The influence of stress factors on selected phenotypic and genotypic features of Listeria monocytogenes - a pilot study.

BACKGROUND: Listeria monocytogenes are Gram-positive rods, widespread in the environment due to their wide tolerance to changing conditions. The apilot study aimed to assess the impact of six various stresses (heat, cold, osmotic, acid, alkali, frozen) on phenotypic features: MIC of antibiotics (penicillin, ampicillin, meropenem, erythromycin, co-trimoxazole; gradient stripes), motility, ability to form a biofilm (crystal violet method) and growth rate (OD and quantitative method), expression level of sigB (stress induced regulator of genes), agrA, agrB (associated with biofilm formation) and lmo2230, lmo0596 (acid and alkali stress) (qPCR) for three strains of L. monocytogenes. RESULTS: Applied stress conditions contributed to changes in phenotypic features and expression levels of sigB, agrA, agrB, lmo2230 and lmo0596. Stress exposure increased MIC value for penicillin (ATCC 19111 - alkaline stress), ampicillin (472CC - osmotic, acid, alkaline stress), meropenem (strains: 55 C - acid, alkaline, o smotic, frozen stress; 472CC - acid, alkaline stress), erythromycin (strains: 55 C - acid stress; 472CC - acid, alkaline, osmotic stress; ATCC 19111 - osmotic, acid, alkaline, frozen stress), co-trimoxazole (strains: 55 C - acid stress; ATCC 19111 - osmotic, acid, alkaline stress). These changes, however, did not affect antibiotic susceptibility. The strain 472CC (a moderate biofilm former) increased biofilm production after exposure to all stress factors except heat and acid. The ATCC 19111 (a weak producer) formed moderate biofilm under all studied conditions except cold and frozen stress, respectively. The strain 55 C became a strong biofilm producer after exposure to cold and produced a weak biofilm in response to frozen stress. Three tested strains had lower growth rate (compared to the no stress variant) after exposure to heat stress. It has been found that the sigB transcript level increased under alkaline (472CC) stress and the agrB expression increased under cold, osmotic (55 C, 472CC), alkali and frozen (472CC) stress. In contrast, sigB transcript level decreased in response to acid and frozen stress (55 C), lmo2230 transcript level after exposure to acid and alkali stress (ATCC 19111), and lmo0596 transcript level after exposure to acid stress (ATCC 19111). CONCLUSIONS: Environmental stress changes the ability to form a biofilm and the MIC values of antibiotics and affect the level of expression of selected genes, which may increase the survival and virulence of L. monocytogenes. Further research on a large L. monocytogenes population is needed to assess the molecular mechanism responsible for the correlation of antibiotic resistance, biofilm formation and resistance to stress factors.

Effect of Radiant Catalytic Ionization and Ozonation on Salmonella spp. on Eggshells.

Three Salmonella enterica strains were used in the study (serovars: S. enteritidis, S. typhimurim and S. virchow). This study evaluated the efficacy of radiant catalytic ionization (RCI) and ozonation against Salmonella spp. on eggshell (expressed as log CFU/egg). The egg surface was contaminated three different bacterial suspension (103 CFU/mL, 105 CFU/mL and 108 CFU/mL) with or without poultry manure. Experiments were conducted at 4 °C and 20 °C in three different time period: 30 min, 60 min and 120 min. Treatment with RCI reduced Salmonella numbers from 0.26 log CFU/egg in bacterial suspension 108 CFU/mL, 4 °C and 20 °C, with manure for 30 min to level decrease in bacteria number below the detection limit (BDL) in bacterial suspension 105 CFU/mL, 20 °C, with or without manure for 120 min. The populations of Salmonella spp. on eggs treated by ozonizer ranged from 0.20 log CFU/egg in bacteria suspension 108 CFU/mL, 20 °C, with manure for 30 min to 2.73 log CFU/egg in bacterial suspension 105 CFU/mL, 20 °C, with manure for 120 min. In all treatment conditions contamination with poultry manure decrease effectiveness the RCI and ozonation. In summary, RCI technology shows similar effectiveness to the ozonation, but it is safer for poultry plant workers and consumers.

Prevalence and antimicrobial susceptibility of Listeria monocytogenes strains isolated from a meat processing plant.

INTRODUCTION AND OBJECTIVE: The ability of L. monocytogenes to create biofilm results in the higher resistance to disinfectants and determines the need to search for effective methods of eradication. The aim of the study was to assess the level of L. monocytogenes contamination in the environment of a meat processing plant. The sensitivity of tested isolates to various antimicrobials used for disinfection purposes was also estimated. MATERIAL AND METHODS: The samples were taken from raw materials, semi-finished and final products, as well as food contact surfaces inthe production hall and deli meat packaging department. The number of L. monocytogenes and the effect of eight different biocides on bacteria planktonic forms and biofilm formed on stainless steel and polypropylene was investigated. The effect of blood and albumin on L. monocytogenes resistance to disinfectants was also analysed. RESULTS: The prevalence of L. monocytogenes on food contact surfaces was estimated at 2.93% (10 of 340 swabs taken). The samples of raw and processed products were not contaminated. Various disinfectants reduced the growth of planktonic L. monocytogenes forms at both tested concentrations 0.5% and 0.1% (irrespective of time exposure). The highest efficacy against L. monocytogenes biofilm was reported for agents containing hydrogen peroxide. The reduction of bacteria number ranged from 6.93-7.21 log CFU × cm-2, and was dependent on the surface type and time of agent application. CONCLUSIONS: In this study, the effectiveness of various disinfectants against planktonic bacteria and Listeria biofilm was observed. For the majority of disinfectants, the extension of time exposure increased bacteria elimination from the biofilm. The presence of blood resulted in reduction of the antilisterial action of most of the disinfectants applied at low concentrations.

Adaptive Response of Listeria monocytogenes to the Stress Factors in the Food Processing Environment.

Listeria monocytogenes are Gram-positive, facultatively anaerobic, non-spore-forming bacteria that easily adapt to changing environmental conditions. The ability to grow at a wide range of temperatures, pH, and salinity determines the presence of the pathogen in water, sewage, soil, decaying vegetation, and animal feed. L. monocytogenes is an etiological factor of listeriosis, especially dangerous for the elderly, pregnant women, and newborns. The major source of L. monocytogenes for humans is food, including fresh and smoked products. Its high prevalence in food is associated with bacterial adaptation to the food processing environment (FPE). Since the number of listeriosis cases has been progressively increasing an efficient eradication of the pathogen from the FPE is crucial. Understanding the mechanisms of bacterial adaptation to environmental stress will significantly contribute to developing novel, effective methods of controlling L. monocytogenes in the food industry.

Effect of Temperatures Used in Food Storage on Duration of Heat Stress Induced Invasiveness of L. monocytogenes.

The unpropitious conditions of the food processing environmenttrigger in Listeria monocytogenes stress response mechanisms that may affect the pathogen's virulence. To date, many studies have revealed that acid, osmotic, heat, cold and oxidative stress modify invasiveness of L. monocytogenes. Nonetheless, there is limited data on the duration of the stress effect on bacterial invasiveness. Since most food is stored at low or room temperatures we studied the impact of these temperatures on the duration of heat stress effect on invasiveness of 8 L. monocytogenes strains. Bacteria were heat-treated for 20 min at 54 °C and then incubated at 5 and 20 °C up to 14 days. A decrease in invasiveness over time was observed for bacteria not exposed to heating. It was found that heat shock significantly reduced the invasion capacity of all strains and the effect lasted between 7 and 14 days at both 5 and 20 °C. In conclusion, 20-min heating reduces invasion capacity of all L. monocytogenes strains; however, the stress effect is temporary and lasts between 7 and 14 days in the food storage conditions. The invasiveness of bacteria changes along with the incubation time and is temperature-dependent.

Effect of Selected Environmental Factors on the Microbicidal Effectiveness of Radiant Catalytic Ionization.

The aim of this study was the assessment of the effect of time exposure, temperature, distance, and organic contaminants on radiant catalytic ionization (RCI) microbicidal effectiveness. The number of all examined bacteria decreased together with time exposure of RCI. The lowest recovery was obtained, both from the rubber surface (6.36 log CFU × cm-2) and steel (6.04 log CFU × cm-2) in the case of Escherichia coli O157:H7. On the other hand, Staphylococcus aureus was isolated in the largest number (rubber: 7.88 log CFU × cm-2, steel: 7.79 log CFU × cm-2). Among the tested environmental conditions, the greatest bacterial population was re-isolated at 4°C (distance: 0.5 m, time: 24 h), whereas the lowest population was found at a distance of 0.5 m (temperature: 20°C, time: 24 h) and on surfaces without contamination. In the samples treated with RCI, the bacterial population was the lowest on non-contaminated surfaces, ranging from 3.76 log CFU × cm-2 (E. coli O157:H7) to 5.58 log CFU × cm-2 (S. aureus) for the rubber, and from 3.26 log CFU × cm-2 (E. coli O157:H7) to 5.20 log CFU × cm-2 (S. aureus) for the stainless steel. The highest bacteria number was isolated from surfaces contaminated with meat and fish pulp. The lowest bacterial reduction caused by RCI was found in the case of rubber contaminated with meat-fish pulp (24 h, 0.5 m, 20°C). The reduction rate was equal to 0.89 log CFU × cm-2 for S. aureus, 1.17 log CFU × cm-2 for Listeria monocytogenes, 1.43 log CFU × cm-2 for Salmonella Enteritidis and 1.61 log CFU × cm-2 for E. coli O157:H7. In turn, the greatest bacterial reduction was found in the case of non-contaminated steel (24 h, 0.5 m, 37°C). The reduction rate was equal to 4.52 log CFU × cm-2 for L. monocytogenes, 3.61 log CFU × cm-2 for S. Enteritidis, 2.98 log CFU × cm-2 for E. coli O157:H7 and 2.77 log CFU × cm-2 for S. aureus. RCI allows the inactivation of pathogens from stainless steel and rubber surfaces. Its efficacy is species-dependent and affected by environmental factors.