TolC but not AcrB is essential for multidrug-resistant Salmonella enterica serotype Typhimurium colonization of chicks.

To study the role of the multidrug efflux system AcrAB-TolC in resistance of multidrug-resistant 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. 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 beta-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 x 10(3) to 1 x 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. 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 x 10(6) and 1.2 x 10(7) cfu for one wild-type S. Typhimurium DT104 strain and the acrB mutant, respectively, and were >1.0 x 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. 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.

Baucheron S ，Mouline C ，Praud K ，Chaslus-Dancla E ，Cloeckaert A ... -  《JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY》

Role of TolC and parC mutation in high-level fluoroquinolone resistance in Salmonella enterica serotype Typhimurium DT204.

To study the role of TolC and of parC mutation in high-level fluoroquinolone resistance in clonal clinical strains of Salmonella enterica serotype Typhimurium phage type DT204 (S. Typhimurium DT204). Deletion of the tolC gene (DeltatolC) was first performed in a susceptible S. Typhimurium DT104 strain lacking target gene mutations involved in fluoroquinolone resistance. P22 transduction was further used to transduce DeltatolC from this strain to a high-level fluoroquinolone-resistant S. Typhimurium DT204 strain carrying several target gene mutations, including one in parC (ciprofloxacin MIC of 32 mg/L). Deletion of tolC in the high-level fluoroquinolone-resistant S. Typhimurium DT204 strain resulted in the same decrease in resistance levels (16- to 32-fold) as shown previously for an acrB mutant of the same strain, suggesting that AcrAB-TolC is the main efflux system involved in high-level fluoroquinolone resistance of S. Typhimurium DT204 strains. In some S. Typhimurium DT204 DeltatolC transductants, concomitant loss of the parC (Ser-80-->Ile) mutation, located approximately 9.3 kb upstream of tolC, resulted in a further 16- to 32-fold decrease in resistance levels to fluoroquinolones and thus a hypersusceptible phenotype (ciprofloxacin MIC of 0.063 mg/L). The AcrAB-TolC efflux system, together with multiple target gene mutations, including the parC mutation, appear essential to confer high-level fluoroquinolone resistance in S. Typhimurium DT204.

Baucheron S ，Chaslus-Dancla E ，Cloeckaert A 《JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY》

Overexpression of the multidrug efflux operon acrEF by insertional activation with IS1 or IS10 elements in Salmonella enterica serovar typhimurium DT204 acrB mutants selected with fluoroquinolones.

High-level fluoroquinolone (FQ) resistance in Salmonella enterica serovar Typhimurium phage type DT204 has been previously shown to be essentially due to both multiple target gene mutations and active efflux by the AcrAB-TolC efflux system. In this study we show that in intermediatly resistant acrB-inactivated serovar Typhimurium DT204 mutants, high-level resistance to FQs can be restored on in vitro selection with FQs. In each FQ- resistant mutant selected from serovar Typhimurium DT204 acrB mutant strains, an insertion sequence (IS1 or IS10) was found integrated upstream of the acrEF operon, coding for AcrEF, an efflux pump highly homologous to AcrAB. In one of the strains, transposition of IS1 caused partial deletion of acrS, the putative local repressor gene of the acrEF operon. Sequence analysis showed that both IS1 and IS10 elements contain putative promoter sequences that might alter the expression of adjacent acrEF genes. Indeed, reverse transcription-PCR experiments showed an 8- to 10-fold increase in expression of acrF in these insertional mutants, relative to their respective parental strain, which correlated well with the resistance levels observed to FQs and other unrelated drugs. It is noteworthy that AcrEF did not contribute to the intrinsic drug resistance of serovar Typhimurium, since acrF deletion in wild-type strains did not result in any increase in drug susceptibility. Moreover, deletion of acrS did not cause any acrF overexpression or any decrease in drug susceptibility, suggesting that acrEF overexpression is mediated solely by the IS1 and IS10 promoter sequences and not by inactivity of AcrS. Southern blot experiments showed that the number of chromosomal IS1 and IS10 elements in the serovar Typhimurium DT204 genome was about 5 and 15 respectively. None were detected in epidemic serovar Typhimurium DT104 strains or in the serovar Typhimurium reference strain LT2. Carrying IS1 and/or IS10 elements in their chromosome may thus be a selective advantage for serovar Typhimurium DT204 strains as opposed to DT104 strains for which no high-level FQ resistance nor insertional mutations were found. Taken together, the results of the present study indicate that the IS1- or IS10- activated AcrEF efflux pump may relay AcrAB in serovar Typhimurium, and underline the importance of transposable elements in the acquisition of FQ and multidrug resistance.

Olliver A ，Vallé M ，Chaslus-Dancla E ，Cloeckaert A ... -  《-》

TolC, but not AcrB, is involved in the invasiveness of multidrug-resistant Salmonella enterica serovar Typhimurium by increasing type III secretion system-1 expression.

The AcrAB-TolC efflux system is involved in multidrug and bile salt resistances. In addition, this pump has recently been suggested to increase the invasion of Salmonella enterica serovar Typhimurium (S. Typhimurium) into host cells in vitro and could therefore have an important clinical relevance for multidrug-resistant strains. The aim of this study was to investigate the role of the TolC outer membrane channel and the AcrB transporter in the interaction of multidrug-resistant S. Typhimurium strains with eukaryotic cells, especially in relation to the expression of the type III secretion system-1 (TTSS-1) required for Salmonella invasion. Deletion of tolC led to a reduced transcription of the Salmonella pathogenicity island-1 genes sipA, invF and hilA, demonstrating that all genes required for TTSS-1 biosynthesis are down-regulated in this mutant. Consequently, tolC mutants secreted smaller amounts of the TTSS-1 effector proteins SipA and SipC, and invasion tests performed with one mutant showed that it was significantly less able to invade HT-29 epithelial cells than its parental strain. This control seems specific to the TTSS-1 among the three TTSS of Salmonella as no down-regulation of expression of TTSS-2 or flagella was observed in this mutant. By contrast, acrB mutants behaved as their parents except that they secrete a slightly greater amount of SipA and SipC proteins. These data indicate that TolC but not AcrB mediates the uptake of multidrug-resistant S. Typhimurium into target host cells. Therefore, this role of TolC in the invasion of the intestine in addition to its role in bile salt resistance reinforces the interest of targeting TolC for fighting multidrug-resistant Salmonella.

Virlogeux-Payant I ，Baucheron S ，Pelet J ，Trotereau J ，Bottreau E ，Velge P ，Cloeckaert A ... -  《-》

AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar typhimurium DT104.

Multidrug-resistant Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) strains harbor a genomic island, called Salmonella genomic island 1 (SGI1), which contains an antibiotic resistance gene cluster conferring resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracyclines. They may be additionally resistant to quinolones. Among the antibiotic resistance genes there are two, i.e., floR and tet(G), which code for efflux pumps of the major facilitator superfamily with 12 transmembrane segments that confer resistance to chloramphenicol-florfenicol and the tetracyclines, respectively. In the present study we determined, by constructing acrB and tolC mutants, the role of the AcrAB-TolC multidrug efflux system in the multidrug resistance of several DT104 strains displaying additional quinolone resistance or not displaying quinolone resistance. This study shows that the quinolone resistance and the decreased fluoroquinolone susceptibilities of the strains are highly dependent on the AcrAB-TolC efflux system and that single mutations in the quinolone resistance-determining region of gyrA are of little relevance in mediating this resistance. Overproduction of the AcrAB efflux pump, as determined by Western blotting with an anti-AcrA polyclonal antibody, appeared to be the major mechanism of resistance to quinolones. Moreover, chloramphenicol-florfenicol and tetracycline resistance also appeared to be highly dependent on the presence of AcrAB-TolC, since the introduction of mutations in the respective acrB and tolC genes resulted in a susceptible or intermediate resistance phenotype, according to clinical MIC breakpoints, despite the presence of the FloR and Tet(G) efflux pumps. Resistance to other antibiotics, ampicillin, streptomycin, and sulfonamides, was not affected in the acrB and tolC mutants of DT104 strains harboring SGI1. Therefore, AcrAB-TolC appears to direct efflux-mediated resistance to quinolones, chloramphenicol-florfenicol, and tetracyclines in multidrug-resistant S. enterica serovar Typhimurium DT104 strains.

Baucheron S ，Tyler S ，Boyd D ，Mulvey MR ，Chaslus-Dancla E ，Cloeckaert A ... -  《-》