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This work was supported by the Institut National pour la Recherche Agronomique (INRA) as an INRA-JSPS (Japan Society for the Promotion of Science) 2010-2012 joint research project.; International audience; Background. Fluoroquinolone (FQ) resistance is increasing worldwide among Salmonella spp. Among the mechanisms involved, increased efflux via the tri-partite AcrAB-TolC efflux system is mainly modulated through control of expression via the ramRA regulatory locus gene products. Interestingly, in some reference strains these have also been experimentally shown to regulate cell invasion-related genes of the type III secretion system-1 (T3SS-1). In this study we investigated whether natural mutations occurring in this locus in FQ-resistant S. enterica serovar Typhimurium (S. Typhimurium) epidemic clones resulted in the same effects. Methods. qRT-PCR and cell invasion assays were used to study three clinical FQ-resistant S. Typhimurium isolates representative of the DT104 and DT204 epidemic clones. For comparison three control reference quinolone-susceptible strains were included. Results. As previously shown, the investigated mutations altering RamR or its DNA binding site increased expression of efflux genes dependently on ramA. However, the decreased expression of T3SS-1 genes previously reported was not always observed and seemed to be dependent on the genetic background the FQ-resistant isolate. Indeed, a ramA-dependent decreased invasion of intestinal epithelial cells was only observed for a particular clinical ramR mutant. Conclusion. ramRA mutations occurring in clinical FQ-resistant S. Typhimurium isolates may negatively modulate their invasiveness but this is strain-dependent.

A multidrug-resistant mutant of Campylobacter jejuni was selected in vitro using increasing concentrations of enrofloxacin. This mutant accumulated less ethidium bromide than the parental strain, suggesting the participation of active efflux as a resistance mechanism. Inactivation of the cmeB gene confirmed active efflux and indicated the involvement of the CmeABC efflux pump in the multidrug resistance of the mutant. Sequencing of the cmeR-cmeA intergenic region revealed a point mutation in the binding site of the CmeR repressor. Transcriptional lacZ fusions showed an increase of transcription of the cmeABC operon in the multidrug-resistant mutant. Gel mobility shift assays and Surface Plasmon Resonance experiments further indicated a decrease in the affinity of the CmeR for the promoting region of the cmeABC operon consecutive to this mutation. Thus, these results showed that the point mutation was responsible, via a lack of binding of the CmeR repressor, for increased expression of the CmeABC efflux pump and consecutive multidrug resistance.

Objectives: To study the role of the multidrug efflux system AcrAB-TolC in resistance of multidrugresistant Salmonella enterica serotype Typhimurium (S. Typhimurium) phage type DT104 and DT204 strains to detergents and bile salts. To evaluate the importance of the inner membrane transporter AcrB and the outer membrane component TolC of this efflux system in the colonization of two multidrug-resistant S. Typhimurium DT104 and one DT204 strain in chicks. Methods: acrB and tolC mutants of multidrug-resistant S. Typhimurium DT104 and DT204 strains were constructed by insertional inactivation of the acrB gene and deletion of the tolC gene. MICs of detergent and bile salts were determined for the wild-type strains, the acrB and the tolC mutant strains, in presence and in absence of the efflux pump inhibitor Phe-Arg b-naphthylamide. The effect of sodium choleate on the in vitro growth of these strains was also evaluated. The LD50s of the strains were measured in a day-old chicken model, inoculated with several doses (1 3 10 3 to 1 3 10 8 cfu) by the oral route, for 7 days post-inoculation. The colonization levels were assessed at the sublethal dose 7 days post-inoculation by determining the number of cfu of Salmonella in the faeces, caecum, spleen and liver. Results: The decrease in resistance levels to bile salts was 64- to 256-fold higher for the tolC mutants than for the acrB mutants relative to those of the parental strains. Addition of choleate in culture medium did not affect the growth of the wild-type strains or that of the acrB mutants, but inhibited completely the growth of the tolC mutants. The LD50s were 1.0 3 10 6 and 1.2 3 10 7 cfu for one wildtype S. Typhimurium DT104 strain and the acrB mutant, respectively, and were >1.0 3 10 8 for the tolC mutants or the S. Typhimurium DT204 strains. In contrast to the acrB mutants, the tolC mutants were unable to colonize the caecum, spleen and liver after 1 week of infection. Moreover, in most chicks, no intestinal excretion was detected for the tolC mutants. The colonization levels of the acrB mutants were not significantly different from those of the wild-type strains. Conclusions: TolC but not AcrB appears to be essential for multidrug-resistant S. Typhimurium DT104 and DT204 colonization of chicks, which is in accordance with their respective roles in resistance to detergents and bile salts. Therefore, TolC could be a better target than AcrB for the development of efflux pump inhibitors.

Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously. We have characterized further the LX1054 mutant strain, the most sensitive of them. The chromosomal DNA segment flanking transposon insertion was cloned and sequenced. The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family. LX1054 exhibited a reduced capacity to colonize the intestinal tract. After passages in mice, the mutant strain lost the sensitive phenotype. In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents. Then, the acquired resistant phenotype seemed stable. The data suggested a role of S. typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization. However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S. typhimurium.

Florfenicol resistance has emerged over the past few years in multidrug-resistant Salmonella enterica serovars Typhimurium, Agona and Paratyphi B. The floR gene encoding florfenicol resistance is chromosomally located in these serovars within a genomic island of 43 kb called SGI1 (Salmonella genomic island 1). In the present study, we characterized florfenicol resistance in a strain of S. enterica serovar Newport isolated from a turkey in 1990 and that lacked SGI1. Florfenicol resistance was mediated by a conjugative plasmid related to R55 from Klebsiella pneumoniae, which was characterized initially in the 1970s and harbours a gene 95% identical to floR.

Sir, A chromosomal genomic island called Salmonella genomic island 1 (SGI1) has been initially described in epidemic multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium phage type DT104 strains (Salmonella Typhimurium DT104). SGI1 contains an antibiotic resistance gene cluster and most often confers resistance to ampicillin, chloramphenicol/ florfenicol, streptomycin/spectinomycin, sulphonamides and tetracyclines. The 43 kb SGI1 is located between the thdF and int2 genes of the chromosome of Salmonella Typhimurium. The int2 gene is part of a retron sequence that has been reported to date only in Salmonella Typhimurium. In other S. enterica serovars, SGI1 is located between the thdF and yidY genes. The antibiotic resistance gene cluster is located near the 30 end of SGI1 and constitutes a complex class 1 integron (Figure 1). At the first attI1 site of this complex integron, the cassette carries the aadA2 gene, which confers resistance to streptomycin and spectinomycin, and a 30-conserved segment (30-CS) with a truncated sul1 (sul1delta) gene is present. At the second attI1 site, the cassette contains the b-lactamase gene blaPSE-1 conferring resistance to ampicillin and the 30-CS comprises a complete sul1 gene conferring resistance to sulphonamides. Flanked by the two cassettes are the floR gene, which confers cross-resistance to chloramphenicol and florfenicol, and the tetracycline-resistance genes tetR and tet(G). SGI1 has been recently demonstrated to be an integrative mobilizable element. Variant SGI1 antibiotic resistance gene clusters have been described in a wide variety of S. enterica serovars. SGI1 variant antibiotic resistance gene clusters were accordingly classified from SGI1-A to SGI1-L. These gene clusters were probably generated after chromosomal recombinational events or by antibiotic resistance gene cassette replacement at one of the attI1 sites. We reasoned that MDR of Salmonella Derby isolated in Taiwan might be due to the spread of SGI1 and the presence of SGI1 antibiotic resistance gene clusters was thus investigated. The 22 MDR isolates of Salmonella Derby isolated in 2003 and 2004 from humans examined in this study were provided by the Center of Disease Control, Taiwan. Detection of SGI1, its location, and mapping of antibiotic resistance genes were performed by PCR on extracted genomic DNA using primers and conditions described previously (Figure 1). Among the 22 Salmonella Derby isolates examined, 13 isolates showed three different MDR resistance phenotypes indicative of the presence of SGI1 antibiotic resistance gene clusters. SGI1 was detected in these 13 isolates based on PCRs using primer pairs corresponding to the left junction and right junction of SGI1 in the Salmonella chromosome. PCR results were positive for the left junction of SGI1 with the chromosomal thdF gene and for the right junction with the chromosomal yidY gene, but negative for the junction with the int2 gene. Thus, the 13 Salmonella Derby strains were shown to harbour SGI1 at the same chromosomal location, i.e. between the thdF and yidY genes, as in non-Typhimurium serovars. In seven Salmonella Derby strains showing the typical Salmonella Typhimurium DT104 MDR phenotype, PCR mapping of the antibiotic resistance gene cluster showed the presence of fragments A, B, C, D, E, F and floR of the sizes expected (Figure 1). These isolates thus carried the SGI1 antibiotic resistance gene cluster classically found in MDR Salmonella Typhimurium DT104. For the five Salmonella Derby strains showing the chloramphenicol/florfenicol, streptomycin/spectinomycin, sulphonamides, tetracycline and trimethoprim MDR phenotype, PCR mapping yielded fragments A–D and floR. However, fragment E specific for the blaPSE-1 gene cassette was negative whereas a PCR using the forward primer of PCR E and the reverse primer of PCR H was positive, with a size slightly different from that expected from an SGI1-carrying Salmonella Typhimurium DT104 control strain, suggesting the presence of another gene cassette(s) at the second attI1 site of the SGI1 antibiotic resistance gene cluster. To confirm this result and to identify the gene cassette(s), PCR amplification of class 1 integrons using primers 50-conserved segment and 30-CS was performed as described previously. This PCR yielded two fragments, one of 1 kb corresponding to the aadA2 gene cassette and one of 1.3 kb, which was sequenced. Nucleotide sequencing identified the dfrA1 (conferring resistance to trimethoprim) and orfC (unknown function) gene cassettes. This array of gene cassettes was previously identified in the SGI1 antibiotic resistance gene cluster called SGI1-I of a Salmonella Derby human isolate from Malaysia. Thus, in these five Salmonella Derby strains the blaPSE-1 gene was replaced by the dfrA1 and orfC gene cassettes and corresponded to the SGI1-I antibiotic resistance gene cluster.