is an innately multidrug-resistant pathogen which is emerging in cystic fibrosis

is an innately multidrug-resistant pathogen which is emerging in cystic fibrosis (CF) patients. species exhibits innate resistance to many antibiotics, including cephalosporins (except ceftazidime), aztreonam, and aminoglycosides (1, 8C10). Clinical strains frequently harbor acquired resistances, especially to ceftazidime, ciprofloxacin, and carbapenems. We have recently described the first resistance-nodulation-cell division (RND)-type multidrug efflux pump in MexAB-OprM efflux pump: AxyABM can extrude cephalosporins (except cefepime), fluoroquinolones, and chloramphenicol. Moreover, AxyABM plays a major role in the innate resistance to aztreonam. Nevertheless, the mechanism(s) leading to aminoglycoside and cefepime resistance remain(s) unknown. It is likely that other efflux systems contribute to the antibiotic resistance of AXX-A strain (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AFRQ01000000″,”term_id”:”339120535″AFRQ01000000). We examined this sequence looking for homology with and from interacts with the periplasmic protein MexX and the outer membrane channel OprM that is encoded by the multidrug efflux operon. The expression of is complex and governed by several regulatory mechanisms. One of them is negative regulation by the product of the gene located upstream from (12, 16). By using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi), we detected 3 putative genes in the AXX-A genome (contig 71, GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AFRQ01000061.1″,”term_id”:”338780815″AFRQ01000061.1), designated gene. Susceptibility testing was performed by Etest (bioMrieux, Marcy l’Etoile, France) and interpreted according to the breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (http://www.eucast.org/clinical_breakpoints/). AXX-A harbors a wild-type antibiotic resistance phenotype, AXX-D an acquired resistance to ceftazidime and fluoroquinolones, and AXX-H an acquired resistance to ceftazidime, fluoroquinolones, and carbapenems (Table 1). In these strains, we have inactivated (nucleotide positions from 1696 to 2450). The 124182-57-6 IC50 PCR product was cloned into the pUC19 vector by using the In-Fusion HD cloning kit (Clontech Laboratories, Mountain View, CA) as recommended by the manufacturer. Newly constructed plasmids (pINA-axyY-AXX-A, pINA-axyY-AXX-D, and pINA-axyY-AXX-H) were used as suicide vectors. They were introduced into each strain by electroporation. Recombinant clones (in each strain was confirmed by PCR and DNA sequencing (primer pairs V-INA-axyY-F/M14R and M14F/V-INA-axyY-R). Table 1 MICs of 22 antibiotics for clinical strains and inactivation led to decreased MICs of aminoglycosides, carbapenems, cefepime, some fluoroquinolones, tetracyclines, erythromycin, and to a lesser extent, ceftazidime (Table 1). The activities of all aminoglycosides tested were substantially enhanced. Susceptibility to tobramycin, amikacin, netilmicin, and gentamicin was restored for all strains. The activities of carbapenems were slightly enhanced in the mutants AXX-A-Y and AXX-D-Y compared with their activities in the original strains, AXX-A and AXX-D. Interestingly, the MICs of meropenem and doripenem were decreased 6-fold and 10-fold, respectively, after disruption in the carbapenem-resistant strain AXX-H. This suggests that AxyXY-OprZ might lead to acquired resistance to carbapenems. Nevertheless, the MIC values 124182-57-6 IC50 of meropenem and doripenem for AXX-H-Y, 2 and 1.5 g/ml, respectively, were 124182-57-6 IC50 still more CD36 elevated than those for AXX-A and AXX-D. It is likely that other mechanisms are involved in the residual carbapenem resistance of AXX-H-Y. Concerning the cephalosporins, we observed that inactivation resulted in a 2-fold decrease of the ceftazidime MIC, whatever the resistance level in parent strains. The activity of cefepime was partially restored in AXX-D-Y but not in AXX-H-Y, suggesting the association of various mechanisms of resistance. Finally, AxyXY-OprZ can also extrude tetracyclines, some fluoroquinolones, and erythromycin, which are also substrates of MexXY/OprM. The restoration of the original drug resistance phenotypes was observed in spontaneous revertants obtained by culturing mutant strains without ticarcillin. AxyXY-OprZ from and MexXY/OprM from have common substrates. This is consistent with the high amino acid sequence similarity observed between the transporters AxyY and MexY (73%), the transporter component being responsible for substrate recognition of the RND-type efflux systems (17). Nevertheless, AxyXY-OprZ confers a much higher level of resistance to aminoglycosides than MexXY-OprM in wild strains. It has recently been reported for some aminoglycoside-resistant strains of that and are linked to the gene in the same operon and that MexXY can utilize either OprM or OprA to form drug efflux complexes (18). Such an operon, including an 124182-57-6 IC50 gene, has been also described in gene that we detected in the three strains studied (primers oprZ-F and oprZ-R) seems to be a homologue of the genes from and (71% nucleotide identity and 57% amino acid identity). We plan to assess the contribution of OprZ to the high level of resistance to aminoglycosides. In 124182-57-6 IC50 conclusion, we have demonstrated that AxyXY-OprZ confers on a broad spectrum of antimicrobial agent resistance. The most interesting finding is that AxyXY-OprZ confers on its intrinsic high level of resistance to aminoglycosides. Our results suggest the involvement of AxyXY-OprZ in acquired resistance to carbapenems and fluoroquinolones that are major antimicrobial components for the treatment of pulmonary infections in CF patients. This will be supported by further studies that will include more clinical isolates. ACKNOWLEDGMENT We acknowledge the contribution of Gael Belliot for revising the manuscript. Footnotes Published ahead of print 22 October 2012.

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