Stenotrophomonas maltophilia from Nepal Producing Two Novel Antibiotic Inactivating Enzymes, a Class A β-Lactamase KBL-1 and an Aminoglycoside 6′-N-Acetyltransferase AAC(6′)-Iap

ABSTRACT Seven drug-resistant strains of Stenotrophomonas maltophilia were isolated from patients at two university hospitals in Nepal. S. maltophilia JUNP497 was found to encode a novel class A β-lactamase, KBL-1 (Kathmandu β-lactamase), consisting of 286 amino acids with 52.98% identity to PSV-1. Escherichia coli transformants expressing blaKBL-1 were less susceptible to penicillins. The recombinant KBL-1 protein efficiently hydrolyzed penicillins. The genomic environment surrounding blaKBL-1 was a unique structure, with the upstream region derived from strains in China and the downstream region from strains in India. S. maltophilia JUNP350 was found to encode a novel 6′-N-aminoglycoside acetyltransferase, AAC(6′)-Iap, consisting of 155 amino acids with 85.0% identity to AAC(6′)-Iz. E. coli transformants expressing aac(6′)-Iap were less susceptible to arbekacin, amikacin, dibekacin, isepamicin, neomycin, netilmicin, sisomicin and tobramycin. The recombinant AAC(6′)-Iap protein acetylated all aminoglycosides tested, except for apramycin and paromomycin. The genomic environment surrounding aac(6′)-Iap was 90.99% identical to that of S. maltophilia JV3 obtained from a rhizosphere in Brazil. Phylogenetic analysis based on whole genome sequences showed that most S. maltophilia isolates in Nepal were similar to those isolates in European countries, including Germany and Spain. IMPORTANCE The emergence of drug-resistant S. maltophilia has become a serious problem in medical settings worldwide. The present study demonstrated that drug-resistant S. maltophilia strains in Nepal harbored novel genes encoding a class A β-lactamase, KBL-1, or a 6′-N-aminoglycoside acetyltransferase, AAC(6′)-Iap. Genetic backgrounds of most S. maltophilia strains in Nepal were similar to those in European countries. Surveillance of drug-resistant S. maltophilia in medical settings in Nepal is necessary.

The present study describes two clinical isolates of S. maltophilia obtained from hospitalized patients in Nepal, one harboring a gene encoding a novel class A b-lactamase, KBL-1, and the other harboring a gene encoding a novel 69-N-aminoglycoside acetyltransferase, AAC(69)-Iap.

RESULTS AND DISCUSSION
Drug susceptibilities of S. maltophilia isolates. Of the seven S. maltophilia isolates, five were resistant to ceftazidime, three were resistant to ticarcillin-clavulanic acid, and one each was resistant to chloramphenicol, levofloxacin, and sulfamethoxazole-trimethoprim (Table 1). All seven isolates had MICs of $64 mg/mL for imipenem, meropenem, and colistin, and six each had MICs of $128 mg/mL for aztreonam, arbekacin, and amikacin.
A novel class A b-lactamase KBL-1. The novel class A b-lactamase KBL-1 consisted of 286 amino acids. A comparison of its sequence to the amino acid sequences of 10 representative class A b-lactamases showed that KBL-1 were closest to PSV-1, with 52.98% sequence identity (Fig. 1). PSV-1 had previously been identified in Pseudovibrio ascidiaceicola, obtained from a species of sponge, Aplysina aerophoba, in Spain (11). Compared with the vector control, E. coli expressing bla KBL-1 showed much higher MIC values (256 to 4,096 mg/mL) toward the penicillins, including ampicillin, amoxicillin,  Table 2). The MICs of these penicillins were significantly reduced by b-lactamase inhibitors combined with penicillins, including amoxicillin-clavulanic acid, ampicillinsulbactam, and piperacillin-tazobactam, which had MICs of 32 to 128 mg/mL. The E. coli expressing bla KBL-1 showed lower MICs for the monobactam aztreonam; the cephalosporins cefepime, cefotaxime, cefoxitin, ceftazidime, cefozopran, cephradine, and moxalactam; and the carbapenems doripenem, imipenem, meropenem, and panipenem. Moreover, except for ceftazidime, there were no significant differences in the MICs of E. coli expressing bla KBL-1 and the vector control for any of these agents. The MIC of ceftazidime for E. coli expressing bla KBL-1 was low (1 mg/mL), but significantly higher than that for the vector control (0.125 mg/mL), suggesting that measurement conditions, such as salinity, temperature, and pH, may affect the hydrolysis of ceftazidime. Recombinant KBL-1 protein had hydrolytic activities against all the b-lactams tested, except for aztreonam (Table 3). Recombinant KBL-1 efficiently hydrolyzed the penicillins, including ampicillin, amoxicillin, penicillin G, and piperacillin with k cat /k m values of 0.422   (7), 83.0% identical to AAC(69)-Iam (9), and 79.1% identical to AAC(69)-Iak (10) (Fig. 2). Compared with vector control, E. coli expressing AAC(69)-Iap showed decreased susceptibilities to arbekacin, amikacin, dibekacin, isepamicin, neomycin, netilmicin, sisomicin, and tobramycin (Table 4). Thin-layer chromatography (TLC) analysis revealed that all the aminoglycosides tested, except for apramycin and paromomycin, were acetylated by AAC(69)-Iap (Fig. 3). These results indicated that aac(69)-Iap is a functional acetyltransferase that modifies the 69-NH 2 position of aminoglycosides and is involved in aminoglycoside resistance. The TLC data for apramycin and paromomycin were consistent with the MICs of the aminoglycosides for E. coli with pSTV28-aac(69)-Iap. Although gentamicin and kanamycin were acetylated by AAC(69)-Iap, the MICs were not higher than those of E. coli harboring pSTV28-aac(69)-Iap. Gentamicin includes gentamicins C1, C2, and C1a, with gentamicin C1 having no amino group at the 69-position, suggesting that gentamicin may only have been partially acetylated by AAC(69)-Iap.
Phylogenetic analysis of S. maltophilia in Nepal. Phylogenetic analysis based on whole genome sequences revealed that S. maltophilia can be divided into three clades, Most S. maltophilia clinical isolates in Nepal were derived from strains in European countries, including Germany and Spain, whereas the S. maltophilia strains JUNP349 and JUNP350 were indigenous to Nepal. Based on SNPs, the genetic backgrounds of the two clonal strains described in this study differed from those of other strains. The present study suggests that most S. maltophilia strains obtained in Nepal had similar genetic background to the wide-distributed strains belonging to Clade D. In several countries, including Australia, Brazil, Germany, Mexico, and the United States, where the wide-distributed strains belonging to Clade D were isolated, the isolation rates of levofloxacin-resistant S. maltophilia were relatively high, according to the SENTRY Antimicrobial Surveillance Program (1997-2016) (2). It is important to continue antimicrobial surveillance of S. maltophilia in Nepal and analyze the genetic backgrounds.

MATERIALS AND METHODS
Bacterial strains. Between April 2018 and November 2019, seven S. maltophilia isolates were obtained from seven patients treated at two hospitals in Kathmandu, Nepal (six isolates from hospital A and one from hospital B). The bacteria were identified using the biochemical API 20 NE test (bio-Mérieux, Marcy L'Etoile, France) and by sequencing their 16S rRNA genes. Of the seven isolates, three were from respiratory tracts, two from pus, one from blood, and one from cerebrospinal fluid. Escherichia coli DH5a (TaKaRa Bio, Shiga, Japan) and E. coli BL21-CodonPlus (DE3)-RIP (Agilent Technologies, Santa Clara, CA) were used as hosts for recombinant plasmids and protein expression, respectively. MICs were determined using a broth microdilution method, with the breakpoints of ceftazidime, chloramphenicol, levofloxacin, minocycline, ticarcillin-clavulanic acid, and trimethoprim-sulfamethoxazole for S. maltophilia determined according to the guidelines of the Clinical and Laboratory Standards Institute (13).
Whole-genome sequencing. Genomic DNA was extracted from each of the seven isolates using DNeasy blood and tissue kits (Qiagen, Tokyo, Japan) and sequenced using the MiSeq platform (Illumina, San Diego, CA) with the Nextera XT DNA library prep kit and MiSeq reagent kit version 3 (600 cycle; Illumina). More than 30-fold coverage was achieved for each isolate. Raw reads of each isolate were  phosphate buffer (pH 7.4) were incubated for 16 h at 37°C. Aliquots of 3 mL of each aminoglycoside mixture were spotted onto the surface of a Silica Gel 60 thin-layer chromatography (TLC) plate (Merck, Darmstadt, Germany), followed by development with a 5% phosphate potassium solution. The aminoglycosides and their acetylated products were detected by spraying the plates with 0.2% ninhydrin in acetone.