Regulation of Smqnrexpression by SmqnrRis strain-specific inStenotrophomonas maltophilia: Table 1.

Sir, One of the elements conferring resistance to quinolones in Stenotrophomonas maltophilia is the quinolone resistance protein SmQnr. Upstream of and divergently transcribed to Smqnr, the genome of S. maltophilia presents a bicistronic operon formed by two genes: SmqnrR, encoding a DeoR-type regulator, and SmtcrA, encoding a putative transporter belonging to the major facilitator superfamily of transmembrane transporters. A recent study, published in this journal, has shown that Smqnr expression is regulated by SmqnrR, which also regulates SmtcrA expression in the strain S. maltophilia KJ. In order to gain deeper insights in the mechanisms of regulation of SmqnrR expression, we have deleted SmqnrR in our model strain S. maltophilia D457. Two homologous regions located upstream and downstream of SmqnrR were obtained by PCR using Expand Long Template (Roche) and 1 mM of each primer [qnrRA (5′-CGGAATTCCCAGCATCGGTGTGTCA TGC-3′, EcoRI site underlined) and qnrR2 (5′-ATGAGCCCCTGACC GTACCGCTCATGTC-3′) for the downstream fragment, and qnrR3 (5′-TCAGGGGCTCATGCTGCAAGTCTGCACG-3′) and qnrRD (5′-CCC AAGCTTTCCAGTCGATTCCCTCGAAC-3′ HindIII site underlined), for the upstream fragment]. PCR consisted of one denaturation step at 948C for 5 min and 10 amplification cycles of 948C for 30 s, 508C for 45 s and 688C for 1 min, followed by a further 15 amplification cycles of 948C for 30 s, 558C for 45 s and 688C for 1 min, plus a final extension step at 688C for 7 min. An overlapping PCR, using upstream and downstream fragments as the template and primers qnrRA and qnrRD, was performed as described above, in this case increasing the time lapse of the amplification step at 688C to 2 min. The PCR product was cloned in pGEM-T, generating the plasmid pBS39, and then sequenced to confirm the sequence. This fragment with upstream and downstream SmqnrR regions was extracted using the enzymes EcoRI and HindIII and cloned in the suicide vector pEX18Tc with the same enzymes, generating the plasmid pBS49. This plasmid was introduced into the conjugative strain Escherichia coli CC118lpir and transferred to S. maltophilia D457 by conjugation. The exconjugants containing integrated pBS49 were selected on LB agar with 10 mg/L tetracycline and 20 mg/L imipenem. These colonies were streaked into LB with 10% sucrose and incubated at room temperature for 72 h. For those colonies growing in sucrose and not in tetracycline, the deletion of SmqnrR was confirmed by PCR with primers external to the SmqnrR gene [qnrR8 (5′-TCGTTGATCCCGGTGCGTGC-3′) and qnrR9 (5′-TTCACCGGCAGTCCAGCCGC-3′)]. One mutant lacking SmqnrR was selected for further study and dubbed MBS697. To study the effect of the deletion of SmqnrR on Smqnr and SmtcrA expression, total RNA was isolated from 30 mL of mid-log-phase cultures performed in LB (A6001⁄40.6 –0.7) using the Qiagen RNeasy Mini Kit (Qiagen), and genomic DNA was removed with TURBO DNA-free DNase (Ambion). RNA was used for reverse transcription using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR (RT-PCR) was performed using Power SYBR Green PCR Master Mix, a 7300 Real-Time PCR System (Applied Biosystems) and primers RTqnr1 and RTqnr2 for Smqnr and RT_tcrAF (5′-GATGCTGAAGAAGGCATGGAA-3′) and RT_tcrAR (5′-GGACTGCACGATGAACGTACA-3′) for SmtcrA. The relative amount of mRNA for Smqnr and SmtcrA in D457 and in the MBS697 mutant was calculated following the 2 method using ftsZ (primers FtsZ1 and FtsZ2) as reference for normalization. Results previously published in this journal using the S. maltophilia KJ strain showed that the mutant lacking SmqnrR presented higher MICs of quinolones and expressed SmqnrR and SmtcrA at higher levels. In the D457 strain, the deletion of SmqnrR also allowed a higher expression of SmtcrA (fold change 6.6+1.72). Nevertheless, and contrary to the situation described for the KJ strain, the lack of SmqnrR neither altered the level of expression of Smqnr nor changed the susceptibility of D457 to quinolones when measured by test strip (Liofilchem) in Mueller– Hinton agar (Pronadisa) (Table 1). Previous work has shown that, in both S. maltophilia KJ and S. maltophilia D457, Smqnr overexpression is involved in acquired resistance. Nevertheless, whereas the deletion of Smqnr increases the susceptibility of the D457 strain to quinolones, this deletion did not alter susceptibility for the KJ strain. Together with the information previously published in this journal, our data indicate that