SmQnr Contributes to Intrinsic Resistance to Quinolones in Stenotrophomonas maltophilia

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 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

Strain	Plasmid	Location of Smqnrb	Mean amt of Smqnr ± SEMc	MIC (mg/liter)a
				LVX	MOX	CIP	OFX	GAT
D457	None	C, none	1.0 ± 0.0	0.5	0.19	0.75	1	0.25
MBS108	pBS12	C, P	10.1 ± 2.5	1	0.75	2	3	0.75
MBS109	pVLT31	C, none	1.2 ± 0.3	0.5	0.19	0.75	1	0.25
MBS82	None	None, none	0.0 ± 0.0	0.25	0.064	0.5	0.5	0.125
MBS101	pBS12	None, P	9.57 ± 2.2	1	0.75	2	3	0.75
MBS100	pVLT31	None, none	0.0 ± 0.0	0.25	0.064	0.5	0.5	0.125

^aLVX, levofloxacin; MOX, moxifloxacin; CIP, ciprofloxacin; OFX, ofloxacin; GAT, gatifloxacin. MICs were determined by epsilon test (at least three determinations were made for each MIC).

^bC, the strain harbors a chromosomally encoded Smqnr gene; P, the strain harbors a plasmid-encoded Smqnr gene.

^cThe amount of Smqnr was calculated by real-time reverse transcription-PCR using SYBR green PCR master mix (Applied Biosystems) and primers RTqnr1 (5′-TTCGAGGGAATCGACTGGAA-3′) and RTqnr 2 (5′-TCGCCCAGATCGGAATTG-3′). Results were normalized to those obtained for the sigma factor rpoD (primers rpoD1 [5′-GGTGCACATGATCGAAACGA-3′] and rpoD2 [5′-GCCGTACTGCTGGAGCATCT-3′]). Five different samples of each strain were analyzed. The relative amounts of mRNA for Smqnr in each case, normalized against an internal control (rpoD), were calculated by the 2^−ΔΔCt method (6). The values are expressed in arbitrary units referred to the expression level in wild-type strain D457.

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 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 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.