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To define the prevalence of antibiotic resistance in Listeria species pathogenic for humans and animals, 1100 isolates (60 from cases of listeriosis and 1040 from food and environment) collected worldwide were screened. Of the 61 tetracycline- and minocycline-resistant strains (37 Listeria monocytogenes), 57 harbored tet(M); 4 non-L. monocytogenes isolates contained tet(S). One Listeria innocua isolate was also resistant to streptomycin and contained the tet(M) and aad6 genes. An L. monocytogenes isolate was trimethoprim-resistant, a characteristic not reported previously in Listeria species, because of the presence of a yet-uncharacterized gene. Three clinical isolates of L. monocytogenes were resistant to low levels of streptomycin. Since the tet(M), tet(S), and aad6 genes are common in enterococci and streptococci, these data suggest transfer from the latter to Listeria species. Uniform susceptibility to tetracycline, minocycline, trimethoprim, and streptomycin cannot be assumed any longer for Listeria species.

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A 1427‐bp DNA fragment containing the kanamycin resistance gene, aphA‐3, of plasmid pIP1433 from Campylobacter coli was inserted into a shuttle vector. Full expression of aphA‐3 was obtained in Bacillus subtilis and in Escherichia coli. This DNA fragment was sequenced in its entirety and the starting point for aphA‐3 transcription in B. subtilis, C. coli and E. coli was determined by S1 nuclease mapping. The sequence of the promoter consists of the hexanucleotides TTGACA and TATAAT, with a spacing of 17 bp. The nucleotide sequence of the aphA‐3 gene from C. coli and from the streptococcal plasmid pJH1 are identical whereas they differ by two substitutions and deletion of a codon from that cloned from the staphylococcal plasmid pSH2. These results indicate a recent extension of the resistant gene pool of Gram‐positive cocci to Gram‐negative bacilli. From an analysis of the DNA sequences surrounding the promoter region, we concluded that the DNA fragment containing the aphA‐3 gene in plasmid pJH1 has evolved by deletions from a sequence similar to that found in plasmid pIP1433.

Five Listeria monocytogenes isolates (CLIP 21369, CLIP 73298, CLIP 74811, CLIP 75679, and CLIP 79372) were found to be resistant to fluoroquinolones during the screening for antibiotic resistance of 488 L. monocytogenes isolates from human cases of listeriosis in France. On the basis of a fourfold or greater decrease in the ciprofloxacin MIC in the presence of reserpine, fluoroquinolone resistance was attributed to active efflux of the drugs. The lde gene (Listeria drug efflux; formerly lmo2741) encodes a 12-transmembrane-segment putative efflux pump belonging to the major facilitator superfamily of secondary transporters that displayed 44% identity with PmrA from Streptococcus pneumoniae. Insertional inactivation of the lde gene in CLIP 21369 indicated that the corresponding protein was responsible for fluoroquinolone resistance and was involved in the level of susceptibility to dyes such as ethidium bromide and acridine orange.Listeria monocytogenes is widely distributed in the environment and can cause serious human infections, such as bacteremia and central nervous system infections, primarily in neonates and immunocompromised adults, and abortions (22). Food-borne transmission is recognized as the main route of acquisition of the infection during epidemic and sporadic listerioses (3, 18). Listeriosis differs from most food-borne diseases by its high fatality rate (20 to 30% of cases), despite the administration of appropriate antibiotics (3, 9). L. monocytogenes is generally susceptible to a wide range of antibiotics but is not susceptible to cephalosporins and fosfomycin (8). However, during the last few years, increasing numbers of strains resistant to one or more antibiotics have been reported (1,2,8,11,19,20).Although indications for the use of fluoroquinolones do not include listeriosis, they can, due to their increasing use for other pathologies such as respiratory tract infections, select resistant Listeria. In gram-positive bacteria, resistance usually results from mutational alterations in the so-called quinolone resistance-determining regions (QRDRs) of the intracellular targets of fluoroquinolones, the type II DNA topoisomerases gyrase and topoisomerase IV, or active export of the drugs via efflux pumps (12). We have detected ciprofloxacin-resistant L. monocytogenes clinical isolates and characterized the efflux pump involved in resistance.(An initial report of this work was presented at the 41st Interscience Conference on Antimicrobial Agents and Chemotherapy [S. Godreuil, M. Galimand, G. Gerbaud, and P. Courvalin, Abstr. 41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. UL-11, 2001].) MATERIALS AND METHODSStrains, plasmids, and growth conditions. Screening of 488 L. monocytogenes isolates responsible for human listeriosis in France for resistance to fluoroquinolones was carried out on brain heart infusion agar (Difco Laboratories, Detroit, Mich.) containing 4 g of ciprofloxacin per ml. Strains that were found to be resistant and susceptible L. monocytogenes EGD were studied ( Table 1)....

Inducible resistance to high levels of the glycopeptide antibiotics vancomycin and teicoplanin (VanA phenotype) in enterococci is mediated by Tn1546 (Fig. 1A) or closely related transposons which are located in self-transferable plasmids (6) or in the chromosome as part of large conjugative elements (13). Resistance to glycopeptides is due to production of peptidoglycan precursors ending in the depsipeptide D-alanyl-Dlactate (D-Ala-D-Lac) instead of the dipeptide D-Ala-D-Ala found in glycopeptide-susceptible bacteria (1,27). This substitution prevents formation of complexes between glycopeptides and peptidoglycan precursors at the cell surface that are responsible for inhibition of cell wall synthesis (9, 22). Tn1546 encodes a dehydrogenase (VanH) and a ligase (VanA) that synthesize D-Ala-D-Lac, and a D,D-dipeptidase (VanX) that hydrolyzes D-Ala-D-Ala and thereby limits synthesis of precursors containing the target of glycopeptides (9, 23, 30). In addition to these enzymes required for resistance, Tn1546 encodes a D,D-carboxypeptidase (VanY) which contributes to vancomycin resistance by hydrolyzing the C-terminal D-Ala residue of late peptidoglycan precursors and a protein of unknown function (VanZ) which confers low-level teicoplanin resistance (2, 4). These last two proteins are necessary for expression of resistance at a high level, since glycopeptides, in particular teicoplanin, remain partly active if the strains synthesize small amounts of D-Ala-D-Ala-ending precursors (3).Analysis of transcriptional fusions indicated that expression of the resistance genes in Tn1546 is activated by a two-component regulatory system (VanR-VanS) in response to the presence of glycopeptides in the culture medium (5). VanR is structurally related to response regulators of the OmpR subclass and is required for transcription initiation at promoter P H , which allows cotranscription of vanH, vanA, and vanX ( Previous attempts to evaluate the role of VanS in the modulation of VanR-mediated promoter activation were unsuccessful, since cloning of the regulatory genes on a multicopy plasmid vector led to constitutive expression of the vanHAX operon (5). In this report, regulation of the van gene cluster carried by natural plasmids was investigated by inserting a chloramphenicol acetyltransferase (cat) reporter gene into vanS or vanA. Strains of Enterococcus faecalis harboring a chromosomal copy of vanR or of vanR and vanS were also constructed to study transactivation of the P H and P R promoters located on plasmids. These complementary approaches

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