Characterization of environmental CTX-M-15-producing Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a widespread environmental microorganism (1) that has emerged as a significant opportunistic pathogen (2) due to its intrinsic resistance to almost all available antibiotics. Apart from native -lactamases L1 and L2 (1), acquired extended-spectrum -lactamases (ESBLs) were also identified (3, 4), pointing to S. maltophilia as a potential reservoir. Furthermore, resistance to trimethoprim-sulfamethoxazole (SXT), a therapy of choice for S. maltophilia, is being increasingly reported (5–8). Forty-one S. maltophilia isolates were recovered from retail shellfish (Mytilus galloprovincialis; 8 isolates) purchased at fish markets in Split, Croatia, and from coastal marine waters near Split (33 isolates) in 2012. Strains were isolated on imipenem (16 g/ml)-containing tryptic soy agar at 30°C and identified using API 20NE. Whole-cell DNA was extracted and used for PCR detection of ESBL genes (9). Nineteen isolates carried blaCTX-M-15, six of which additionally harbored blaTEM-116, and one carried blaTEM-127 (Table 1). Metallo-lactamase genes were not detected (10). TEM116 is usually associated with environmental Enterobacteriaceae (11) and Pseudomonas spp. (12) and has never before been identified in S. maltophilia. More importantly, CTX-M-15 was previously identified only in an S. maltophilia clinical strain from France (4). S1 nuclease pulsed-field gel electrophoresis (PFGE) of plasmid DNA followed by Southern blotting (9) showed that blaCTX-M-15 was located on large plasmids of various sizes (Table 1). Conjugation transfer of blaCTX-M-15 using Escherichia coli J53 (13) at 37°C and 27°C and using azide (100 g/ml) and cefotaxime (8 g/ml)-containing Luria-Bertani agar failed even after repeated attempts. Only a 160-kb plasmid (isolate 248) was successfully transferred into E. coli JM109 using heat shock transformation. For PCR-based replicon typing (14), plasmid DNA from the transformant was used, while for other isolates, each plasmid band was cut from the gel and, after confirmation as blaCTX-M-15 positive by PCR, was used as a template. Interestingly, all plasmids belonged to the IncFIB incompatibility group. IncFII, IncFIA, and IncFIB blaCTX-M-15-bearing plasmids have been previously reported in Croatia (9, 15). A possible explanation for the unusual lack of IncF plasmid replication in E. coli recipients could be that plasmids adapted to S. maltophilia, altering their host range and specificity of replication traits (16). Further studies are needed to better characterize these resistance plasmids. PFGE of XbaI-digested genomic DNA (17, 18) showed heterogeneity among CTX-M-15-producing isolates (data not shown). Isolates were further investigated for class 1, 2, and 3 integrases,