Mechanism of Nisin, Pediocin 34, and Enterocin FH99 Resistance in Listeria monocytogenes

Nisin-, pediocin 34-, and enterocin FH99-resistant variants of Listeria monocytogenes ATCC 53135 were developed. In an attempt to clarify the possible mechanisms underlying bacteriocin resistance in L. monocytogenes ATCC 53135, sensitivity of the resistant strains of L. monocytogenes ATCC 53135 to nisin, pediocin 34, and enterocin FH99 in the absence and presence of different divalent cations was assessed, and the results showed that the addition of divalent cations significantly reduced the inhibitory activity of nisin, pediocin 34, and enterocin FH99 against resistant variants of L. monocytogenes ATCC 53135. The addition of EDTA, however, restored this activity suggesting that the divalent cations seem to affect the initial electrostatic interaction between the positively charged bacteriocin and the negatively charged phospholipids of the membrane. Nisin-, pediocin 34-, and enterocin-resistant variants of L. monocytogenes ATCC 53135 were more resistant to lysozyme as compared to the wild-type strain both in the presence as well as absence of nisin, pediocin 34, and enterocin FH99. Ultra structural profiles of bacteriocin-sensitive L. monocytogenes and its bacteriocin-resistant counterparts revealed that the cells of wild-type strain of L. monocytogenes were maximally in pairs or short chains, whereas, its nisin-, pediocin 34-, and enterocin FH99-resistant variants tend to form aggregates. Results indicated that without a cell wall, the acquired nisin, pediocin 34, and enterocin FH99 resistance of the variants was lost. Although the bacteriocin-resistant variants appeared to lose their acquired resistance toward nisin, pediocin 34, and enterocin FH99, the protoplasts of the resistant variants appeared to be more resistant to bacteriocins than the protoplasts of their wild-type counterparts.     

Enhanced Control of Listeria monocytogenes by In Situ-Produced Pediocin during Dry Fermented Sausage Production

[This corrects the article on p. 887 in vol. 58.].     

Glutamate Decarboxylase-Mediated Nisin Resistance in Listeria monocytogenes

Analysis of a complete set of glutamate decarboxylase (gad) mutants of Listeria monocytogenes strain LO28 (ΔgadD1, ΔgadDT1, ΔgadD2, ΔgadT2, and ΔgadD3 mutants) revealed that the ΔgadD1 mutant is impaired in its ability to tolerate exposure to both sublethal and lethal levels of the lantibiotic nisin. gadD1 is strain variable and is found only in approximately 50% of L. monocytogenes strains. Growth and survival experiments revealed that possession of gadD1 correlates with a higher degree of tolerance to nisin. Significantly, a similar finding using a gadB mutant of L. lactis IL1403 implies that this may be a general phenomenon in Gram-positive bacteria. Our findings thus suggest that the specific inhibition of GAD activity or a reduction in the levels of free glutamate may prevent the growth of otherwise resistant GAD⁺ bacteria in foods where low pH and/or nisin is used as a preservative.Listeria monocytogenes is a food-borne pathogen that is the causative agent of listeriosis, an opportunistic infection associated with high rates of morbidity and mortality (18). The microorganism has also been the cause of significant commercial losses, being responsible for 71% of all recalls of food products due to bacterial contamination in the United States between 1993 and 1998 (25). The ubiquitous nature of L. monocytogenes, together with its ability to tolerate a variety of environmental extremes, including high salt concentrations and low pH, and the ability to grow at refrigeration temperatures, makes control of the bacterium in foods difficult (10). Hence it is not altogether surprising that the food industry invests considerable effort into developing strategies to prevent the survival and growth of this pathogen. One such strategy involves the utilization of bacteriocins. Bacteriocins are antimicrobial peptides produced by one bacterium that inhibit the growth of other bacteria and have been used as “natural” preservatives to control undesirable microbiota in food (5). The most extensively studied bacteriocin is nisin A (here referred to as nisin), a 34-amino-acid class I bacteriocin (lantibiotic) produced by Lactococcus lactis strains that is currently approved for use in foods in over 50 countries. Nisin functions by binding lipid II, an essential precursor of cell wall peptidoglycan biosynthesis. Binding to lipid II also facilitates the formation of pores within the cytoplasmic membrane leading to the release of ATP and other small cytoplasmic contents, resulting in depolarization of the membrane potential and ultimately cell death (13).The molecular mechanisms employed by L. monocytogenes to cope with nisin are poorly understood. To date, loci that have been implicated in nisin tolerance include the alternative stress sigma factor SigB, the class three stress gene regulator CtsR, the two-component systems LisRK and HK1027, and a penicillin binding protein, Pbp (2, 6, 11, 15, 21). In addition, several studies have uncovered a link between the acid stress response of L. monocytogenes and nisin resistance (3, 17, 24). Several systems are employed by L. monocytogenes to withstand low pH stress, but the glutamate decarboxylase (GAD) system is probably the most important (an overview of the GAD system is in Fig. ​Fig.1).1). Mutation of specific gad genes renders cells exquisitely sensitive to ex vivo porcine and synthetic human gastric fluid and significantly impairs growth and survival in low-pH foods (4, 7, 8). Given the link between acid and nisin resistance phenotypes, the present study was initiated in order to investigate the contribution, if any, of gad genes to the nisin tolerance of L. monocytogenes.Open in a separate windowFIG. 1.An overview of the L. monocytogenes glutamate decarboxylase (GAD) system. L. monocytogenes possesses five gad genes. gadD1, gadD2, and gadD3 encode decarboxylases which catalyze the conversion of glutamate to γ-amino butyrate (GABA) and carbon dioxide (CO₂). gadT1 and gadT2 encode glutamate-GABA antiporters. Nisin functions by binding to lipid II, an essential precursor of cell wall peptidoglycan synthesis. Binding to lipid II facilitates the formation of pores within the cytoplasmic membrane leading to the release of ATP and may ultimately result in cell death. We suggest that under certain conditions gadD1 may contribute to intracellular ATP pools and hence tolerance of nisin.     

单核细胞增多性李斯特菌在体外模拟消化道环境中的抗性

[目的]通过测定存活率及细胞内pH(pHi)变化,分析单核细胞增多性李斯特菌(单增李斯特菌)在体外模拟消化道中的抗性.[方法]模拟唾液、胃液和小肠液根据其主要组成成分配制,按试验设计顺次加入后获得模拟的消化道各段混合液(包括相应的pH及其可能的范围).平板计数法测定单增李斯特菌在模拟消化液中的存活率,并用荧光比例成像显微镜(fluorescence ratio imaging microscopy,FRIM)测定细菌的pHi.[结果]单增李斯特菌在唾液中存活率＞90％;经pH≤3.0的胃液处理后,其在胃液和胃-肠混合液中的存活率低于0.05％;提高胃液pH至3.5,细菌存活率开始上升;在胃液pH4.0时,两株单增李斯特菌在模拟胃肠液中存活率显著提高(11.2％-85.9％).FRIM研究表明,单增李斯特菌在模拟唾液中的pHi与对照组相近.经过pH为3.5和4.0的胃液和胃-肠混合液处理后,pHi值仍维持在较高水平(＞7.75).[结论]单增李斯特菌在经过pH≥3.5胃液后,能够维持菌体细胞内的pH稳态,且存活率较高,表明其细胞膜仍保持完整.     

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.     

Specificity of Acquired Resistance Produced by Immunization with Listeria monocytogenes and Listeria Fractions

Mice were immunized with 1.0 mg of an attenuated strain of Listeria monocytogenes to determine the period of protection afforded by this strain when the mice were challenged intravenously with 5 MLD of listeria. Protection appeared 2 days after immunization and was still apparent 4 weeks after immunization. If the challenge dose was decreased to 1 MLD, protection was apparent at 10 weeks. Mice immunized with a comparable dose of mycobacterial cells and challenged intravenously with 1 MLD of listeria showed no protection at 10 weeks. The magnitude of the immune response to listeria challenge was not increased in mice immunized with the same virulent strain as that used for challenge. It was also found that resistance to listeria challenge appeared early after listeria immunization if the immunizing dose was large. As the immunizing dose was decreased and the challenge dose increased, resistance appeared later. Listeria killed by heat or ultraviolet irradiation, living but nonmultiplying streptomycin-dependent listeria, or listeria ribosomal fraction gave no protection against listeria challenge. The magnitude of the immune responses after listeria immunization to listeria challenge and to mycobacteria challenge were compared. It was found that protection after listeria challenge was of longer duration. In addition, a 100-fold larger vaccinating dose was required to give comparable protection against tuberculous infection.     

Resistance of heat-shocked cells of Listeria monocytogenes to mano-sonication and mano-thermo-sonication

Heat shocks did not increase the resistance of Listeria monocytogenes to an ultrasonication treatment under pressure (Mano-Sonication; MS). While heat-shocked cells (180 min, 45 degrees C) became sixfold more heat resistant than native cells (D62 = 1.8 min vs D62 = 0.24 min), the resistance of native and heat-shocked cells to MS (200 kPa, 117 microns) was the same (DMS = 1.6 min). The inactivation rate of non-heat-shocked cells of L monocytogenes by a combined heat/ultrasonication treatment under pressure (Mano-Thermo-Sonication; MTS) was an additive effect. On the contrary, on heat-shocked cells, the inactivation rate of MTS was greater than that of heat added to the inactivation rate of MS (synergistic effect) in the range 62-68 degrees C.     

Autolysis of Listeria monocytogenes

Physiological conditions that could provide maximal rates of autolysis of Listeria monocytogenes were examined. L. monocytogenes was found to be refractory to most treatments that promote rapid autolysis in other bacteria. Best rates of autolysis were obtained after resuspending the cells in Tris-hydrochloride buffer at 37 degrees C with the pH optimum at 8.0. Autolysis was also efficiently promoted by the surfactant Triton X-100. Antibiotics that interfere with the biosynthesis of the cell wall murein (peptidoglycan) caused death of the cells without autolysis after prolonged incubation in the presence of the drug. Only nisin, which has been shown to bind in vitro to the murein precursors lipid I and lipid II brings about autolysis of L. monocytogenes cells, although with slower kinetics than in the case of Tris-HCl and Triton.     

Autolysis of Listeria monocytogenes

Autolytic curves of five representative strains of Listeria monocytogenes are described. Of 24 strains so far examined, the majority are unstable in vitro.     

Frequency of Bacteriocin Resistance Development and Associated Fitness Costs in Listeria monocytogenes

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in Listeria monocytogenes to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10⁻⁶. However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10⁻⁷ to 10⁻². Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10°C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 × 10⁻⁸). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5°C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5°C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.     

Postadaptational Resistance to Benzalkonium Chloride and Subsequent Physicochemical Modifications of Listeria monocytogenes

Many studies have demonstrated that bacteria, including Listeria monocytogenes, are capable of adapting to disinfectants used in industrial settings after prolonged exposure to sublethal concentrations. However, the consequent alterations of the cell surface due to sanitizer adaptation of this pathogen are not fully understood. Two resistant and four sensitive L. monocytogenes strains from different sources were progressively subcultured with increasing sublethal concentrations of a surfactant, benzalkonium chloride (BC). To evaluate the effects of acquired tolerance to BC, parent and adapted strains were compared by using several morphological and physiological tests. Sensitive strains showed at least a fivefold increase in the MIC, while the MIC doubled for resistant strains after the adaptation period. The hydrophobicities of cells of parent and adapted strains were similar. Serological testing indicated that antigen types 1 and 4 were both present on the cell surface of adapted cells. The data suggest that efflux pumps are the major mechanism of adaptation in sensitive strains and are less important in originally resistant isolates. A different, unknown mechanism was responsible for the original tolerance of resistant isolates. In an originally resistant strain, there was a slight shift in the fatty acid profile after adaptation, whereas sensitive strains had similar profiles. Electron micrographs revealed morphological differences after adaptation. The changes in cell surface antigens, efflux pump utilization, and fatty acid profiles suggest that different mechanisms are used by resistant and sensitive strains for adaptation to BC.     

Fatty Acids Regulate Stress Resistance and Virulence Factor Production for Listeria monocytogenes

Fatty acids (FAs) are the major structural component of cellular membranes, which provide a physical and chemical barrier that insulates intracellular reactions from environmental fluctuations. The native composition of membrane FAs establishes the topological and chemical parameters for membrane-associated functions and is therefore modulated diligently by microorganisms especially in response to environmental stresses. However, the consequences of altered FA composition during host-pathogen interactions are poorly understood. The food-borne pathogen Listeria monocytogenes contains mostly saturated branched-chain FAs (BCFAs), which support growth at low pH and low temperature. In this study, we show that anteiso-BCFAs enhance bacterial resistance against phagosomal killing in macrophages. Specifically, BCFAs protect against antimicrobial peptides and peptidoglycan hydrolases, two classes of phagosome antimicrobial defense mechanisms. In addition, the production of the critical virulence factor, listeriolysin O, was compromised by FA modulation, suggesting that FAs play a key role in virulence regulation. In summary, our results emphasize the significance of FA metabolism, not only in bacterial virulence regulation but also in membrane barrier function by providing resistance against host antimicrobial stress.     

Listeria monocytogenes in Nature

Samples from 12 farms were examined during two successive spring and early autumn seasons. L. monocytogenes was isolated from vegetation or soil taken from 11 of the 12 farms and from 6 of the 7 nonagricultural sites. A total of 27 strains were isolated from the 19 sites. The organism was not isolated from any of the autumn collections.     

Survival and Heat Resistance of Listeria monocytogenes after Exposure to Alkali and Chlorine

A strain of Listeria monocytogenes isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59°C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21°C. Cells of L. monocytogenes incubated at 37°C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56°C than were cells stored in TPB at pH 7.3. The alkaline-stress response in L. monocytogenes may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 ± 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log₁₀ CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log₁₀ CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56°C than cells treated for 5 min. Contamination of foods by L. monocytogenes cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of L. monocytogenes against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log₁₀-unit reductions is not compromised in these cells.