Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentationnegative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.

ABSTRACTThe persistence ofListeria monocytogenesin food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1,cadA2, andcadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated:cadA1was more common in isolates of serotypes 1/2a and 1/2b than 4b, whilecadA2was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups ofL. monocytogenes, including exposures to heavy metals and disinfectants.

The atypical hemolytic Listeria innocua strains PRL/NW 15B95 and J1-023 were previously shown to contain gene clusters analogous to the pathogenicity island (LIPI-1) present in the related foodborne gram-positive facultative intracellular pathogen Listeria monocytogenes, which causes listeriosis. LIPI-1 includes the hemolysin gene, thus explaining the hemolytic activity of the atypical L. innocua strains. No other L. monocytogenesspecific virulence genes were found to be present. In order to investigate whether any other specific L. monocytogenes genes could be identified, a global approach using a Listeria biodiversity DNA array was applied. According to the hybridization results, the isolates were defined as L. innocua strains containing LIPI-1. Surprisingly, evidence for the presence of the L. monocytogenes-specific inlA gene, previously thought to be absent, was obtained. The inlA gene codes for the InlA protein which enables bacterial entry into some nonprofessional phagocytic cells. PCR and sequence analysis of this region revealed that the flanking genes of the inlA gene at the upstream, 5-end region were similar to genes found in L. monocytogenes serotype 4b isolates, whereas the organization of the downstream, 3-end region was similar to that typical of L. innocua. Sequencing of the inlA region identified a small stretch reminiscent of the inlB gene of L. monocytogenes. The presence of two clusters of L. monocytogenes-specific genes makes it unlikely that PRL/NW 15B95 and J1-023 are L. innocua strains altered by horizontal transfer. It is more likely that they are distinct relics of the evolution of L. innocua from an ancestral L. monocytogenes, as postulated by others.Listeria innocua is a species that is ubiquitously distributed in the natural environment, and unlike L. monocytogenes, represents an example of a nonharmful, nonhemolytic saprophytic Listeria sp. L. innocua has been isolated from a variety of environmental sources, including surface water, soil, sewage, vegetation, and food-processing plants. According to current knowledge, L. innocua does not carry the virulence-associated genes or clusters present and described in the genomes of the pathogenic Listeria species, L. monocytogenes and L. ivanovii. However, several unusual L. monocytogenes-like hemolytic L. innocua strains were isolated and phenotypically and genetically characterized (19). An example is the food isolate PRL/NW 15B95, a naturally but atypically hemolytic L. innocua strain (19). We have shown previously that this L. innocua strain contains the L. monocytogenes PrfA-regulated pathogenicity island 1 (LIPI-1) gene cluster. LIPI-1 of strain PRL/NW 15B95 is inserted in a background in which L. innocua genes preponderate. The initial characterization of this strain did not allow a definitive conclusion about how it evolved, although the results indicated horizontal transfer of this island, as other, non-LIPI-1 genes of L. monocytogenes were absent. Thus, importation of the cluster into an ancestral L. innocua strain rather than v...

Spores of Bacillus and Clostridium species were subjected to oxidizing and reducing agents known to rupture protein disulphide bonds and to irradiation (ultraviolet ; high speed electron). These treatments caused no loss of brightness of spores when viewed by phase-contrast microscopy. However, lysozyme or hydrogen peroxide caused phase darkening of treated spores and loss of dipicolinic acid typical of normal germination except that hydrogen peroxide eventually caused almost complete lysis of the spores. Under certain conditions, spore viability was unaffected during treatment with reducing agents and during subsequent phase darkening in lysozyme. Spores made susceptible to lysozyme by reducing agents became insusceptible after storage in aerated water. These reactions are compatible with Vinter's observation of the high content of disulphide bonds in the coat fraction of spores (Vinter, 1960) and with the chemical or physical rupture of these bonds. Rupture of disulphide bonds allows action of lysozyme or hydrogen peroxide on previously protected substrates. The disulphide bonds are therefore probably important in the resistance of spores to enzymes and irradiation, and rupture of these bonds may be involved in the germination process. The probable location of the lysozyme substrate in mucopeptide of the spore cortex indicates the importance of the integrity of cortex structure in maintaining the phase brightness of spores.

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