PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
 
Antimicrob Agents Chemother. 2010 January; 54(1): 580–581.
Published online 2009 October 19. doi:  10.1128/AAC.00496-09
PMCID: PMC2798493

SmQnr Contributes to Intrinsic Resistance to Quinolones in Stenotrophomonas maltophilia[down-pointing small open triangle]

It has been recently shown that Stenotrophomonas maltophilia contains a chromosomally encoded qnr gene (Smqnr) which confers low-level resistance to quinolones upon its expression in a heterologous host (11, 12). However, although several studies have shown that plasmid-encoded Qnr determinants, acquired by horizontal gene transfer, contribute to quinolone resistance in different bacterial species (8), the role of these determinants in the susceptibility of their original hosts to quinolones remains unknown. Here, we analyzed the role of SmQnr in the intrinsic resistance of S. maltophilia to quinolones by comparing the susceptibility of a markerless Smqnr deletion mutant with that of its isogenic wild-type strain.

To generate an Smqnr deletion mutant, two DNA fragments homologous to the upstream and downstream regions of the Smqnr gene were obtained by PCR using primers qnr1 (5′-CGGAATTCCGGCCGGCAGCTGCTGGG-3′ [EcoRI site underlined]) and qnr2 (5′-GCTCTAGACATGCCA[T/A]GTGCCCCGCC-3′ [XbaI site underlined]) for the upstream fragment and qnr3 (5′-GCTCTAGATAG[A/G]CATCA[T/C]CCGGCGTACG-3′ [XbaI site underlined]) and qnr4 (5′-CCCAAGCTTGAGGCGCA[A/G]CCGGGCTGG-3′ [HindIII site underlined]) for the downstream fragment. The PCR products were cloned into pGEM-T, generating pGEM-T12 (upstream region) and pGEM-T34 (downstream region). pGEM-T12 was EcoRI-XbaI digested. The upstream fragment was cloned into the suicide vector pEX18Tc, yielding pBS6. pGEM-T34 was XbaI-HindIII digested, and the downstream fragment was cloned into the same restriction sites in pBS6, yielding pBS8, which was introduced into Escherichia coli CC118λpir and mobilized afterwards into S. maltophilia D457 by tripartite conjugation (5). The exconjugants containing pBS8 were selected on LB agar containing 10 μg/ml tetracycline and 20 μg/ml imipenem. Tetracycline-resistant colonies were streaked onto 10% sucrose-LB agar to select double recombinants presenting the Smqnr deletion. Deletion in the selected colonies was confirmed by PCR.

As shown in Table Table1,1, deletion of Smqnr renders an increased susceptibility to quinolones in S. maltophilia, demonstrating that SmQnr contributes to intrinsic resistance to these drugs in this bacterial species. Our article is the first demonstration of a role for chromosomally encoded Qnr determinants in intrinsic bacterial resistance to quinolones.

TABLE 1.
Quinolone susceptibilities of different S. maltophilia strains and their levels of Smqnr expression

To further confirm that the phenotype of the Smqnr mutant is due to Smqnr deletion, this gene was obtained by SacI/SphI digestion from pBS3.D457 (11) and cloned into the same restriction sites of pUC19, generating pBS10. pBS10 was digested with EcoRI/HindIII, and Smqnr was cloned into the same restriction sites of pVLT31 (4), rendering pBS12. Introduction of pBS12 restored resistance to quinolones in the Smqnr-defective mutant, whereas introduction of the control vector pVLT31 did not produce any change in susceptibility to these drugs (Table (Table1),1), confirming that SmQnr is involved in the intrinsic resistance of S. maltophilia to quinolones. Introduction of pBS12 into both the wild-type strain and the ΔSmqnr mutant increased resistance to quinolones to levels higher than those observed in wild-type S. maltophilia strain D457. This is consistent with a gene dosage effect of SmQnr similar to that described for other mechanisms of resistance (7, 9). To confirm this, the expression of Smqnr was analyzed in these strains by real-time RT-PCR.

As shown in Table Table1,1, Smqnr expression was higher when the gene was plasmid encoded (strains MBS108 and MBS101). This high-level expression is very similar, independently of whether the chromosomally encoded Smqnr gene is present or absent, showing that the level of expression of chromosomally encoded Smqnr is low in comparison with the expression of the plasmid-encoded gene. The fact that SmQnr overexpression reduced the susceptibility of the wild-type S. maltophilia strain to quinolones indicates that SmQnr is present at a limiting concentration in this bacterial species, in such a way that increased SmQnr amounts can protect more topoisomerase molecules and further preclude the activity of quinolones.

In contrast to what has been described for other bacteria, quinolone-resistant S. maltophilia mutants do not present mutations in bacterial topoisomerases (10, 13). One of the causes of this situation might be that the efflux pump SmeDEF confers high-level quinolone resistance upon its overexpression (1-3). The finding that SmQnr can also increase resistance when overexpressed suggests that Smqnr overexpression might also be involved in acquired, as well as intrinsic, resistance in S. maltophilia.

Acknowledgments

This work has been supported by grant BIO2008-00090 from the Spanish Ministerio de Ciencia e Innovación and grants LSHM-CT-2005-518152, LSHM-CT-2005-018705, and KBBE-227258 (BIOHYPO) from the European Union.

Footnotes

[down-pointing small open triangle]Published ahead of print on 19 October 2009.

REFERENCES

1. Alonso, A., and J. L. Martinez. 2000. Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 44:3079-3086. [PMC free article] [PubMed]
2. Alonso, A., and J. L. Martinez. 2001. Expression of multidrug efflux pump SmeDEF by clinical isolates of Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 45:1879-1881. [PMC free article] [PubMed]
3. Alonso, A., and J. L. Martinez. 1997. Multiple antibiotic resistance in Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 41:1140-1142. [PMC free article] [PubMed]
4. de Lorenzo, V., L. Eltis, B. Kessler, and K. N. Timmis. 1993. Analysis of Pseudomonas gene products using lacIq/Ptrp-lac plasmids and transposons that confer conditional phenotypes. Gene 123:17-24. [PubMed]
5. de Lorenzo, V., and K. N. Timmis. 1994. Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. Methods Enzymol. 235:386-405. [PubMed]
6. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402-408. [PubMed]
7. Martinez, J. L., E. Cercenado, M. Rodriguez-Creixems, M. F. Vincente-Perez, A. Delgado-Iribarren, and F. Baquero. 1987. Resistance to beta-lactam/clavulanate. Lancet 2:1473. [PubMed]
8. Martinez-Martinez, L., A. Pascual, and G. A. Jacoby. 1998. Quinolone resistance from a transferable plasmid. Lancet 351:797-799. [PubMed]
9. Reguera, J. A., F. Baquero, J. C. Perez-Diaz, and J. L. Martinez. 1991. Factors determining resistance to beta-lactam combined with beta-lactamase inhibitors in Escherichia coli. J. Antimicrob. Chemother. 27:569-575. [PubMed]
10. Ribera, A., A. Domenech-Sanchez, J. Ruiz, V. J. Benedi, M. T. Jimenez de Anta, and J. Vila. 2002. Mutations in gyrA and parC QRDRs are not relevant for quinolone resistance in epidemiological unrelated Stenotrophomonas maltophilia clinical isolates. Microb. Drug Resist. 8:245-251. [PubMed]
11. Sanchez, M. B., A. Hernandez, J. M. Rodriguez-Martinez, L. Martinez-Martinez, and J. L. Martinez. 2008. Predictive analysis of transmissible quinolone resistance indicates Stenotrophomonas maltophilia as a potential source of a novel family of Qnr determinants. BMC Microbiol. 8:148. [PMC free article] [PubMed]
12. Shimizu, K., K. Kikuchi, T. Sasaki, N. Takahashi, M. Ohtsuka, Y. Ono, and K. Hiramatsu. 2008. Smqnr, a new chromosome-carried quinolone resistance gene in Stenotrophomonas maltophilia. Antimicrob. Agents Chemother. 52:3823-3825. [PMC free article] [PubMed]
13. Valdezate, S., A. Vindel, J. A. Saez-Nieto, F. Baquero, and R. Canton. 2005. Preservation of topoisomerase genetic sequences during in vivo and in vitro development of high-level resistance to ciprofloxacin in isogenic Stenotrophomonas maltophilia strains. J. Antimicrob. Chemother. 56:220-223. [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)