Smqnr, a New Chromosome-Carried Quinolone Resistance Gene in Stenotrophomonas maltophilia

Abstract

A new chromosome-carried quinolone resistance gene from Stenotrophomonas maltophilia, Smqnr, was characterized. The gene was present in type strain CCUG 5866 and was also detected in 24 clinical isolates and showed some allelic diversity. The expression of Smqnr in Escherichia coli decreased the susceptibilities of the E. coli isolates to several fluoroquinolones.

qnr (later termed qnrA1), the first plasmid-mediated quinolone resistance determinant identified, was first reported in 1998 from Klebsiella pneumoniae (7). QnrA1 belongs to the pentapeptide repeat family and protects DNA gyrase and topoisomerase IV from the inhibitory actions of quinolones (10, 11, 12). QnrA1 confers resistance to quinolones and increases the MICs of fluoroquinolones up to 32-fold (8). Since then, several proteins belonging to the Qnr family have been described in members of the family Enterobacteriaceae (6). The source of the plasmid-mediated qnrA genes has been identified in the chromosome of Shewanella algae (9), while the possible source of the plasmid-mediated qnrS determinants was identified in the chromosome of Vibrio splendidus (3).

Stenotrophomonas maltophilia is a nonfermentative gram-negative environmental species that can cause nosocomial infections and that is characterized by intrinsic resistance to several antibiotics (2, 5). In silico analysis of the recently released genome sequence of S. maltophilia strain R551-3 (GenBank accession no. NZ_AAVZ0100006) revealed an open reading frame (SmalDRAFT_0855) coding for a 219-amino-acid protein that shares 61.5% and 61% amino acid identities with QnrB1 and QnrB2, respectively. In this study, we cloned and sequenced the S. maltophila qnr homologue, designated Smqnr, from S. maltophilia CCUG 5866^T and 24 epidemiologically unrelated clinical isolates (which mainly originated from respiratory specimens).

The species identities of the S. maltophilia clinical isolates were confirmed by species-specific PCR (13). The nucleotide sequences of the Smqnr alleles were determined by a PCR-based strategy. Primer pair SmQnrX-F (5′-ACACAGAACGGCTGGACTGC-3′) and SmQnrX-R (5′-TTCAACGACGTGGAGCTGTT-3′) amplified an 817-bp fragment containing almost all the Smqnr alleles evaluated in this study. Other primer pairs were also used. Primer pair SmQnrY-F (5′-GATCGGAGCTCATGCTGCAA-3′) and SmQnrY-R (5′-GCAGCGCGCGATCGAAGCAA-3′) amplified a 970-bp fragment containing the Smqnr-1 gene, and primer pair SmQnrZ-F (5′-TCTATGGATCGGCCTCG-3′) and SmQnrZ-R (5′-TTCAGCTTCAAGGGCTGGG-3′) amplified a 745-bp fragment containing the Smqnr-10 gene. The nucleotide sequences and the deduced amino acid sequences were analyzed with GENETYX-MAC software (version 13; GENETYX Corporation, Tokyo, Japan) and the information available on the National Center for Biotechnology Information website (www.ncbi.nih.gov). Multiple-protein-sequence alignments were carried out with the program CLUSTALW (http://clustalw.ddbj.nig.ac.jp/top-j.html).

Sequences related to the Smqnr gene from strain R551-3 were detected both in the type strain and in each of the clinical isolates, suggesting that the gene is ubiquitous in S. maltophilia. Some allelic variability was observed among the Smqnr genes. A total of 11 alleles were identified, and these encoded proteins that differed by up to 20 amino acids at 219 possible sites (9.1%) (Fig. 1).

FIG. 1.

Comparison of amino acid sequences of 11 SmQnr alleles, SmQnr of S. maltophilia R511-3, and plasmid-mediated QnrB1. SmQnr1 from S. maltophilia JCSC 7117; SmQnr2 was from S. maltophilia JCSC 7107; SmQnr3 was from S. maltophilia JCSC 7099; SmQnr4 was from S. maltophilia JCSC 7102; SmQnr5 was from S. maltophilia JCSC 7103; SmQnr6 was from S. maltophilia JCSC 7095, JCSC 7100, JCSC 7101, JCSC 7110, JCSC 7111, and JCSC 7112; SmQnr7 was from S. maltophilia CCUG 5866^T; SmQnr8 was from S. maltophilia JCSC 7097, JCSC 7098, JCSC 70104, JCSC 7106, JCSC 7114, JCSC 7115, and JCSC 7119; SmQnr9 was from S. maltophilia JCSC 7108, JCSC 7113, and JCSC 7120; SmQnr10 was from S. maltophilia JCSC 7109; and Smqnr11 was from S. maltophilia JCSC 7105 and JCSC 7116. Symbols: asterisks, identical amino acids; colons, strongly similar amino acids; periods, weakly similar amino acids. The amino acid differences among all the SmQnr amino acid sequences are boxed.

The amplified PCR fragments containing the 11 different Smqnr alleles identified in this work were cloned into plasmid pCR-4 I-TOPO (Invitrogen, Life Technologies, Carlsbad, CA) to yield recombinant plasmids pSmQnr1, pSmQnr2, pSmQnr3, pSmQnr4, pSmQnr5, pSmQnr6, pSmQnr7, pSmQnr8, pSmQnr9, pSmQnr10, and pSmQnr11, respectively. All recombinant plasmids carried the cloned genes in the same orientation under the transcriptional control of the plasmid P_lac promoter flanking the multiple-cloning site. Then, Escherichia coli TOP10 (Invitrogen) derivatives carrying each of these plasmids were used to determine the MICs of quinolone and fluoroquinolones by Etest (AB Biodisk, Solna, Sweden). The MICs were interpreted according to the guidelines of the CLSI (4).

The results are shown in Table 1. Different quinolones and fluoroquinolones MICs were observed among the different allelic variants. These findings are suggestive of the correlation between the amino acid substitutions and the degree of quinolone resistance, but it is still unclear which amino acid substitutions contribute to quinolone resistance.

TABLE 1.

MICs of quinolone and fluoroquinolones against E. coli TOP10 harboring recombinant plasmids pSmQnr1 to pSmQnr11^a

Strain	MIC (μg/ml)b
	NAL	NOR	LVX	CIP	SPX	GAT	MXF
E. coli TOP10	1.5	0.032	0.004	0.002	<0.002	<0.002	<0.002
E. coli TOP10/pSmQnr1	3.0	0.064	0.023	0.008	0.004	0.008	0.012
E. coli TOP10/pSmQnr2	2.0	0.047	0.012	0.004	<0.002	<0.002	<0.002
E. coli TOP10/pSmQnr3	1.5	0.032	0.006	0.003	<0.002	0.003	0.002
E. coli TOP10/pSmQnr4	1.5	0.032	0.006	0.002	<0.002	0.002	0.002
E. coli TOP10/pSmQnr5	2.0	0.032	0.008	0.002	<0.002	<0.002	0.003
E. coli TOP10/pSmQnr6	2.0	0.064	0.023	0.016	0.125	0.064	0.047
E. coli TOP10/pSmQnr7	2.0	0.047	0.023	0.008	0.008	0.023	0.008
E. coli TOP10/pSmQnr8	2.0	0.047	0.012	0.004	<0.002	0.002	0.002
E. coli TOP10/pSmQnr9	1.5	0.032	0.008	0.003	<0.002	0.002	<0.002
E. coli TOP10/pSmQnr10	2.0	0.032	0.023	0.004	0.002	0.003	0.004
E. coli TOP10/pSmQnr11	2.0	0.032	0.012	0.006	<0.002	0.003	0.002

^aMICs of quinolone and fluoroquinolones against E. coli TOP10 and E. coli TOP10 harboring recombinant plasmids pSmQnr1, pSmQnr2, pSmQnr3, pSmQnr4, pSmQnr5, pSmQnr6, pSmQnr7, pSmQnr8, pSmQnr9, pSmQnr10, and pSmQnr11.

^bNAL, nalidixic acid; NOR, norfloxacin; LVX, levofloxacin; CIP, ciprofloxacin; SPX, sparfloxacin; GAT, gatifloxacin; MXF, moxifloxacin.

SmQnr is clearly capable of decreasing quinolone and fluoroquinolone susceptibilities, similar to the other Qnr determinants. However, further studies are required to assess the contribution of Smqnr to the quinolone susceptibility of S. maltophilia, which is also known to be affected by efflux-based mechanisms (1).

Nucleotide sequence accession numbers.

The following nucleotide sequences described in this paper have been submitted to the EMBL/GenBank/DDBJ database under the indicated accession numbers: Smqnr1, AB430839; Smqnr2, AB430840; Smqnr3, AB430841; Smqnr4, AB430842; Smqnr5, AB430843; Smqnr6, AB430849; Smqnr7, AB430845; Smqnr8, AB430850; Smqnr9, AB430846; Smqnr10, AB430847; and Smqnr11, AB430848.