A simple chemiluminescence-based method for rapid enumeration of Listeria spp. microcolonies.

AIMS: Listeria monocytogenes is capable, under certain conditions, of producing chemiluminescence which is amplified by luminol. This property was used to detect and count microcolonies of Listeria spp. in a few hours, without the use of a microscope. METHODS AND RESULTS: After trapping Listeria cells on polyvinylidene fluoride membranes, a chemiluminescence mixture was sprayed onto the membrane. The chemiluminescent spots emitted were analysed by a charge-coupled device camera connected to a data-processing system, which restored the intensity of the signals into three dimensional images. The intensity of the luminescence of microcolonies was improved by addition of cellobiose, and by brief exposure to u.v. light. CONCLUSION: Microcolonies of Listeria spp. can be imaged and counted by luminol-enhanced chemiluminescence with a photon-counting system. SIGNIFICANCE AND IMPACT OF THE STUDY: This method can be applied to the rapid detection and counting of Listeria spp. in raw milk.

Effect of siderophores, catecholamines, and catechol compounds on Listeria spp. Growth in iron-complexed medium.

Almost all bacteria require iron for growth and virulence expression. However, Listeria spp. do not produce any siderophore for iron acquisition. Representative strains of each of the six species of Listeria were examined for their ability to use various compounds as iron suppliers in iron-restricted medium. Here we show that L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, and L. grayi were able to use exogenous siderophores and various catechol ligands, including catecholamines, to overcome growth inhibition induced by tropolone, an iron chelating agent. In contrast, no growth promoting effect was observed with normetanephrine or 4-hydroxy-3-methoxyphenylglycol-piperazine salt, which indicates that the o-diphenol function of the ligand must be free to allow iron acquisition. Furthermore, we demonstrate that catecholamines do not act through specific bacterial receptors, because no difference in growth stimulation was observed between [+]- and [-]-norepinephrine. These results show that utilization of a variety of catechol compounds to acquire iron is a general phenomenon in the genus Listeria.

Utilization of iron-catecholamine complexes involving ferric reductase activity in Listeria monocytogenes.

Listeria monocytogenes is a ubiquitous potentially pathogenic organism requiring iron for growth and virulence. Although it does not produce siderophores, L. monocytogenes is able to obtain iron by using either exogenous siderophores produced by various microorganisms or natural catechol compounds widespread in the environment. In the presence of tropolone, an iron-chelating agent, growth of L. monocytogenes is completely inhibited. However, the growth inhibition can be relieved by the addition of dopamine or norepinephrine under their different isomeric forms, while the catecholamine derivatives 4-hydroxy-3-methoxyphenylglycol and normetanephrine did not relieve the inhibitory effect of tropolone. Preincubation of L. monocytogenes with chlorpromazine and yohimbine did not antagonize the growth-promoting effect of catecholamines in iron-complexed medium. In addition, norepinephrine stimulated the growth-promoting effect induced by human transferrin in iron-limited medium. Furthermore, dopamine and norepinephrine allowed 55Fe uptake by iron-deprived bacterial cells. The uptake of iron was energy dependent, as indicated by inhibition of 55Fe uptake at 0 degrees C as well as by preincubating the bacteria with KCN. Inhibition of 55Fe uptake by L. monocytogenes was also observed in the presence of Pt(II). Moreover, when assessed by a whole-cell ferric reductase assay, reductase activity of L. monocytogenes was inhibited by Pt(II). These data demonstrate that dopamine and norepinephrine can function as siderophore-like compounds in L. monocytogenes owing to their ortho-diphenol function and that catecholamine-mediated iron acquisition does not involve specific catecholamine receptors but acts through a cell-bound ferrireductase activity.

Esculetin antagonizes iron-chelating agents and increases the virulence of Listeria monocytogenes.

Iron is an essential compound for the growth and virulence of Listeria monocytogenes. In extracellular environments, iron often requires a siderophore to be acquired by microorganisms. Although it does not produce siderophores, L. monocytogenes can use some exogenous bacterial or fungal siderophores as well as a number of animal or plant o-diphenol compounds to overcome growth inhibition by the iron-chelating agents tropolone and 8-hydroxyquinoline. Esculin, a plant glycoside, can be hydrolysed by L. monocytogenes to the o-diphenol aglucon, esculetin. The latter neutralized in vitro growth inhibition induced by the iron-chelating agents. Furthermore, when injected into infected mice, esculetin enhanced mortality in a dose-dependent manner and increased bacterial counts in spleen induced by sublethal doses of L. monocytogenes. Esculetin apparently functioned as a siderophore for L. monocytogenes in murine tissues.

Utilization of exogenous siderophores and natural catechols by Listeria monocytogenes.

Listeria monocytogenes does not produce siderophores for iron acquisition. We demonstrate that a number of microbial siderophores and natural iron-binding compounds are able to promote the growth of iron-starved L. monocytogenes. We suggest that the ability of L. monocytogenes to use a variety of exogenous siderophores and natural catechols accounts for its ubiquitous character.

Luminol-enhanced chemiluminescence by Listeria monocytogenes: a test for rapid assessment of antimicrobial agents.

Listeria monocytogenes produces chemiluminescence in brain heart infusion broth at 37 degrees C in the presence of carbonate ions and acetaldehyde. This phenomenon can be enhanced by the use of luminol rather than acetaldehyde. Furthermore, there is direct relationship between the extent of growth and the level of luminescence which culminates at the end of the exponential growth. This property was used to study the susceptibility of this bacterium to two antiseptics, cetrimonium bromide and chlorhexidine, and to two antibiotics, ampicillin and chloramphenicol. Inhibition of chemiluminescence was proportional to the antimicrobial agents' concentrations and was complete at their minimal inhibitory concentrations.

Purification and properties of mercuric reductase from Yersinia enterocolitica 138A14.

A mercuric ion-reducing flavoprotein was purified from Yersinia enterocolitica 138A14 using dye matrix affinity chromatography. The purified enzyme had a characteristic absorption spectrum similar to those of flavin compounds, and FAD was detected as a part of the purified enzyme by thin-layer chromatography. Freshly purified preparations of the enzyme showed a single band on SDS polyacrylamide gel electrophoresis with a molecular weight of 70,000. The isolated enzyme had a molecular weight of about 200,000 as determined by gel filtration and disc gel electrophoresis. These results suggest an apparently trimeric structure of the enzyme. Dithiothreitol treatment disrupted the trimer into a dimeric structure of 140,000. Along with ageing, as well as limited proteolytic digestion, the enzyme evolved to give a dimeric molecule of 105,000 composed of two identical subunits of 52,000. The combination of the purified enzyme with HgCl2, or unexpectedly with merthiolate, oxidised the NADPH, which was followed spectrophotometrically. The Km for HgCl2 was dependent on the concentration of exogenous thiol compounds. A comparison of physical properties as well as kinetic characteristics indicated that the enzyme from Y. enterocolitica 138A14 is similar to mercuric reductases isolated from other mercury-resistant bacteria.

Essential arginines in mercuric reductase isolated from Yersinia enterocolitica 138A14.

The mercuric reductase from Yersinia enterocolitica 138A14 was inactivated by the arginine modifying reagents 2,3-butanedione and phenylglyoxal. The inactivation by 2,3-butanedione exhibited second order kinetics with rate constant of 32 min-1 M-1. In the case of phenylglyoxal, biphasic kinetics were observed. The oxidized coenzyme (NADP+) prevented inactivation of the enzyme by the alpha-dicarbonyl reagents, whereas the reduced coenzyme (NADPH) enhanced the inactivation rate. The loss of enzyme activity was related to the incorporation of [2-14C] phenylglyoxal; when two arginines per subunit were modified the enzyme was completely inactivated.

Relationships between beta-lactamase production and beta-lactam susceptibility of environmental strains of Yersinia kristensenii.

Strains of Yersinia kristensenii display high susceptibility to carbenicillin (MIC90 less than 8 micrograms/ml) in comparison with the majority of environmental strains of Yersinia closely related to Y. enterocolitica which are resistant to this antibiotic (MIC90 greater than 256 micrograms/ml). beta-lactamases of 39 strains of Y. kristensenii isolated from foods were analysed by isoelectric focusing and gel electrophoresis of ultrasonically disrupted uninduced cultures. beta-lactamase patterns showed the presence of only one out of three classes of enzymes of pI 6.7, 7.6 and 8.2, respectively, by strain. One beta-lactamase showed electrophoretic mobility different (EM + 2.0 cm/h) from that of all the other enzymes (EM + 1.6 cm/h) belonging to the class of pI 7.6. Induction by cefoxitin revealed the existence of inducible beta-lactamases in two out of eight selected strains. The substrate profile of these enzymes, which are probably chromosomally mediated, showed a predominant cephalosporinase activity. None of the type A and B beta-lactamases described by Cornelis and Abraham in Y. enterocolitica were found in any of the strains examined. The lack of beta-lactamase A (a penicillinase) accounts for the carbenicillin susceptibility of Y. kristensenii strains.

Characteristics of the yellow pigment from a strain of Yersinia enterocolitica biovar 1.

Yersinia enterocolitica 195A14J, a milk-isolated strain of biovar 1, produces a non-diffusible yellow pigment and forms star-shaped colonies when grown on egg-yolk agar at 28 degrees C. Solubility properties and in situ Raman spectrum of the pigment support evidence that it is not a carotenoid, although it contains a 9 (+/- 1) double-bond polyenic chain. Pigmentless variants, also star-shaped, appeared with a frequency of ca.10(-3) when bacteria were grown at 38 degrees C. Agarose gel electrophoresis of DNA extracted from the pigmented strain revealed the presence of a 42-kb plasmid which was lost in pigmentless variants.

Isolation of Yersinia enterocolitica and related species from foods in France.

The occurrence of Yersinia enterocolitica and related species (Y. intermedia, Y. frederiksenii, Y. kristensenii) in foods from France was investigated by using different enrichment procedures. Initially, seven procedures were evaluated with pork products. These methods included a cold preenrichment in yeast extract-rose bengal broth or in phosphate-sorbitol-bile medium, followed by selective enrichment either in Pastone-sucrose-Tris-azide broth, in modified Rappaport broth, or in bile-oxalate-sorbose broth, and then isolation onto Hektoen or cefsulodin-irgasan-novobiocin agar with or without KOH pretreatment. The best enrichment procedure in terms of percentage of positive samples obtained within the shortest time was the combination of phosphate-sorbitol-bile and bile-oxalate-sorbose with alkali treatment before isolation onto cefsulodin-irgasan-novobiocin agar. This system was then used to analyze foods other than pork. An average contamination rate of 33.5% was observed for 666 samples analyzed; pork products were by far the most contaminated, especially the so-called tartinette (96.8% of positive samples) which contained up to five different strains of Yersinia spp. Environmental serogroups of Y. enterocolitica O:5, O:39,41, O:6, and O:7,8 were predominant, but no isolate of either human pathogenic type (O:3 or O:9) was obtained.

Antimicrobial susceptibilities of food-isolated strains of Yersinia enterocolitica, Y. intermedia, Y. frederiksenii, and Y. kristensenii.

The in vitro antimicrobial susceptibility of Yersinia enterocolitica and newly related species isolated from foods was examined. Only 4 of 375 isolates displayed resistance to non-ss-lactam antibiotics. MICs of ampicillin and carbenicillin determined by agar dilution with respect to 125 isolates showed the high susceptibility of Y. kristensenii and biovar 3 of Y. enterocolitica to carbenicillin (MIC for 90% of the strains, less than or equal to 8 micrograms/ml).

Characterization of Tn3926, a new mercury-resistance transposon from Yersinia enterocolitica.

A new transposon coding for mercury resistance (HgR), Tn3926, has been found in a strain of Yersinia enterocolitica, YE138A14. The element has a size of 7.8 kb and transposes to conjugative plasmids belonging to different incompatibility groups. A restriction map has been established. DNA-DNA hybridization indicates that Tn3926 displays homology with both Tn501 and Tn21; the greatest homology is shown with the regions of these transposons that encode HgR. Weaker homology is observed between Tn3926 sequences and those regions of Tn501 and Tn21 that encode transposition functions. Complementation experiments indicate that the Tn3926 transposase mediates transposition of Tn21, albeit somewhat inefficiently, but not of Tn501, while the resolvase mediates resolution of transposition cointegrates formed via Tn21, Tn501, or Tn1721.

Mercury resistance in Yersinia enterocolitica.

First isolation of a mercury-resistant strain of Yersinia enterocolitica is reported. This strain, named 138-A14, is resistant to mercuric chloride and merbromin, but sensitive to phenylmercuric borate and sodium merthiolate. The mercury resistance of 138-A14 is not transferable spontaneously to Escherichia coli K12 by conjugation.

Incidence of Yersinia enterocolitica in raw milk in eastern France.

A total of 75 raw milk samples collected from a central dairy or from retailers in Alsace, France, were analyzed for the presence of Yersinia enterocolitica. Three procedures were used: enrichment at 4 degrees C for 1 month; enrichment in modified Rappaport medium at room temperature for 72 h after a preenrichment at 4 degrees C for 1 month; and enrichment in a new medium containing sucrose, tris(hydroxymethyl)aminomethane, sodium azide, and ampicillin (PSTA) at 28 degrees C for 48 h after a preenrichment at 4 degrees C for 1 month. Isolation of Y. enterocolitica was made on Hektoen medium plus ampicillin. Sixty-one samples were positive (81.4%), but the PSTA medium produced the greatest number of isolates. Biochemical, serological, and phage typing of 40 isolates showed that chemotype 1 and serogroup O:5 were predominant. In seven cases, two different strains were obtained from the same samples. Most of the 66 isolates tested for their antimicrobial susceptibility were resistant to ampicillin and carbenicillin, and all were sensitive to tetracycline, chloramphenicol, streptomycin, sulfonamides, and mercuric ions.

[Influence of temperature, pH and glucidic substrates on the growth and conidiogenesis of Arthroderma (trichophyton) simii Stockdale]. / Influence de la température, du pH et du substrate glucidique sur la croissance et la conidiogenese de l'Arthroderma (Trichophyton) simii Stockdale.

Growth and conidiogenesis of the (+) IMI 101 693 and (-) IMI 101 695 strains of Trichophyton simii were compared at several temperatures (20 to 40 degrees C) and various pH (pH 4 to 9) and were correlated with the concentration of glucose (20 to 100 g/l) or other glucidic substrates (10 g/l). Although the development of both strains was optimal between 25 to 33 degrees C at pH 6 and with 10 g/l of glucose, the (-) strain always growed less than the (+) strain. Conidiogenesis was inhibited by sucrose in the (-) strain and by lactose in the (+) strain.

Incidences of simple and transferable drug resistance in Escherichia coli and Salmonella isolated from various foods: identification of a R plasmid in S. saint-paul.

A study of 1,043 strains of E. coli and 22 strains of Salmonella isolated from various foods in eastern France between the dates of June 1974 and December 1976 showed that 164 (15.6%) of E. coli and nine Salmonella were resistant to one or several drugs. Only 18 of the 83 resistant E. coli strains were able to transfer their resistance to tetracycline and some other drug resistance to E. coli K12 in vitro. A conjugative R plasmid has been identified by transfer experiments, incompatibility testing and agarose gel electrophoresis in a strain of multiresistant S. saint-paul.