Plasmid Location and Molecular Heterogeneity of the L1 and L2 β-Lactamase Genes of Stenotrophomonas maltophilia

Abstract

An approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia. This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 ␤-lactamase genes. The location of L1 and L2 on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems. Sequence analysis of the 10 L1 genes revealed three novel genes, L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1 (L1a), respectively. The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250-and 100-fold lower k cat /K m ratios respectively). L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a. Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences. Three novel L2 genes (L2b, L2c, and L2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275 L2 (L2a) by 4, 9, and 25%, respectively. Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found. It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.In recent years there have been major increases in the frequencies with which certain, previously rare bacterial species have been identified as the causes of hospital-acquired bacteremias (26). Three principle factors have combined to bring about this change: (i) increased numbers of hospitalized patients who are severely immunosuppressed; (ii) an increase in complicated surgical procedures, such as transplant and oncology surgery, and the use of intravenous catheters; and (iii) the prophylactic use of antibiotics, particularly ␤-lactams (26). A prime example of such an emergent pathogen is Stenotrophomonas maltophilia (6,11,25,26,28). Its tolerance to silverlined catheters (28) and its inherent resistance to many antibacterial drugs, including most, if not all, ␤-lactams (1, 26, 27), give it a survival advantage over other potential pathogens in the hospital environment. Its incidence as a cause of nosocomial bacteremias caused by gram-negative organisms is now second only to that of bacteremia caused by Pseudomonas aeruginosa, and the frequency of its isolation is increasing (25). It is also a significant cause of bacterial infection among young adults with cystic fibrosis (10).The mechanisms of antibacterial drug resistance in S. maltophilia have not been studied in detail, but it is expected that many of the acquired mechanisms found in P. aeruginosa and other gram-negative bacteria are likely to be present. Strains that are resistant to all known aminoglycosides, quinolones, ␤-lactams, chloramphenicol, ri...

Characterization of Monomeric L1 Metallo-β-lactamase and the Role of the N-terminal Extension in Negative Cooperativity and Antibiotic Hydrolysis

“…After confirmation of the sequence, the mutated pL1PUC19 plasmid was digested with NdeI and HindIII, and the 900 bp, mutated L1 gene was gel-purified and ligated into pET26b to create the mutant overexpression plasmids. E. coli BL21(DE3)pLysS cells were transformed with the mutated overexpression plasmids, and large scale (4 liter) preparations of the L1 M140D mutant enzyme were performed as described previously (34). Protein purity was assessed by SDS-PAGE.…”

“…Steady State Kinetics-␤-Lactamase assays and steady state kinetics were carried out as described previously (34). Extinction coefficients and wavelengths for the ␤-lactams used were: nitrocefin, 17 (at 233 nm).…”

“…The presence of recombinant L1 clones was confirmed by PCR. The L1 mutant proteins were overexpressed and purified by fast protein liquid chromatography (FPLC), as described previously (34). The gel filtration column was calibrated using immunoglobulin G (BioRad Laboratories) (molecular mass, 150,000 Da; elution volume, 260 ml), bovine serum albumin (Sigma) (molecular mass, 67,000 Da; elution volume, 305 ml), and carbonic anhydrase (Sigma) (molecular mass, 29,000 Da; elution volume, 340 ml).…”

Molecular Characterization of Virulence Determinants of Stenotrophomonas Maltophilia Strains Isolated from Patients Affected by Cystic Fibrosis

“…All S. maltophilia strains, herein designated as OBG, were isolated from respiratory secretions of CF patients at the Hospital "Bambin Gesu" of Rome. S. maltophilia LMG959, an environmental isolate, and K279a, a clinical blood isolate, were used as reference strains (9). Bacteria were routinely grown at 37°C in Brain Heart Infusion (BHI) broth, Trypticase Soy Broth (TSB) or in Luria-Bertani (LB) broth, unless otherwise indicated.…”

Occurrence and features of chromosomally encoded carbapenemases in Gram-negative bacteria in farm animals sampled at slaughterhouse level

“…The Pseudomonas alcaligenes isolate was analyzed for the presence of blaPAM-1 (Suzuki et al, 2014) using the newly designed primers PAM-1F 5'-CGT TTC CTC GCC AGC CTC G-3' and PAM-1R 5'-CGC GAC TTT GCC TGC TCG-3'. The Stenotrophomonas malto philia isolate was tested for bla L1 and blaL2 with primers published previously (Avison et al, 2001). Amplicons were visualized by electrophoresis with 1% agarose gels stained with ethidium bromide under ultraviolet light.…”