cells became less sensitive, especially to QACs-based sanitizers, an increase in the percentage of sublethally injured cells was observed to the levels dependent upon sanitizer concentration. Confocal laser scanning microscopy (CLSM) analysis revealed that biofilm cells experienced cell membrane damage when exposed to QACs-based and phenolic-based sanitizers, providing more protection to cells located inside the biofilm matrix. This study highlights the ongoing need for improvement in intervention methods to control L. monocytogenes in food processing plants.     

Surveillance of listeriosis and its causative pathogen,Listeria monocytogenes

To manage the problem of foodborne listeriosis requires an understanding of the burden of the disease on a worldwide scale as foods that are prone to contamination are eaten widely domestically and many are traded globally. Surveillance of the disease, caused by Listeria monocytogenes, is typically restricted to developed countries, but many of these do not consider listeriosis as a notifiable disease and estimate the numbers by other means. Incidence rates range from 0.3 to 1.3 per 100,000, but most are in the 0.3–0.5 range, irrespective of the regulatory system and industry control programmes that have been in place. Ready-to-eat foods are the vehicle for transmission of the Listeria through contamination somewhere in the food chain. Meat, poultry and dairy products have been most frequently implicated, but other foods including produce may also have been vehicles of transmission. Large outbreaks are usually linked to errors in food processing plants, such as contaminated slicing machines, followed by opportunities for growth of the pathogen. Less is known about home-generated illnesses but incorrect use of refrigerators can allow cross-contamination and growth of the pathogen to levels that can cause infections. In the U.S., door-to-door salesmen have sold contaminated Hispanic soft cheeses that have led to outbreaks and stillbirths. In addition to outbreak investigation, case-control studies, and the use of experts, risk assessments, and food attribution studies can help focus on areas of greatest risk for prevention and control measures throughout the food chain.     

Temperature distribution in Spanish domestic refrigerators and its effect on Listeria monocytogenes growth in sliced ready-to-eat ham

In order to evaluate the increase of L. monocytogenes concentration during home storage, a low level of the pathogen (<10 CFU/g) was inoculated in sliced ham. Two storage temperatures (5 °C and 9 °C) were selected from a previous study in 33 home refrigerators. Three days of storage or less were necessary to reach the regulated limited concentration (100 CFU/g), at both temperatures. When storage time was extended to 5 days, the pathogen achieved risky values (>10³ CFU/g).     

Adaptive response of Listeria monocytogenes to heat and its impact on food safety

Listeria monocytogenes is an important food associated pathogen because of its relatively high heat resistance and ability to multiply in refrigeration temperatures. Its thermotolerance can be increased when its cells are subjected to heat shock. One- to eight-fold increase of D values of L. monocytogenes have been reported, depending on the heat shock duration, the temperature and the heating menstrum. This acquisition of heat tolerance is related to the induction of the synthesis of heat shock proteins (HSPs).The adaptive response of food pathogens has important consequences on the safety of thermally processed foods. It is believed that this is responsible for the frequent occurrence of deviations (tails and shoulders) during heat treatments that are observed in the exponential model of microbial inactivation. These deviations from log-linear kinetic especially encountered under mild heat treatments, mean that prediction of food safety can no longer rely upon D and z values. Adaptive response to heat must be considered when quantifying and modeling microbial inactivation during thermal processing in order to achieve microbiologically safe products without overly conservative heat processes. Therefore a more mechanistic approach is needed for more accurate predictions of thermal inactivation. Prerequisite to this model are thorough studies to understand how L. monocytogenes and other pathogens adapt their cellular physiology to overcome heat and other stresses.     

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.     

Combined effect of carvacrol and citral on the growth of Listeria monocytogenes and Listeria innocua and on the occurrence of damaged cells

The aim of this study was to evaluate the growth kinetics of Listeria innocua Serovar 6a (CECT 910) and Listeria monocytogenes Serovar 4b (CECT 4032) exposed to combinations of carvacrol and citral (0.0 μL/mL (control), 0.050 μL/mL of carvacrol and 0.075 μL/mL of citral, 0.050 μL/mL of carvacrol and 0.125 μL/mL of citral, 0.085 μL/mL of carvacrol and 0.075 μL/mL of citral, and 0.085 μL/mL of carvacrol and 0.125 μL/mL of citral), with two initial inoculum concentrations, and also the occurrence of sublethal damage in these cell populations. The terpene combinations exhibited antibacterial activity against L. innocua and L. monocytogenes and the effects were dependent on the concentration of terpenes present in the culture medium (p ≤ 0.05). When terpene-treated L. innocua and L. monocytogenes were incubated in TSB, significant differences in lag phase and growth rate were observed between low and high inoculum concentrations (p ≤ 0.05), indicating that the inoculum level should be taken into account in modeling studies. When bacterial cells were exposed to terpenes the proportion of sublethally injured cells increased with the increase in the terpene dose (p ≤ 0.05). In conclusion, all of these results show that carvacrol and citral can be used in combination at 25% of the MIC in order to control Listeria growth.     

Persister cell formation of Listeria monocytogenes in response to natural antimicrobial agent nisin

The persistence of Listeria monocytogenes (L. monocytogenes) can be defined by its prolonged existence in food environments that poses a risk to food safety. The persistence of cells exposed to antibiotics and chemical sanitisers is well recorded however the persistence following treatment with natural antimicrobials like bacteriocins has not been determined. This study used two L. monocytogenes isolates, one from food and one from an animal origin, to obtain a persistent subpopulation following exposure to the natural antimicrobial nisin. Overnight cultures of L. monocytogenes were treated with 75 μg/ml of nisin over 24 h, and a biphasic killing pattern was observed for both strains. Persister cells became a population of cells showing tolerance to the high concentrations of nisin in our tests and proved not to be resistant with nisin re-exposure tests. XTT assays showed that the reduced level of metabolism maybe an important requirement for the L. monocytogenes persistence and the persister population which could survive at high concentration of nisin was probably related to less negative cell surface charges. The current data will be initial evidence to understand the mechanism of persistence through gene expression and in the future to give more reliable clues in developing strategies to control this food safety risk.     

Antibacterial mechanism of Myagropsis myagroides extract on Listeria monocytogenes

The ethanol extract of Myagropsis myagroides had antimicrobial activity against Gram-positive bacteria. The extract was fractionated through liquid–liquid extraction; the chloroform fraction had strong antimicrobial activity against Listeria monocytogenes (minimum inhibitory concentration (MIC) 0.063 mg/mL), and Clostridium perfringens (MIC 0.031 mg/mL). The chloroform fraction was separated into 22 sub-fractions using silica gel column chromatography, with the fourth fraction (CH4) possessing the strongest antimicrobial activity against Gram-positive bacteria. Leakage of 260 nm-absorbing material and ATP was observed in CH4-treated cells and morphological alterations were observed by electron microscopy. These results indicate that the cytoplasmic membrane may be a target of the CH4 fraction.     

The challenge of setting risk-based microbiological criteria for Listeria monocytogenes

After more than 20 years of work with discussing the setting of microbiological criteria for Listeria monocytogenes in foods, Codex Alimentarius on Food Hygiene has finalised a proposal that was recently adopted by the Codex Alimentarius Commission. The effort of developing procedures for making the microbiological criteria risk-based to the greatest extent possible has challenged scientists and managers during this long time period. Yet, the establishment of microbiological criteria for L. monocytogenes is still being discussed and several approaches are possible. Setting of microbiological criteria continues to be a risk management decision – even when using mathematical modelling to enhance the scientific level of a risk-based approach.     

Influence of Listeria innocua on the attachment of Listeria monocytogenes to stainless steel and aluminum surfaces

Listeria monocytogenes is an important foodborne pathogen that may be transmitted from the food-processing environment to food; however, the ecology and interaction of this organism with microbial residents on surfaces within the food industry is not well understood. The current study was undertaken to investigate the influence of Listeria innocua on the growth and attachment of L. monocytogenes to stainless steel or aluminum surfaces at 23 °C. When grown in broth as a mixed culture, L. innocua reached a higher cell count at 24 h than did L. monocytogenes. Attachment was evaluated by placing an aliquot containing 10³ CFU/ml of L. innocua and 10³ CFU/ml of L. monocytogenes on the coupons and by quantifying attached cells after 24 and 72 h. Attachment of L. monocytogenes was decreased by the presence of L. innocua. When compared to L. monocytogenes alone, there was a significant reduction of attachment of L. monocytogenes at 24 and 72 h on stainless steel and 72 h on aluminum surface when L. innocua was added at the same time. L. innocua exhibited an effect on the attachment of L. monocytogenes, increasing our knowledge of the behavior of L. monocytogenes in the presence of another Listeria species.     

Influence of temperature on alkali stress adaptation in Listeria monocytogenes

Listeria monocytogenes cells may induce alkali stress adaptation when exposed to sublethal concentrations of alkaline cleaners and sanitizers that may be frequently used in the food processing environment. In the present study, the effect of temperature on the induction and the stability of such alkali stress adaptation in L. monocytogenes were investigated. Our results demonstrate that temperature plays a critical role in the induction of alkali stress adaptation in L. monocytogenes under sublethal alkaline conditions. Pre-exposure to pH 9.0 tryptic soy broth supplemented with 0.6% yeast extract (TSB-YE) at 37 °C induced pronounced alkali stress adaptation whereas sublethal alkaline pre-exposure at 4 °C failed to induce any alkali stress adaptation. Furthermore, this pattern of alkali stress adaptation in L. monocytogenes was not dependent on the length of pre-exposure time, the concentration of sublethal alkali, the types of alkaline agents and the growth phases of cells. In addition, alkali stress adaptation induced at 37 °C was completely reversed in pH 7.2 TSB-YE within 2 h at 37 °C or within 4 h at 22 °C. However, once it was induced at a higher temperature, alkali stress adaptation in L. monocytogenes remained stable at 4 °C for at least 4 h. Our findings suggest that even though cold temperatures do not induce alkali stress adapted phenotypes, but it can maintain the previously acquired alkali stress adaptation much longer in L. monocytogenes.