Search

D.J.-M. VIDON, S. DONZE, C. MULLER, A. ENTZMANN AND P. ANDRE. 2001. 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. Conclusions: 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.

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.

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.

Ninety samples of honey produced in eastern France, and 26 samples of honey originating from various countries, were analyzed for the presence of Clostridium botulinum spores. Although eight samples were found positive when using the mouse toxicity test, none of the strains isolated from the positive samples were identified as C. botulinum. This result is consistent with the hitherto rare occurrence of cases of infant botulism reported in France.

All Enterococcus spp., isolated from environmental water samples (n = 81), emitted a high chemiluminescence signal in the presence of luminol (10 � 2 M). Kinetic studies of chemiluminescence show a close correlation between chemiluminescence and growth curves during the exponential phase, with a maximum chemiluminescence reached just before bacterial growth entered in the stationary phase. On the other hand, genera closely related to Enterococcus such as Streptococcus or Lactococcus produced a very weak chemiluminescent signal. Chemiluminescence of enterococci could therefore offer a rapid test, in aiding the identification of the genus Enterococcus and in the survey of the microbiological quality of water supplies. � 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Listeria monocytogenes and Listeria innocua are able, under certain conditions, to produce chemiluminescence (CL), which is amplified by luminol. Kinetic studies of CL by L. monocytogenes and L. innocua show a close parallelism between CL and growth curves during the exponential phase, with a maximum of CL reached just before entrance of bacteria into the stationary phase. CL is tightly correlated with the release of oxygen compounds. The reactive oxygen species scavengers tryptophan, mannitol, and tiron, as well as cellobiose and high temperature, were assessed with regard to CL in the two Listeria species. Only tiron strongly reduced the CL emitted by L. monocytogenes and L. innocua. On the other hand, charcoal pretreatment of the growth medium inhibited the CL, whereas ferric citrate strongly increased the CL of L. monocytogenes and L. innocua. These data suggest that iron and superoxide radical are implicated in the CL produced by these bacteria, but this phenomenon is not correlated to virulence.

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.

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.Key words: Yersinia enterocolitica, mercury resistance, mercuric reductase.

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.

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.

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.