To identify putative new virulence factors of avian pathogenic Escherichia coli (APEC) strains, a genomic subtraction was performed between the APEC strain MT512 and the non-pathogenic E. coli strain of avian origin EC79. Seventeen DNA fragments were cloned that were specific for the APEC strain. Among them, nine were identified that were more frequent among pathogenic than non-pathogenic isolates in a collection of 67 avian E. coli. Chromosome or plasmid location, and the nucleotide sequence of these nine fragments were characterized. Four fragments were plasmid-located. The nucleotide sequence of two of them exhibited identity with the sequence of the RepF1B replicon of E. coli plasmids, and the amino-acid deduced sequences from the two other fragments exhibited similarity to the products of genes sitA of Salmonella Typhimurium and iroD of E. coli, which are involved in iron metabolism. Of the five chromosome-located fragments, three were predicted to encode parts of proteins that were significantly homologous to previously described proteins: TktA (transketolase) of Haemophilus influenzae, a FruA (fructokinase) homologue of Listeria innocua and Gp2 (large terminal subunit) of phage 21. The putative products of the two other chromosome-located fragments were homologous to proteins with unknown functions: Z0255 of E. coli strain EDL933 (EHEC) and RatA of Salmonella Typhimurium strain LT2. Both these chromosomal fragments, whose presence is correlated with serogroups O1 and O2 and to the virulence of APEC strains belonging to these serogroups, are good candidates for being part of novel virulence determinants of APEC. Moreover, several fragments were shown to be located close to tRNA selC, asnT or thrW, which suggests they could be part of pathogenicity islands. Six fragments that were shown to be part of whole ORFs present in the APEC strain MT 512 were also present in extra-intestinal pathogenic E. coli (ExPEC) strains of human and animal origin. Thus, the putative novel virulence factors identified in this study could be shared by ExPEC strains of different origins.

A new chloramphenicol resistance gene from Salmonella typhimurium DT104, designated floR, also conferring resistance to florfenicol, was characterized. Sequence analysis of the deduced FloR protein suggested that it belongs to the 12-TMS (transmembrane segments) multidrug efflux pumps family. The floR gene, and the downstream sequenced tetR and tetA tetracycline resistance genes, were surrounded by two class 1 integrons. The first one contained the resistance gene aadA2 and a deleted sulI resistance gene. The second one contained the beta-lactamase gene pse1 and a complete sulI gene. Thus, the floR gene is included in a multiresistance locus of at least 12.5 kb. Its particular organization and chromosomal location could be involved in the antibioresistance pattern stability of the DT104 Salmonella typhimurium strains.

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The prevalence of resistance to florfenicol, a phenicol drug newly introduced in veterinary therapy, was determined in 86 chloramphenicol-resistant Salmonella Typhimurium isolates from cattle collected during 1985-1995. All were highly resistant to chloramphenicol (MICs >128 mg=L) and 38 were simultaneously resistant to florfenicol (MICs >16 mg=L) and to â-lactam agents, spectinomycin, streptomycin, sulphonamides and tetracyclines. The isolates susceptible to florfenicol harboured the chloramphenicol acetyl transferase gene, cat of type I. All the florfenicol-resistant isolates harboured the floR resistance gene and the characteristic multiple resistance genetic locus, previously characterised in a S. Typhimurium DT104 strain and identified by a multiplex PCR. Plasmid profiles and ribotype patterns were determined for all the isolates. The florfenicol-resistant isolates were grouped into the same ribotyping pattern and presented similar plasmid profiles, whereas the florfenicol-susceptible isolates showed a wider genetic diversity that is usual for S. Typhimurium. Thus, the florfenicolresistant isolates could represent a clonal cluster, closely related to, if not of DT104 phage type, which appeared in 1989 and is now predominant within chloramphenicolresistant S. Typhimurium. The multiplex PCR provided a useful tool to survey further evolution of multiresistant S. Typhimurium strains.

Listeria monocytogenes is a food-borne pathogen with the ability to grow under conditions of high osmolarity. In a previous study, we reported the identification of 12 proteins showing high induction after salt stress. One of these proteins is highly similar to the general stress protein Ctc of Bacillus subtilis. In this study, induction of Ctc after salt stress was confirmed at the transcriptional level by using RNA slot blot experiments. To explore the role of the ctc gene product in resistance to stresses, we constructed a ctc insertional mutant. No difference in growth was observed between the wild-type strain LO28 and the ctc mutant either in rich medium after osmotic or heat stress or in minimal medium after heat stress. However, in minimal medium after osmotic stress, the growth rate of the mutant was increased by a factor of 2. Moreover, electron microscopy analysis showed impaired morphology of the mutant grown under osmotic stress conditions in minimal medium. Addition of the osmoprotectant glycine betaine to the medium completely abolished the osmotic sensitivity phenotype of the ctc mutant. Altogether, these results suggest that the Ctc protein of L. monocytogenes is involved in osmotic stress tolerance in the absence of any osmoprotectant in the medium.Listeria monocytogenes is a food-borne pathogen widely distributed in the environment. This microorganism is of particular concern in the food industry because of its ability to survive and frequently to grow under a wide range of adverse conditions used to preserve food such as low temperature, low pH, and high osmolarity (8). Growth of L. monocytogenes has been reported at NaCl concentrations as high as 10% (21).Most bacteria cope with elevated osmolarity in the environment by intracellular accumulation of compatible solutes, called osmolytes (32). Among the compatible solutes efficient in L. monocytogenes, two quaternary amines, glycine betaine and carnitine, are the most effective (5, 34). The accumulation of these osmoprotectants in L. monocytogenes occurs through osmotic activation of their transport from the medium rather than through de novo synthesis. Accumulation of glycine betaine and carnitine occurs via at least two glycine betaine transporters encoded by the betL gene and the gbu operon (18, 30) and one carnitine transporter encoded by the opuC operon (2, 9, 33). A betL knockout mutant and a mutant of gbu obtained by transposition were significantly affected in their abilities to accumulate glycine betaine and were unable to withstand concentrations of salt as high as the isogenic parent strain can withstand (18,30). Similarly, a mutant with an insertional inactivation of opuCA was defective in the uptake of carnitine and had impaired growth at high osmolarity (9, 33). Proline has been identified as an osmolyte for L. monocytogenes. The proline transport mechanism has not been characterized yet. However, the proBA operon, coding for the enzymes that catalyze the two first steps of proline biosynthesis, has recently been identified. Disru...