Smqnr variants in clinical isolates of Stenotrophomonas maltophilia in Brazil.

Stenotrophomonas maltophilia contains a novel chromosomally-encoded qnr gene named Smqnr that contributes to low intrinsic resistance to quinolone. We described Smqnr in 13 clinical isolates of S. maltophilia from two Brazilian hospitals, over a 2-year period. The strains were identified by API 20 NE (bioMérieux, France). Susceptibility by microdilution method to trimetroprim/sulfamethoxazole, ciprofloxacin, levofloxacin, minocycline, ceftazidime, chloramphenicol and ticarcillin/clavulanate was performed according to CLSI. PCR detection of Smqnr gene was carried out. The sequence of Smqnr was compared with those deposited in GenBank. Pulsed-field gel electrophoresis (PFGE) of all strains was performed. Thirteen Smqnr positives isolates were sequenced and three novel variants of Smqnr were identified. All 13 Smqnr isolates had distinguishable patterns by PFGE. This is the first report of Smqnr in S. maltophilia isolated in Brazil.

INTRODUCTION

Stenotrophomonas maltophilia, a non-fermentative Gram-negative bacillus that is ubiquitous in the environment, has emerged as an important opportunistic pathogen . This microorganism exhibits intrinsic and acquired resistance to a wide variety of antimicrobial agents and few options of treatment are available . So far, trimethoprim/sulfamethoxazole is the drug of choice to treat infections caused by this microorganism, however, during the past few years increased resistance to this antibiotic has been reported . The new fluoroquinolones such as levofloxacin and moxifloxacin showed promising in vitro activity against S. maltophilia . Resistance to these new fluoroquinolones, among S. matophilia, is rare and needs to be further researched.

S. maltophilia contains a novel chromosomally-encoded S. maltophilia qnr gene named Smqnr with 219 amino acids with two classic pentapeptide repeat motifs separated by a glycine residue, which confers low level resistance to quinolone antibiotics as showed in vitro experiments . The role of Smqnr on quinolones resistance, however, is controversial and there is a lack of research evaluating its association with levofloxacin resistance in S. maltophilia.

We describe the characterization of Smqnr genes in clinical isolates of S. maltophilia susceptible and resistant to ciprofloxacin and levofloxacin.

MATERIAL AND METHODS

Clinical samples of S. maltophilia isolates from two Brazilian teaching hospitals, over a 2-year period were evaluated. Isolates were identified by API 20 NE (bioMérieux, France). Susceptibility by microdilution method to trimethoprim/sulfamethoxazole, ciprofloxacin, levofloxacin, minocycline, ceftazidime, chloramphenicol and ticarcillin/clavulanate was performed according to the CLSI (CLSI 2011) . Tigecycline MIC was interpreted following the Food and Drug Administration (FDA) recommendation for Enterobacteriaceae. Endonuclease-digested genomic DNAs were separated by pulsed-field gel electrophoresis (PFGE) using a CHEF-DR III system (Bio-Rad, USA). Genomic DNA was digested with 10U of SpeI (fermentas, USA). Running conditions were 21 h at 14 °C, with and initial switching time of one s and final time of 30 s, at 6 V/cm.

PCR for the Smqnr gene was carried out using five different set of specific sequence primers QnrM+ (5′-CTTGGCATGGAATCCC TGAT-3′)/QnrM- (5′-TGATGCCTACGGCACCAC-3′), QnrMR55+ (5′-CATGGCATGGAATCCCCGAT-3′)/QnrMR55- (5′-TGATG TCTACGGCACCAC-3′), qnrA (F:5′-CTCGAATGCCTGGCGCG TGTTT-3′) (R: 5′- AAGAGATTTCTCAGCCAGG-3′), qnrB (F: 5′-TGCCAGGCACAGATCTTGAC-3′) (R: AGGMATHGAAATTCG CCACTG-3′) and qnrS (F: 5′- TTTGCYGYYCGCCAGTCGAA-3′) (R:5′:GCAAGTTCATTGAACAGGGT-3′) and was performed in accordance with SANCHEZ et al. (2008) and ROBICSEK et al. (2006) . We used five set of primers because the regions around qnr are different in the sequences of S. maltophilia strains K279a, R551-3 and qnr A, B, S of Enterobacteriaceae species.

The nucleotide sequences and the deduced amino acid sequence were analyzed using the biological sequence aligment editor and CLUSTALW (www.mbio.ncsu.edu/bioedit/bioedit) (CA, USA).

This study was approved by the Ethics Committee of the two hospitals.

RESULTS

Thirteen S. maltophilia isolates harboring Smqnr were studied, eight resistant to ciprofloxacin and two to levofloxacin. QnrM gene was detected only using primers derived from S. maltophilia strain K279a; qnr A, B and S genes of Enterobacteriaceae were not detected.

All 13 isolates showed distinguishable patterns by PFGE (Table 1). The distribution of isolates occurred evenly in different units and with different clonal profiles during the study period, which ruled out the possibility of an outbreak.

Table 1

Characteristics and antimicrobial susceptibilities of 13 clinical isolates of S. maltophilia

Isolates	Source	PFGE	MIC (mg/L)
			SMX	LEV	CIP	MIN	TIG	CAZ	CLO	TIC
LIM7	Blood	A	0.5	1	8	<0.25	0.5	64	8	8
LIM9	Blood	B	2	2	8	0.5	1	32	8	8
LIM11	Blood	C	2	<0.25	1	0.25	0.25	32	8	>128
LIM14	CVC	D	<0.25	<0.25	0.5	0.25	0.25	>128	8	32
LIM31	CVC	E	<0.25	1	2	<0.25	0.5	4	32	32
LIM33	CVC	F	1	16	64	2	4	16	32	64
LIM35	CVC	G	0.5	0.25	4	<0.25	0.25	>128	16	128
LIM37	CVC	H	0.25	0.5	8	<0.25	2	128	16	32
LIM39	CVC	I	0.5	4	16	4	2	8	64	128
LIM41	CVC	J	8	8	32	2	8	64	128	32
LIM45	BAL	K	4	0.5	2	0.5	2	64	>128	128
LIM47	Blood	L	0.5	1	1	<0.25	2	4	32	32
LIM49	Blood	M	1	4	16	<0.25	1	8	128	>128

MIC, microdilutional method; BAL, Bronchoalveolar lavage; CVC, cateter venous central; PFGE, Pulsed field gel electrophoresis; SXT, trimethoprim/sulfamethoxazole; LEV, levofloxacin; CIP, ciprofloxacin; MIN, minocycline; TIG, tigecycline; CAZ, ceftazidime; CLO, chloramphenicol; TIC, ticarcillin/clavulanate. PFGE: 13 distinguishable patterns (letter A to M).

Two of the 13 isolates were resistant (MIC 8 and 16 mg/L) and two showed increased MIC to levofloxacin (MIC 4 mg/L). Eight isolates were resistant and one exhibited increased MIC to ciprofloxacin (MIC ≥ 2 mg/L). Two isolates were resistant to trimethoprim/sulfamethoxazole (MIC 4 and 8 mg/L). Two isolates were resistant to tigecycline (MIC 4 and 8 mg/L) and all isolates were susceptible to minocycline (MIC ≤ 4 mg/L) (Table 1).

The Smqnr peptide sequences of the 13 isolates were compared with the known Smqnr 1-27 subtypes in GenBank. Sequence analysis showed that seven isolates were identical to the equivalent sequence of Smqnr6 from Japan (AB430849), the other isolates were distributed as followed: one Smqnr4 (GenBank AB430842), one Smqnr12 (GenBank AB430844) and one Smqnr1 (Genbank AB430839) identified in Japan. Three novel variants were observed, the subtype SmqnrLIM31 have six amino acid residues differences, the subtype SmqnrLIM39 have four amino acid residues differences and subtype SmqnrLIM45 showed two amino acids alteration (Fig. 1).

Fig. 1

Aminoacid sequence alignments of 13 SmQnr proteins from Brazil, SmQnr1 (SHIMIZU et al.) and SmQnr 13 (GORDON et al). Asterisks, identical aminoacids, colons, strongly similar aminoacids (conserved substitutions); full stops, weakly similar amino acids (semi-conserver substitutions); spaces, variable aminoacids. Amino acid differences are shown in redbold.

DISCUSSION

S. maltophilia strains display high ciprofloxacin resistance, mainly due to several efflux systems . However, in vitro, susceptibility testing to levofloxacin is recommended by CLSI (CLSI 2009), and levofloxacin and moxifloxacin are used to treat infections caused by this pathogen. Resistance to levofloxacin and moxifloxacin is still rare among S. maltophilia . Two recent studies of clinical isolates of S. maltophilia that evaluated 102 isolates of bloodstream infection and 377 isolates (majority from the respiratory tract and blood) showed respectively 92.9% and 79.6% of susceptibility to levofloxacin . In our study two isolates showed resistance and two increased MIC to levofloxacin. All isolates were susceptible to minocycline and two were resistant to trimethoprim/sulfamethoxazole. Despite good activity in vitro, the experience of the clinical use of minocycline to treat infections caused by S. maltophilia is restricted to anecdotal reports .

The Smqnr plasmid mediated genes are pentapeptides repeat proteins that confer low-level resistance to quinolone by protecting DNA gyrase. The potential source of qnr is believe to be horizontal transfer by integrons and mobile genetic elements from chromosome of aquatic or environmental bacterial, such Shewanella algae, Aeromonas spp., Psychromonas spp and Vibrionaceae .

The qnr genes in S. maltophilia isolates have been studied by some authors . In our study, among 13 isolates harboring Smqnr; two were resistant (MIC 8 and 16 mg/L) and two exhibited increased MIC to levofloxacin (MIC 4 mg/L) and eight isolates exhibited resistant to ciprofloxacin. Three new Smqnr variants were identified. Two (LIM31 and LIM45) of them presented high levofloxacin MIC. The isolates were polyclonal, showing that they did not have a clonal relationship. This is the first study that reports Smqnr in S. maltophilia clinical isolates in Brazil.

One important limitation of our study is that we were not able to perform cloning and transformation assays to confirm the role of Smqnr on fluorquinolone resistance in S. maltophilia.

The role of Smqnr on quinolones resistance among S. maltophilia, remains controversial, and appears to be associated with the clonality of strains and varies with the hospital and country. A recent study conducted in China, evaluated 442 clinical isolates of S. maltophilia from nine hospitals. The resistance against co-trimoxazole was 48.6%, and a high susceptibility was shown to levofloxacin, only 6.1% of strains were resistant to levofloxacin . Smqnr genes were detected in 114 (26%) isolates in similar frequency in both quinolones sensitive and nonsensitive strains. Twenty new variants of Smqnr genes were identified and called Smqnr (28-47) . An in vitro study, showed that overexpression of Smqnr upon deletion increased modestly the MIC of nalidixic acid and moxifloxacin . And finally, a study conducted in the UK, identified two new variants of Smqnr that when expressed in E. coli top10 showed reduced susceptibility to several quinolone including levofloxacin and moxifloxacin .

In conclusion, this is the first report of the presence of Smqnr in isolates of S. matophilia resistant or with high levofloxacin MIC in Brazil. Three new Smqnr variants were identified. These findings alert the clinicians to the emergence of resistance to this antibiotic that is widely used in the treatment of infections by this agent, and strengthens the role of Smqnr with levofloxacin resistance. In addition, minocycline presented good activity in vitro against multidrug resistant strains of S. maltophilia and, in the future, may be an option for the treatment of infections caused by this agent.