Evaluation of ALOA plating medium for its suitability to recover high pressure-injured Listeria monocytogenes from ground chicken meat

AIMS: To evaluate a chromogenic plating medium for the isolation of sublethally injured cells of Listeria monocytogenes from processed foods. METHODS AND RESULTS: The inactivation of L. monocytogenes at pressures up to 400 MPa and 12 degrees C in ground chicken meat was employed to examine the recovery of high-pressure injured cells. Before and after different repair incubation periods at 30 degrees C in a nonselective broth, samples were plated onto a selective and differential agar [Agar Listeria according to Ottaviani and Agosti (ALOA)] and in the same medium supplemented with 4% sodium chloride (ALOA-S), and incubated at 37 degrees C. Sublethally injured cells were able to grow when directly plated onto the ALOA medium, without a previous repair incubation period. However, only uninjured cells grew on the ALOA-S medium. CONCLUSIONS: Sublethally injured cells of L. monocytogenes can be quantified by subtracting counts on ALOA-S medium from counts on ALOA medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Possible applications include direct enumeration on ALOA of stressed cells of L. monocytogenes in foods with more than 100 colony forming units per gram.     

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.     

Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria

Minimum growth temperatures and those of decreased growth were determined for 100 strains of listerias. The ability of 78 strains of Listeria monocytogenes isolated from animals and 22 non-haemolytic strains to grow at low temperatures was studied, using a flooding technique, in a plate-type continuous temperature gradient incubator at temperatures between -1.6 and 14.5 degrees C. The mean minimum temperature for L. monocytogenes was +1.7 +/- 0.5 degrees C. The growth of non-haemolytic listerias was unobservable at +1.7 +/- 0.5 degrees C. The L. monocytogenes strains grew at about 0.6 degrees C lower than the non-pathogenic strains. No differences in growth temperatures were observed among L. monocytogenes strains isolated from different sources. The serovars with the OI antigen grew at lower temperatures (+1.0 +/- 0.3 degrees C) than the other common serovar 4b (+1.3 +/- 0.4 degrees C). The results indicate that L. monocytogenes grows better than non-haemolytic strains under cold conditions. The possible role of haemolysins as growth factors is also discussed.     

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.     

Plasmids in Listeria monocytogenes and other Listeria species.

One hundred and twenty-two food, clinical, and veterinary strains of Listeria monocytogenes were examined for the presence of plasmids. Twenty-five (20%) contained plasmids, which varied from 1.3 to 66 MDa in size. Of 10 strains of other Listeria species (L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, L. grayi, and L. murrayi) examined, seven (70%) contained plasmids, varying from 38 to 53 MDa. No strains with multiple plasmids were found. Plasmids of identical size were isolated from related strains in some, although not all, cases. The presence of a plasmid in a strain was not related to phenotypic characters of known extrachromosomal inheritance.     

Immobilization and Detection of Listeria monocytogenes

A procedure was developed for immobilization of Listeria monocytogenes cells on metal hydroxides coupled with detection and enumeration using an automated optical system. The results of the immobilization procedure (<1 h) and detection during overnight incubation agreed with calculated plate counts, and this technique is simple and rapid and provides samples that are ready for confirmation of the presence of the pathogen by rapid methods.     

Immobilization and detection of Listeria monocytogenes

A procedure was developed for immobilization of Listeria monocytogenes cells on metal hydroxides coupled with detection and enumeration using an automated optical system. The results of the immobilization procedure (<1 h) and detection during overnight incubation agreed with calculated plate counts, and this technique is simple and rapid and provides samples that are ready for confirmation of the presence of the pathogen by rapid methods.     

Increased ATPase activity is responsible for acid sensitivity of nisin-resistant Listeria monocytogenes ATCC 700302

The growth of the foodborne pathogen Listeria monocytogenes can be controlled by nisin, an antimicrobial peptide. A spontaneous mutant of L. monocytogenes shows both resistance to nisin and increased acid sensitivity compared to the wild type. Changes in the cell membrane correlated with nisin resistance, but the mechanism for acid sensitivity appears unrelated. When hydrochloric or lactic acid is added to cultures, intracellular ATP levels drop significantly in the mutant (P < 0.01) compared to the results seen with the wild type. Characterization of the F(0)F(1) ATPase, which hydrolyzes ATP to pump protons from the cell cytoplasm, shows that the enzyme is more active in the mutant than in the wild type. These data support a model in which the increased activity of the mutant ATPase upon acid addition depletes the cells' supply of ATP, resulting in cell death.     

Increased ATPase Activity Is Responsible for Acid Sensitivity of Nisin-Resistant Listeria monocytogenes ATCC 700302

The growth of the foodborne pathogen Listeria monocytogenes can be controlled by nisin, an antimicrobial peptide. A spontaneous mutant of L. monocytogenes shows both resistance to nisin and increased acid sensitivity compared to the wild type. Changes in the cell membrane correlated with nisin resistance, but the mechanism for acid sensitivity appears unrelated. When hydrochloric or lactic acid is added to cultures, intracellular ATP levels drop significantly in the mutant (P < 0.01) compared to the results seen with the wild type. Characterization of the F₀F₁ ATPase, which hydrolyzes ATP to pump protons from the cell cytoplasm, shows that the enzyme is more active in the mutant than in the wild type. These data support a model in which the increased activity of the mutant ATPase upon acid addition depletes the cells' supply of ATP, resulting in cell death.     

单核细胞增生李斯特菌抗酸应激机制

单核细胞增生李斯特菌(Listeria monocytogenes,简称单增李斯特菌)是重要的人畜共患食源性病原,在青贮饲料、发酵食品、宿主胃内以及巨噬细胞吞噬体内都会遭遇酸应激。该菌有多种抗酸应激系统,如F0F1-ATPase、谷氨酸脱羧酶(Glutamate decarboxylase system,GAD)、精氨酸脱亚胺酶(Arginine deiminase,ADI)、鲱氨酸脱亚胺酶(Agmatine deiminase,Ag DI)系统等。在环境pH(pHex)4.5条件下可维持其细胞内pH(pHi)稳态,在pHex 3.5时仍能存活;用温和酸应激(pHex 4.5)预处理单增李斯特菌,可以通过酸耐受反应(Acid tolerance response)提高其在致死性酸性环境中的存活率,这一过程受σB正调控,即σB激活可以保护单增李斯特菌应对多种环境应激。因此,σB可以作为新型抗菌药物的靶标。更为重要的是,弱酸性发酵食品要严格控制李斯特菌的污染,以降低消费者的感染风险。     

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.     

Biodiversity of the species Listeria monocytogenes and the genus Listeria

This review describes the Listeria monocytogenes genome sequences available today and their comparison with that of Listeria innocua and Listeria welshimeri by highlighting their characteristic features and common traits. The diversity present among them is analysed with emphasis on putative virulence and host-pathogen interaction related functions. Then large-scale studies comparing gene content of Listeria and how these studies contributed to typing applications will be discussed. Finally, evolutionary conclusions and future perspectives in Listeria genomics are presented.