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...

It has been reported that Stenotrophomonas maltophilia L1 and L2 beta-lactamase expression is coordinated. We have isolated S. maltophilia mutants where (i) L1 is constitutively hyper-expressed and L2 is inducible; (ii) L2 is hyper-expressed and L1 is inducible; and (iii) L1 and L2 are constitutively hyper-expressed. The frequency of isolating type 1 and 2 mutants is c. 10(-7), indicating that promoter mutations are probably not involved and providing strong evidence that L1 and L2 expression is not, after all, coordinated. The frequency of isolating type 3 mutants is c. 10(-9), however, implying that there is a significant overlap between the regulatory mechanisms.

A constitutively expressed beta-lactamase gene from a clinical isolate of Stenotrophomonas maltophilia, J675Ia, has been cloned. Its DNA sequence is almost identical to that of bla(TEM2) (one nucleotide change) and the expressed enzyme is a Bush type 2a penicillinase with an amino acid sequence identical to that of TEM-2. The bla(TEM) gene was present within a novel Tn1/Tn3-type transposon in the genome of isolate J675Ia and the transposon was able to mobilize bla(TEM) on to the broad host-range conjugative plasmid, R388. When transferred to an Escherichia coli recipient, R388::Tn conferred high-level ampicillin resistance. This represents the first identification of a TEM beta-lactamase in S. maltophilia and the first evidence that this important clinical pathogen is able to act as a reservoir for mobile beta-lactamase genes in the hospital environment.