Heat resistance of different serovars of Listeria monocytogenes

Twelve Listeria monocytogenes strains representing seven serovars were heat-treated in physiological saline by a glass capillary tube method. Five strains were treated at 58°, 60° and 62°C, three at 60°, 62° and 64°C and four at 60°C. Heat-treated bacteria were recovered on blood agar in two ways: (1) incubation at 37°C for 7 d; and (2) preincubation at 4°C for 5 d, followed by incubation at 37°C for 7 d. D and z values were determined. Better average recovery and higher D values were obtained when the preincubation procedure was used. The final evaluations of the heat resistance properties of the strains were therefore based on values for preincubated samples. D values recorded at 58°, 60°, 62° and 64°C for preincubated samples were 1.7–3.4, 0.72–3.1, 0.30–1.3 and 0.33–0.68 min, respectively. z values determined were 5.2–6.9°C. D values were compared statistically. Significant differences in heat resistance were noted both between serovars and between strains belonging to the same serovar.     

Heat resistance of Listeria monocytogenes in dairy products as affected by the growth medium

Listeria monocytogenes strains 1151 and Scott A were grown in broth at 30 °C and transferred to half cream, double cream and butter stored at 5 °C to determine the influence of dairy product composition on heat resistance at 52, 56, 60, 64 and 68 °C. Strain 1151 showed a higher heat resistance than strain Scott A. The heat resistance of both strains was higher in the dairy products than in broth, particularly at lower temperatures. A significant difference was observed between log 10 of the D -values in the different dairy products. The D -values obtained for both strains resuspended in all the dairy products would result in efficient elimination of the pathogen at 72·7 °C for 15 s. The highest D -value was 11·30 s at 68 °C and by using a z -value of 6·71 °C it can be determined that at 72·7 °C the D -value would be 1·5 s. The 15 s process would therefore achieve 10 log reductions. The effect of growth conditions on the heat resistance at 60 °C of L. monocytogenes Scott A was also investigated. When the cells were grown in the dairy products themselves, and particularly butter, the heat resistance of Scott A was enhanced; for example, the D -values were 7·15 times higher than in broth. Further studies are required to investigate if this protection against heating exists at higher temperatures, in which case the efficiency of pasteurization treatments or other heat treatments would be considerably lowered.     

Antimicrobial resistance of Listeria monocytogenes

Listeria monocytogenes is an opportunistic pathogen that causes rare but frequently fatal infections, termed listerioses. In general, strains of L. monocytogenes are susceptible to a wide range of antibiotics, except for the cephalosporins, fluorochinolones and fosfomycin (Hof, 1991). The current therapy of choice is a combination of ampicillin and aminoglycoside, usually gentamicin (Lorber, 1997). In cases when it is not possible to use a beta-lactam antibiotic, second-choice therapy involves the use of an association of trimethoprim with a sulfonamide, such as in co-trimoxazole, in which the more active in the combination seems trimethoprim, synergized by the sulfa compound. Other second line agents for listeriosis include erythromycin and vancomycin (Temple and Nahata, 2000). The first strains of L. monocytogenes resistant to antibiotics were reported in 1988 (Poyart-Salmeron et al. 1990) The present paper reviews the current state of affairs with regard to the resistance of L. monocytogenes isolated from food products and clinical material to different antibiotics, with particular emphasis on those used in the therapy of listeriosis.     

The heat resistance of Listeria monocytogenes

Heat resistance data for Listeria monocytogenes are reviewed. The organism is appreciably more resistant than common Salmonella serotypes but less resistant than Salmonella senftenberg 775W. Reports that the organism can survive heating at 80°C have not been substantiated and are incompatible with carefully determined D and z values in milk and a range of foods. Cooking food to an internal temperature of 70°C for 2 min is adequate to ensure destruction of L. monocytogenes. Normal pasteurization procedures will inactivate L. monocytogenes in milk but the margin of safety is greater for vat pasteurization than for high temperature short time treatment.     

Ecogenetics of antibiotic resistance in Listeria monocytogenes

The acquisition process of antibiotic resistance in an otherwise susceptible organism is shaped by the ecology of the species. Unlike other relevant human pathogens, Listeria monocytogenes has maintained a high rate of susceptibility to the antibiotics used for decades to treat human and animal infections. However, L. monocytogenes can acquire antibiotic resistance genes from other organisms’ plasmids and conjugative transposons. Ecological factors could account for its susceptibility. L. monocytogenes is ubiquitous in nature, most frequently including reservoirs unexposed to antibiotics, including intracellular sanctuaries. L. monocytogenes has a remarkably closed genome, reflecting limited community interactions, small population sizes and high niche specialization. The L. monocytogenes species is divided into variants that are specialized in small specific niches, which reduces the possibility of coexistence with potential donors of antibiotic resistance. Interactions with potential donors are also hampered by interspecies antagonism. However, occasional increases in population sizes (and thus the possibility of acquiring antibiotic resistance) can derive from selection of the species based on intrinsic or acquired resistance to antibiotics, biocides, heavy metals or by a natural tolerance to extreme conditions. High-quality surveillance of the emergence of resistance to the key drugs used in primary therapy is mandatory.     

Screening of benzalkonium chloride resistance in Listeria monocytogenes strains isolated during cold smoked fish production

AIMS: To determine the susceptibility to disinfectants and cross-resistance to antibiotics in Listeria monocytogenes strains isolated from fish products and the fish-processing environment. METHODS AND RESULTS: Minimal inhibitory concentration assessment, using the agar dilution method, showed 108 of 255 L. monocytogenes isolates with low susceptibility to benzalkonium chloride (BC), commonly used in food industries. Most of them are from raw products of farmed fish during processing, while the remaining resistant isolates were mainly from the environment and finished products irrespective of the fish species. Two BC-resistant isolates were resistant to ethidium bromide (EB). The conservation of resistance after plasmid curing suggested that the resistance genes are not plasmid associated. EB accumulation assays demonstrated that the two BC(R) EB(R) isolates used an efflux pump to expel these substrates whereas a different mechanism was probably used by the majority of the strains with BC(R) EB(S) pattern. No cross-resistance was found with antibiotics. CONCLUSIONS: This study highlights the difference in susceptibilities to BC for L. monocytogenes strains isolated from fish-processing plants and in resistance mechanisms to BC developed by these bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of BC resistant L. monocytogenes strains could contribute to their adaptation and so explained their survival and persistence in the fish-processing environment.     

Mechanistic study of membrane concentration and recovery of Listeria monocytogenes

Detection of the foodborne pathogen Listeria monocytogenes requires that food samples be processed to remove proteins and lipids, concentrate microorganisms to a detectable concentration, and recover the concentrated cells in a small volume compatible with micron-scale biochips. Mechanistic considerations addressed in this research include the roles of membrane structure, pore size, and detergents in maximizing recovery of cells from a complex biological fluid. The fluid in this case was a food sample (hotdog extract) inoculated with L. monocytogenes. This study showed how membrane filtration using a syringe filter is able to concentrate L. monocytogenes by 95x with up to 95% recovery of living microorganisms by concentrating 50 mL of food sample into a volume of 500 microL. Tween 20 was added to the sample to prevent irreversible adsorption of the microorganism to the membrane and thereby help to ensure high recovery. Comparison of polycarbonate, mixed cellulose, nylon, and PVDF membranes with 0.2 to 0.45 microm pores showed the 0.2 microm polycarbonate membrane with straight through, mono-radial pores gives the highest recovery of living microorganisms. The mixed cellulose, nylon, and PVDF membranes have a fibrous structure whose characteristic openings are much larger than their effective pore size cut-offs of 0.22 or 0.45 microm. We define conditions for rapid membrane-based cell concentration and recovery that has the potential to supplant enrichment steps that require a day or more. This approach has the added benefit of facilitating examination of a large amount of fluid volume by reducing its volume to a range that is compatible with the microliter scales of biochip or other biosensor detection systems.     

Methods used for the detection and subtyping of Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen responsible for non-invasive and invasive diseases in the elderly, pregnant women, neonates and immunocompromised populations. This bacterium has many similarities with other non-pathogenic Listeria species which makes its detection from food and environmental samples challenging. Subtyping of L. monocytogenes strains can prove to be crucial in epidemiological investigations, source tracking contamination from food processing plants and determining evolutionary relationships between different strains. In recent years there has been a shift towards the use of molecular subtyping. This has led to the development of new subtyping techniques such as multi-locus variable number tandem repeat analysis (MLVA) and multi-locus sequence based typing (MLST). This review focuses on the available methods for Listeria detection including immuno-based techniques and the more recently developed molecular methods and analytical techniques such as matrix-assisted laser desorption/ionisation time-of-flight based mass spectrometry (MALDI-TOF MS). It also includes a comparison and critical analysis of the available phenotypic and genotypic subtyping techniques that have been investigated for L. monocytogenes.     

Methods for improved recovery of Listeria monocytogenes from cheese

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.     

Modelling the effect of the redox potential and pH of heating media on Listeria monocytogenes heat resistance

Aims:  Study the effect of redox potential and pH of the heating media on Listeria monocytogenes heat resistance and model its action at fixed temperature.
Methods and Results:  The heat resistance of Listeria monocytogenes at 58°C was studied in Brain Heart Infusion broth as a function of pH (from 5·0 to 7·0) and redox potential ( E _h7). The media redox was adjusted with nitrogen gas, potassium ferricyanide and dithiothreitol. A Weibull model was used to fit survival curves. The heat resistance parameter (δ_58°C) was estimated from each inactivation curve. A major effect of pH was observed. Bigelow model was used to describe the effect of redox potential on the apparent L. monocytogenes heat resistance. The highest δ_58°C values have been obtained at pH 7·0 and oxidizing conditions.
Conclusions:  The developed model indicates that the E _h7 has a significant effect and varied depending on the pH of the heating media. The z _redox values, calculated from δ_58°C allowed quantifying the influence of heating media redox potential on L. monocytogenes thermal inactivation.
Significance and Impact of the Study:  The obtained model shows the action of redox potential on L. monocytogenes thermal destruction and might be useful to take into account in food thermal processes.     

Heat resistance and growth of Salmonella enteritidis, Listeria monocytogenes and Aeromonas hydrophila in whole liquid egg

Salmonella enteritidis ATCC,13067, Listeria monocytogenes ATCC,19116 and Aeromonas hydrophila ATCC,7965 strains were evaluated for growth and thermal resistance in liquid whole egg (LWE). Each strain grew well in LWE at temperatures between 4 and 30 degrees C, except S. enteritidis which grew weakly at 4 and 10 degrees C. Maximum populations for each strain increased with increasing growth temperature. The thermal destruction of each strain was determined in six liquid products. The egg products used were LWE, LWE with 5, 10 and 15% NaCl and LWE with 5 and 10% sucrose. L. monocytogenes tended to be more heat resistant than S. enteritidis and A. hydrophila. The highest kill rates were noted in LWE, while survival was best in those products supplemented with NaCl. Radiation D10 values of strains in LWE were 0.18, 0.39 and 0.49 kGy for A. hydrophila, S. enteritidis and L. monocytogenes, respectively.     

Selective plating medium for quantitative recovery of food-borne Listeria monocytogenes

A new plating medium (lithium chloride-ceftazidime agar [LCA]) was designed to quantitatively recover food-borne Listeria monocytogenes in the form of large colonies while inhibiting most other food-borne microorganisms. This medium included brain heart infusion agar as the nutritive agar base and a combination of selective agents (LiCl, glycine anhydride, and ceftazidime). Comparison of LCA and lithium chloride-phenylethanol-moxalactam agar (LPM) indicated that both were equally effective for the enumeration of the cold-tolerant pathogen in artificially and naturally contaminated foods. However, LCA was more effective than LPM in the recovery of sublethally heat-injured cells. Moreover, Listeria colonies on LCA exhibited a more distinct bluish hue than those on LPM when viewed by the Henry oblique transillumination technique.     

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.     

Methods for improved recovery of Listeria monocytogenes from cheese.

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.     

Mathematical modelling of the heat resistance of Listeria monocytogenes

The heat resistance of Listeria monocytogenes phagovar 2389/2425/3274/2671/47/108/340 (1992 French outbreak strain) in broth was studied at 55, 60 and 65 °C. Experiments were carried out on bacterial cultures in three different physiological states: cultures at the end of the log phase, cultures heat-shocked at 42 °C for 1 h, and subcultures of cells resistant to prolonged heating. Survivor curves were better fitted using a sigmoidal equation than the classical log-linear model. This approach was justified by the existence of heat resistance distributions within the bacterial populations. Peaks (log₁₀ of heating time) of heat resistance distributions of untreated, heat-shocked, and selected cultures at 55, 60 and 65 °C were 0·34, −0·90 and −1·84 min, 0·74, −0·51 and −1·24 min, and 0·17, −0·94 and−1·45 min, respectively. The widths of the distributions are proportional to 0·29, 0·36and 0·41 min^0·5, 0·26, 0·36 and 0·41 min^0·5, and 0·34, 0·44 and 0·41 min^0·5. An increase in thethermal tolerance could then be induced by sublethal heat shock or by selection of heatresistant cells.     

Expression of ferritin-like protein in Listeria monocytogenes after cold and freezing stress

The cold shock protein family consists of the transfer of the foodborne pathogen Listeria monocytogenes from 37 to 4 and ?20?°C and was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein ferritin-like protein (Flp) has an important role in regulation of various microbial physiological processes. Flp have a molecular mass of about 18?kDa, as observed on SDS?CPAGE. The purification procedure including ammonium sulfate fractionation was used. Monospecific polyclonal antibodies raised in rabbits against the purified new Flp immunostained a single 18-kDa Flp band in extracts from different cytoplasmic proteins blotted onto nitrocellulose. A 411-bp cDNA fragment that corresponds to an internal region of an flp gene was obtained by RT-PCR. Our result indicated a surexpression of major cold shock protein and an important increase in flp mRNA amount after a downshift temperature especially at ?20?°C.