Insertional knock-out of protein translocation systems common for Yersinia enterocolitica and Listeria monocytogenes

To carry out efficient insertional mutagenesis in Listeria monocytogenes and to facilitate the characterisation of disrupted genes, a novel derivative of plasmid pACYC 184 was constructed, pLIV virA3, carrying a fragment from the virA region of the of Y. enterocolitica plasmid pYVe 0:9. After transformation of this plasmid into L. monocytogenes it was possible to select for its integration into the host DNA at 42 degrees C. Insertional mutants of L. monocytogenes obtained by using pLIV vector containing plasmid DNA fragments from Y. enterocolitica were constructed and are described.     

Growth of Listeria monocytogenes and Yersinia enterocolitica colonies under modified atmospheres at 4 and 8 degrees C using a model food system

The growth of Listeria monocytogenes and Yersinia enterocolitica colonies was studied on solid media at 4 and 8 degrees C under modified atmospheres (MAs) of 5% O2: 10% CO2: 85% N2 (MA1), 30% CO2: 70% N2 (MA2) and air (control). Colony radius, determined using computer image analysis, allowed specific growth rates (mu) and the time taken to detect bacterial colonies to be estimated, after colonies became visible. At 4 degrees C both MAs decreased the growth rates of L. monocytogenes by 1.5- and 3.0-fold under MA1 (mu = 0.02 h(-1)) and MA2 (mu = 0.01 h(-1)), respectively, as compared with the control (mu = 0.03 h(-1)). The time to detection of bacterial colonies was increased from 15 d (control) to 24 (MA1) and 29 d (MA2). At 8 degrees C MA2 decreased the growth rate by 1.5-fold (mu = 0.04 h(-1)) as compared with the control (mu = 0.06 h(-1)) and detection of colonies increased from 7 (control) to 9 d (MA2). At 4 degrees C both MAs decreased the growth rates of Y. enterocolitica by 1.5- and 2.5-fold under MA1 (mu = 0.03 h(-1)) and MA2 (mu = 0.02 h(-1)), respectively, as compared with the control (mu = 0.05 h(-1)). At 8 degrees C identical growth rates were obtained under MA1 and the control (mu = 0.07 h(-1)) whilst a decrease in the growth rate was obtained under MA2 (mu = 0.04 h(-1)). The detection of colonies varied from 6 (8 degrees C, aerobic) to 19 d (4 degrees C, MA2). Refrigerated modified atmosphere packaged foods should be maintained at 4 degrees C and below to ensure product safety.     

Light butter: natural microbial population and potential growth of Listeria monocytogenes and Yersinia enterocolitica

Yarrowia lipolytica, Bacillus polymyxa and Enterococcus faecium were the most frequent yeast and bacterial spoilage species associated with commercial light butter. Following inoculation and incubation at 4°C, strains of Y. enterocolitica and L. monocytogenes exhibited growth rates higher than those of the naturally occurring microflora. Listeria monocytogenes displayed a higher aptitude to proliferate in such food while the cell increment of Y. enterocolitica was limited.     

The effect of culture growth phase on induction of the heat shock response in Yersinia enterocolitica and Listeria monocytogenes

The effect of culture growth phase on induction of the heat shock response in Yersinia enterocolitica and Listeria monocytogenes, was examined. Exponential or stationary preconditioned cultures were heat shocked and survivor numbers estimated using selective and overlay/resuscitation recovery techniques. The results indicate that prior heat shock induced increased heat resistance in both micro-organisms to higher heat treatments. Heat-shocked cells of each micro-organism were able to survive much longer than non-heat-shocked cells when heated at 55 degrees C. The size of the change in heat resistance between heat-shocked and non-heat-shocked cells was greatest for exponential cultures (X:X). Results indicate that the overall relative thermal resistance of each pathogen was dependent on cell growth phase. Stationary cultures (S:S) were significantly (P < 0.01) more thermotolerant than exponential cultures (X:X) under identical processing conditions. Under most conditions, the use of an overlay/resuscitation recovery medium resulted in higher D-values (P < 0.05) compared with a selective recovery medium.     

Synergistic effect of heat and sodium lactate on the thermal resistance of Yersinia enterocolitica and Listeria monocytogenes in minced beef

The effect of sodium lactate (NaL) (0, 2.4 or 4.8%), in heating and recovery media, on Yersinia enterocolitica and Listeria monocytogenes numbers recovered from minced beef heated at 55 degrees C, was examined. Survivors were enumerated on selective media at pH 5.7/7.4 (Y. enterocolitica) or pH 5.7/7.2 (L. monocytogenes). Recovery of the organisms depended on the pH and NaL levels in the recovery medium. The heat resistance of Y. enterocolitica (P < 0.001) and L. monocytogenes (P < 0.01) decreased as the concentration of NaL in the minced beef increased from 0 to 2.4% or 4.8%. The thermal destruction of pathogens in foods processed using mild temperatures may be enhanced by the addition of 2.4% NaL.     

Biocontrol of the Food-Borne Pathogens Listeria monocytogenes and Salmonella enterica Serovar Poona on Fresh-Cut Apples with Naturally Occurring Bacterial and Yeast Antagonists

Fresh-cut apples contaminated with either Listeria monocytogenes or Salmonella enterica serovar Poona, using strains implicated in outbreaks, were treated with one of 17 antagonists originally selected for their ability to inhibit fungal postharvest decay on fruit. While most of the antagonists increased the growth of the food-borne pathogens, four of them, including Gluconobacter asaii (T1-D1), a Candida sp. (T4-E4), Discosphaerina fagi (ST1-C9), and Metschnikowia pulcherrima (T1-E2), proved effective in preventing the growth or survival of food-borne human pathogens on fresh-cut apple tissue. The contaminated apple tissue plugs were stored for up to 7 days at two different temperatures. The four antagonists survived or grew on the apple tissue at 10 or 25°C. These four antagonists reduced the Listeria monocytogenes populations and except for the Candida sp. (T4-E4), also reduced the S. enterica serovar Poona populations. The reduction was higher at 25°C than at 10°C, and the growth of the antagonists, as well as pathogens, increased at the higher temperature.     

Behavior of Yersinia enterocolitica in the Presence of the Bacterivorous Acanthamoeba castellanii

Free-living protozoa play an important role in the ecology and epidemiology of human-pathogenic bacteria. In the present study, the interaction between Yersinia enterocolitica, an important food-borne pathogen, and the free-living amoeba Acanthamoeba castellanii was studied. Several cocultivation assays were set up to assess the resistance of Y. enterocolitica to A. castellanii predation and the impact of environmental factors and bacterial strain-specific characteristics. Results showed that all Y. enterocolitica strains persist in association with A. castellanii for at least 14 days, and associations with A. castellanii enhanced survival of Yersinia under nutrient-rich conditions at 25°C and under nutrient-poor conditions at 37°C. Amoebae cultivated in the supernatant of one Yersinia strain showed temperature- and time-dependent permeabilization. Intraprotozoan survival of Y. enterocolitica depended on nutrient availability and temperature, with up to 2.8 log CFU/ml bacteria displaying intracellular survival at 7°C for at least 4 days in nutrient-rich medium. Transmission electron microscopy was performed to locate the Yersinia cells inside the amoebae. As Yersinia and Acanthamoeba share similar ecological niches, this interaction identifies a role of free-living protozoa in the ecology and epidemiology of Y. enterocolitica.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

Incidence of Listeria spp. and Listeria monocytogenes in a Poultry Processing Environment and in Poultry Products and Their Rapid Confirmation by Multiplex PCR

Volume 60, no. 12, p. 4602: Fig. 1 should appear as shown below. FIG. 1. Incidence of L. monocytogenes and other Listeria spp. in a poultry processing environment and in raw and cooked poultry products from March to September 1992. (A) Raw (R) and cooked (C) areas within the processing environment. (B) Raw and cooked poultry products. (box), L. monocytogenes; (box), Listeria spp. (not L. monocytogenes); (symbl), no Listeria spp. [This corrects the article on p. 4600 in vol. 60.].     

Inhibition of Listeria monocytogenes in Cooked Ham by Virulent Bacteriophages and Protective Cultures

Protective cultures can be used successfully as an additional hurdle together with phages to reduce growth of Listeria monocytogenes on sliced cooked ham. Addition of phages resulted in a rapid 10-fold reduction of L. monocytogenes. After 14 to 28 days of storage, a 100-fold reduction was observed in samples with phages and protective culture compared to results for samples with phages alone.Listeriosis in Europe has an average incidence between 2 and 10 reported cases per million population per year (7). Listeria monocytogenes is found in raw and ready-to-eat (RTE) products, poultry, seafood, and dairy products. A review of the incidence and transmission of L. monocytogenes in RTE products has been published by Lianou and Sofos (11). The USDA has implemented a “zero-tolerance” policy for L. monocytogenes in RTE products (2). In the European Union, the limit for common RTE foods is 100 CFU/g (1). Recently, Codex Alimentarius adopted new standards for L. monocytogenes in RTE foods, with a limit of 100 CFU/g in foods where L. monocytogenes cannot grow and absence in foods where the bacterium can grow. However, an alternative approach is accepted. Competent authorities may choose to establish and implement other validated limits (http://www.codexalimentarius.net/web/archives.jsp?lang=en, Alinorm 09/32/REP and Alinorm 09/32/13). L. monocytogenes in cooked products is connected with cross-contamination after heat treatment (11, 12). Bacteriophages have been successfully applied to a number of food products to reduce the level of contaminating L. monocytogenes (6, 8-10, 13, 14). The effect of phages varies with the type of product and is strongly dose dependent (6, 8). Active phages can be recovered from foods after long storage, but the phage particles appear to become immobilized soon after addition to nonliquid foods and therefore, due to limited diffusion, cannot infect bacteria (8). Bacteria surviving phage treatment can later grow in the product. Additional hurdles should therefore be present to inhibit later outgrowth of L. monocytogenes.We have previously employed Lactobacillus sakei TH1 as a protective culture against L. monocytogenes (4, 5). Here we examine the combined use of phages and protective culture to reduce outgrowth of L. monocytogenes on cooked ham.Rifampin (rifampicin)-resistant mutants of L. monocytogenes 2230/92 serotype 1, implicated in a listeriosis outbreak in Norway (12), and L. monocytogenes 167 serotype 4b were grown overnight in brain heart infusion (Difco Laboratories, Detroit, MI) at 37°C without shaking and stored at 4°C for 24 h (3-5). Cells were diluted in 0.9% NaCl and plated on brain heart infusion agar with 200 μg/ml rifampin. L. sakei TH1 was grown at 30°C in MRS (de Man, Rogosa, Sharpe) broth (CM 359; Oxoid, Hampshire, England) (pH 6.2) and plated on MRS agar (5). Listex P100 phages, 2 × 10¹¹ PFU/ml, were from EBI Food Safety (Wageningen, The Netherlands).Ten-gram slices of hams with 2.3% NaCl and 0.01% disodium diphosphate (pH 6.2; a_w > 0.97), made at Nofima''s pilot plant (Aas, Norway), were inoculated with a cold-adapted 1:1 mixture of L. monocytogenes 2230/92 and 167. Bacteria were spread in 100 μl 0.9% NaCl over the 80-cm² surface area of each slice to 10³ CFU/cm² using a bent glass rod. After 1 h at 20°C, phages (5 × 10⁷ PFU/cm² in a total volume of 100 μl) were spread over the same surface. After one additional hour, 10³ CFU/cm² L. sakei TH1 in 100 μl 0.9% NaCl was added where appropriate. The slices were vacuum packed and stored at 10°C. Growth was measured before and after spiking and at 0, 3, 7, 14, and 28 days after homogenizing the slices in 100 ml 0.9% NaCl in a Stomacher homogenizer. No lactic acid bacteria were detected in uninoculated samples. Experiments were performed with three parallel samples. L. monocytogenes alone grew from 10⁴ CFU/g at the onset of the experiment to 10⁷ CFU/g the first 7 days, reached 2 × 10⁸ CFU/g after 14 days, and remained unchanged thereafter (Fig. ​(Fig.1).1). In samples with both L. monocytogenes and phages, a rapid 1-log reduction in L. monocytogenes was observed. Surviving L. monocytogenes, however, grew as well as that in the phage-free controls, reaching >10⁷ CFU/g after 14 days. In samples where both P100 phages and L. sakei TH1 were added, the same initial reduction of L. monocytogenes was observed, but the later outgrowth was reduced by the fast-growing lactic acid bacteria and the L. monocytogenes levels were 2 logs lower than those with P100 phages alone after 28 days of incubation. The phages did not influence the growth and survival of L. sakei TH1. During the 28 days of storage, the pH changed from 6.20 to 6.05 in samples with L. monocytogenes and to 6.00 in samples with both L. monocytogenes and L. sakei TH1. The results were reproduced in a separate repetition of the experiment at 10°C (not shown).Open in a separate windowFIG. 1.Inhibition of L. monocytogenes in cooked ham with bacteriophages and protective culture at 10°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫), L. monocytogenes and 5 × 10⁷ PFU/cm² P100 phages (▪), or L. monocytogenes, 5 × 10⁷ PFU/cm² P100 phages, and 10³ CFU/cm² (approximately 10⁴ CFU/g) protective-culture L. sakei TH1 (▴) and stored at 10°C. Growth of L. sakei TH1 is shown by the broken line.The effect of the protective culture was dose dependent when 10⁴ CFU/g and 10⁶ CFU/g of L. sakei TH1 were added to slices of ham (Fig. ​(Fig.2).2). L. monocytogenes alone grew to 2 × 10⁸ CFU/g after 14 days. When L. sakei TH1 was added at a low concentration (10⁴ CFU/g), L. monocytogenes grew to approximately 4 × 10⁶ CFU/g, while when L. sakei TH1 was added at a high concentration, L. monocytogenes levels were 1 to 2 logs lower. The pHs in the low- and high-inoculum hams were reduced from the initial 6.20 to 6.16 and 6.02, respectively, at day 28. For hams stored at 4°C, slow growth of L. monocytogenes occurred between days 14 and 28 from 10⁴ to 10⁵ CFU/g (P = 0.003) (Fig. ​(Fig.3).3). With phages and L. sakei TH1 added, a rapid 1-log reduction of L. monocytogenes was observed due to the phage attack, and no growth was observed during the 28-day storage period. The L. sakei TH1 strain showed a longer lag phase at this low temperature but nevertheless reached 10⁷ CFU/g at day 14 and thereby inhibited any growth of L. monocytogenes.Open in a separate windowFIG. 2.Inhibition of L. monocytogenes in cooked ham inoculated with large or small amounts of protective culture at 10°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫), L. monocytogenes and 10⁶ CFU/g (10⁵ CFU/cm²) L. sakei TH1 (▴), or L. monocytogenes and 10⁴ CFU/g (10³ CFU/cm²) L. sakei TH1 (▪) and stored at 10°C. Growth of L. sakei TH1 is shown by broken lines. The L. monocytogenes control is the same control as in Fig. ​Fig.11.Open in a separate windowFIG. 3.Inhibition of L. monocytogenes in cooked ham with bacteriophages and protective culture at 4°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫) or L. monocytogenes, 5 × 10⁷ PFU/cm² P100 phages, and 10⁴ CFU/g (10³ CFU/cm²) protective-culture, L. sakei TH1 (▴) and stored at 4°C. Growth of L. sakei TH1 is shown by the broken line.Since L. sakei TH1 grows well at low temperatures, prevents growth of L. monocytogenes, and has no negative influence on the organoleptic properties of ham (4, 5), it can successfully be employed as an additional hurdle together with phages.We here chose to perform the storage experiments under “worst-case” conditions. Generally, the contamination levels of L. monocytogenes are lower than in our setup, in the range of 10 to 100 CFU/g (see reference 11 and references therein). Since L. sakei TH1 grows well at low temperatures (Fig. ​(Fig.3),3), its selective advantage will be greater at 4°C than at abuse temperatures. From the above, it is evident that it is possible to optimize L. monocytogenes inhibition by increasing both the phage titer and the starting amount of protective culture. An enhanced effect may also be experienced by modifying phage application, e.g., by using larger liquid volumes (6, 8).Emergence of resistant L. monocytogenes may be a potential problem when treating foods with phages. No emergence of resistance has been detected after phage treatment (6, 8). Such strategies as use of phage mixtures, phage rotation schemes, and treatment of products immediately prior to packaging may reduce eventual resistance problems (8). Some L. monocytogenes strains are naturally phage resistant (6). In these cases, a protective culture still constitutes a powerful hurdle.In conclusion, we have shown here that by applying phages and protective culture as two independent hurdles, it is possible to both reduce the number of L. monocytogenes bacteria on a product and inhibit outgrowth of eventual remaining surviving cells. This is a general method that can potentially be applied to different foods where there is a potential risk for growth of L. monocytogenes, provided a suitable protective culture is available.     

Autonomous Growth of Isolated Single Listeria monocytogenes and Salmonella enterica Serovar Typhimurium Cells in the Absence of Growth Factors and Intercellular Contact

The aim of this study was to observe growth of isolated single bacterial cells in the absence of growth factors and intercellular contact. In order to exclude stochastic uncertainties induced by dilution series, a new micromanipulation method was developed to ensure explicit results under visual control. This was performed with particular care for production of single prokaryotic cells and subsequent investigation of their autonomous growth. Over 450 single isolated Listeria monocytogenes and Salmonella enterica subsp. enterica serovar Typhimurium cells in lag, log, and stationary growth phases were investigated by this method, which included thoroughly washing the cells. The proportion of living cells within the initial cultures was compared to the proportion of positive samples after enrichment of the separated single cells. This resulted in P values of ≥0.05 using the chi-square test for statistical analysis, indicating no significant difference, and clearly demonstrates reproduction of isolated single bacterial cells without the need for growth factors or intercellular contact. Ease of handling of the apparatus and good performance of the cleaning procedures were achieved, as was validation of the method, demonstrating its suitability for routine laboratory use.The possibility of independent growth of isolated single prokaryotic cells has been discussed recently and remains controversial (15). Undoubtedly, cell-to-cell communication plays a key role in the establishment and development of bacterial communities. Both physical and chemical factors influence the organization of biofilms, sporulation, and resuscitation of bacterial populations. The analogy of the chemical factors to eukaryotic pheromones, as well as their role in bacterial cell division, has been postulated (34). These facts are evident and have been well investigated, and, in summary, the necessity of intercellular contacts, population effects, and intrinsic growth factors such as resuscitating promoting factor (rpf) is generally supposed (24, 36). Nevertheless, when it comes to cell division and growth of low inocula of prokaryotic cells, some questions appear to remain open. Considering evolutionary developments, asexual reproduction is a prerequisite for survival of single organisms in the environment and one of the necessities for the success of the prokaryotic kingdom. Taken the other way, asexual reproduction also suggests the possibility of independent growth of isolated single bacterial cells. The scientific community remains divided over this possibility, not least because of the possible heterogeneity of bacterial cultures in terms of the physiological status of every single cell and the effect of a Poisson distribution in highly diluted cultures, which influence experimental setups and results (17).If one viable prokaryotic cell is to be shown to be capable of generating a population of daughter cells, a prerequisite is its isolation and physical manipulation as an individual cell. Simple serial dilution protocols, such as the most probable number (MPN) method, achieve this task, but they suffer from a lack of certainty that the diluted solution indeed contains only one viable cell, free from any adherent growth factors. In such cases the accuracy of the data obtained by multiple dilution procedures becomes uncertain regarding the Poisson distribution of highly diluted bacterial cell (≤10 CFU/ml) suspensions (11, 32).Combined microinjection and micromanipulation methods are alternative applications. These are widespread routine techniques that have been developed recently for large eukaryotic cells (14, 38). However, application of these methods to prokaryotic cells leads to new physical conditions defined by the technical equipment secondary to the size of the handled cells (≤1 μm). These include limitations of fluorescence microscopy, general microscopic analysis, and photographic documentation. Over the past 30 years several attempts to improve the management of single prokaryotic cells using micromanipulation techniques have been made (10). The core issue of these studies has been isolation of single microbial cells from mixed populations under direct visual control (9, 13).In contrast, the aim of this study was to develop a method capable of serial manipulation of single prokaryotic cells under visual control. This was accomplished to produce series of single prokaryotic cells and subsequently to investigate their autonomous growth. The growth of single Listeria monocytogenes and Salmonella enterica subspecies enterica serovar Typhimurium cells was investigated in highly diluted buffer systems without facultative growth factors being transferred from the primary enrichment. In this study over 450 single cells isolated from different growth phases (lag phase, log phase, and stationary phase) were manipulated using a novel single bacterial cell manipulation (SBCM) technique.     

一多重耐药单增李斯特菌株的生长特性及毒力分析

为了解单增李斯特菌株耐药后可能发生的生物学变化,以哈市生肉中分离到的1株对17种抗生素耐受的单增李斯特菌株L.M.B8为研究对象,对其生长及毒力特性进行研究。结果显示,L.M.B8的生长及毒力特性均与标准菌株有明显差异。在NaC l浓度为0.5%~5%、pH值为4.0~10.0及温度为20~45℃范围内,L.M.B8的生长速度均明显高于标准菌株。L.M.B8对高浓度盐的敏感性高于标准菌株,且对温度的适应能力强于标准菌株。从生长曲线看,L.M.B8的对数生长期与稳定期均较标准菌株提前2~3 h,且其稳定期较标准菌株明显缩短。L.M.B8小鼠腹腔注射半数致死量（LD50）较标准菌株明显降低。该研究为进一步探讨单增李斯特菌的耐药性与其他生物学特性的相关性奠定基础。     

Thermal inactivation of Listeria monocytogenes and Yersinia enterocolitica in minced beef under laboratory conditions and in sous-vide prepared minced and solid beef cooked in a commercial retort

D-values were obtained for Listeria monocytogenes and Yersinia enterocolitica at 50, 55 and 60 degrees C in vacuum-packed minced beef samples heated in a laboratory water-bath. The experiment was repeated using vacutainers, which allowed heating of the beef to the desired temperature before inoculation. D-values of between 0.15 and 36.1 min were obtained for L. monocytogenes. Pre-heating the beef samples significantly affected (P < 0.05) the D60 value only. D-values for Y. enterocolitica ranged from 0.55 to 21.2 min and all the D-values were significantly different (P < 0.05) after pre-heating. In general, the D-values obtained for core inoculated solid beef samples were significantly higher (P < 0.05) than those generated in minced beef when heated in a Barriquand Steriflow commercial retort.     

Influence of Temperature and Growth Phase on Expression of a 104-Kilodalton Listeria Adhesion Protein in Listeria monocytogenes

Interaction of Listeria monocytogenes with mammalian intestinal cells is believed to be an important first step in Listeria pathogenesis. Transposon (Tn916) mutagenesis provided strong evidence that a 104-kDa surface protein, designated the Listeria adhesion protein (LAP), was involved in adherence of L. monocytogenes to a human enterocyte-like Caco-2 cell line (V. Pandiripally, D. Westbrook, G. Sunki, and A. Bhunia, J. Med. Microbiol. 48:117–124, 1999). In this study, expression of LAP in L. monocytogenes at various growth temperatures (25, 37, and 42°C) and in various growth phases was determined by performing an enzyme-linked immunoassay (ELISA) and Western blotting with a specific monoclonal antibody (monoclonal antibody H7). The ELISA and Western blot results indicated that there was a significant increase in LAP expression over time only at 37 and 42°C and that the level of LAP expression was low during the exponential phase and high during the stationary phase. In contrast, there were not significant differences in LAP expression between the exponential and stationary phases at 25°C. Examination of the adhesion of L. monocytogenes cells from exponential-phase (12-h) or stationary-phase (24-h) cultures grown at 37°C to Caco-2 cells revealed that there were not significant differences in adhesion. Although expression of L. monocytogenes LAP was different at different growth temperatures and in different growth phases, enhanced expression did not result in increased adhesion, possibly because only a few LAP molecules were sufficient to initiate binding to Caco-2 cells.     

Effect of cultivation temperature on nucleic acid level in bacteria Listeria monocytogenes and Yersinia pseudotuberculosis

The relationship between the multiplication of bacteria, the content of nucleic acid and the specific rate of their growth during their batch cultivation in nutrient broth and mineral medium at temperatures of 37 degrees C and 4-6 degrees C was studied in the causative agents of saprozoonotic infections with L. monocytogenes and Y. pseudotuberculosis used as typical representatives of such bacteria. The content of DNA was shown to remain practically unchanged after the alteration of cultivation temperature and the conditions of nutrition. The linear relationship between the content of RNA and specific growth rate was registered both at 37 degrees C and 4-6 degrees C. However a higher content of RNA at low temperatures was found to correspond to one and the same specific growth rate, which was linked with the additional synthesis of this nucleic acid.     

Studies on a haemolytic substance in Yersinia enterocolitica

Yersinia enterocolitica serotype O3 was found to produce a haemolytic substance which could be released from the bacterial cells by sonic disintegration. The substance was non-dialysable, thermolabile, antigenic, and sensitive to trypsin. Chromatographic studies indicated a high molecular weight. Erythrocytes from different mammalian species differed in sensitivity to the haemolytic substance. Y. enterocolitica serotypes O8 and O9 produced no haemolytic substance.     

Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola

AIMS: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. METHODS AND RESULTS: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3.5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10(3)-10(7) CFU ml(-1)). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine- or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B12 biosynthesis and propanediol and ethanolamine utilization. CONCLUSIONS: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.     

Modeling the Growth of Listeria monocytogenes in Soft Blue-White Cheese

The aim of this study was to develop a predictive model simulating growth over time of the pathogenic bacterium Listeria monocytogenes in a soft blue-white cheese. The physicochemical properties in a matrix such as cheese are essential controlling factors influencing the growth of L. monocytogenes. We developed a predictive tertiary model of the bacterial growth of L. monocytogenes as a function of temperature, pH, NaCl, and lactic acid. We measured the variations over time of the physicochemical properties in the cheese. Our predictive model was developed based on broth data produced in previous studies. New growth data sets were produced to independently calibrate and validate the developed model. A characteristic of this tertiary model is that it handles dynamic growth conditions described in time series of temperature, pH, NaCl, and lactic acid. Supplying the model with realistic production and retail conditions showed that the number of L. monocytogenes cells increases 3 to 3.5 log within the shelf life of the cheese.