Stenotrophomonas maltophilia is an emerging nosocomial pathogen that displays high-level intrinsic resistance to a variety of structurally unrelated antimicrobial agents. Efflux mechanisms are known to contribute to acquired multidrug resistance in this organism, and indeed, one such multidrug efflux system, SmeDEF, was recently identified. Still, the importance of SmeDEF to intrinsic antibiotic resistance in S. maltophilia had not yet been determined. Reverse transcription-PCR confirmed expression of the smeDEF genes in wild-type S. maltophilia, and deletion of smeE or smeF in wild-type strains rendered the mutants hypersusceptible to several antimicrobials, suggesting that SmeDEF contributes to intrinsic antimicrobial resistance in this organism. Expression of smeDEF was also enhanced in an in vitro-selected multidrug-resistant mutant, although deletion of smeF but not of smeE in these mutants compromised antimicrobial resistance. Apparently, hyperexpressed SmeF is capable of functioning with additional multidrug efflux components to promote multidrug resistance in S. maltophilia.
The presence of the multidrug efflux pump SmeDEF was assessed in a collection of clinical isolates of Stenotrophomonas maltophilia. All isolates encoded this pump, as demonstrated by PCR. Forty-seven percent of the strains overproduced a protein of the same size that was immunoreactive against an anti-SmeF antibody, and 33% overexpressed the gene semD when they were tested by reverse transcription-PCR. A correlation between smeDEF overexpression and antibiotic resistance was observed.
The wide utilization of biocides poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that biocides can select, at least in laboratory experiments, antibiotic resistant bacteria. This situation has raised concerns, not just on scientists and clinicians, but also on regulatory agencies, which are demanding studies on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases and on human health. In the present article, we explored the possibility that the widely used biocide triclosan might induce antibiotic resistance using as a model the opportunistic pathogen Stenotrophomonas maltophilia. Biochemical, functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic- and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. To our knowledge this is the first crystal structure obtained for a transcriptional regulator bound to triclosan. This work provides the molecular basis for understanding the mechanisms allowing the induction of phenotypic resistance to antibiotics by triclosan.
The wide utilization of biocides for different purposes, including toothpastes, soaps, house-hold compounds surfaces' disinfectants and even their use as additives of different materials (from textiles to concrete used in germ-free buildings) to avoid their colonization by microorganisms, poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that such biocides can select, at least in laboratory experiments, bacteria resistant to antibiotics. This situation has raised concerns on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases. In the present article we study whether biocides can induce phenotypic resistance to antibiotics, a process that would be barely detectable unless purposely searched out. In the article, we present functional, biochemical and structural data showing that the widely used biocide triclosan induces antibiotic resistance, mediated by the binding of the biocide to SmeT, the transcriptional regulator of the expression of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF, which can extrude an ample range of antibiotics. Our study provides an unambiguous link between the presence of this biocide and the increased efflux of antibiotics by the opportunistic pathogen S. maltophilia.
The possibility that triclosan selects Stenotrophomonas maltophilia mutants overexpressing the multidrug resistance pump SmeDEF is analyzed. Five out of 12 triclosan-selected mutants were less susceptible to antibiotics than the wild-type strain and overproduced SmeDEF. Results are discussed in relation to current debates on the potential selection of antibiotic-resistant bacteria by household biocides.
We report on the cloning of the gene smeT, which encodes the transcriptional regulator of the Stenotrophomonas maltophilia efflux pump SmeDEF. SmeT belongs to the TetR and AcrR family of transcriptional regulators. The smeT gene is located upstream from the structural operon of the pump genes smeDEF and is divergently transcribed from those genes. Experiments with S. maltophilia and the heterologous host Escherichia coli have demonstrated that SmeT is a transcriptional repressor. S1 nuclease mapping has demonstrated that expression of smeT is driven by a single promoter lying close to the 5′ end of the gene and that expression of smeDEF is driven by an unique promoter that overlaps with promoter PsmeT. The level of expression of smeT is higher in smeDEF-overproducing S. maltophilia strain D457R, which suggests that SmeT represses its own expression. Band-shifting assays have shown that wild-type strain S. maltophilia D457 contains a cellular factor(s) capable of binding to the intergenic smeT-smeD region. That cellular factor(s) was absent from smeDEF-overproducing S. maltophilia strain D457R. The sequence of smeT from D457R showed a point mutation that led to a Leu166Gln change within the SmeT protein. This change allowed overexpression of both smeDEF and smeT in D457R. It was noteworthy that expression of wild-type SmeT did not fully complement the smeT mutation in D457R. This suggests that the wild-type protein is not dominant over the mutant SmeT.
We have determined that the mutational inactivation of the SmeDEF efflux pump and the SmQnr quinolone resistance protein widens the mutant selection windows for ofloxacin and ciprofloxacin of Stenotrophomonas maltophilia by reducing their MICs. Resistant mutants arising from a strain lacking SmeDEF and SmQnr presented levels of susceptibility similar to those of the wild-type strain. This indicates that inactivation of intrinsic resistance determinants might increase the chances for selecting resistant mutants at low antibiotic concentrations.
Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, which causes infections that are often difficult to manage because of the inherent resistance of the pathogen to a variety of antimicrobial agents. In this study, we analyzed the expressions of smeABC and smeDEF and their correlation with antimicrobial susceptibility. We also evaluated the genetic relatedness and epidemiological links among 33 isolates of S. maltophilia.
In total, 33 S. maltophilia strains were isolated from patients in a tertiary hospital in Daejeon. Minimum inhibitory concentrations (MICs) of 11 antimicrobial agents were determined by using agar dilution method and E-test (BioMérieux, France). Real-time PCR analysis was performed to evaluate the expression of the Sme efflux systems in the S. maltophilia isolates. Additionally, an epidemiological investigation was performed using multilocus sequence typing (MLST) assays.
The findings of susceptibility testing showed that the majority of the S. maltophilia isolates were resistant to β-lactams and aminoglycosides. Twenty-one clinical isolates overexpressed smeABC and showed high resistance to ciprofloxacin. Moreover, a high degree of genetic diversity was observed among the S. maltophilia isolates; 3 sequence types (STs) and 23 allelic profiles were observed.
The smeABC efflux pump was associated with multidrug resistance in clinical isolates of S. maltophilia. In particular, smeABC efflux pumps appear to perform an important role in ciprofloxacin resistance of S. maltophilia. The MLST scheme for S. maltophilia represents a discriminatory typing method with stable markers and is appropriate for studying population structures.
Stenotrophomonas maltophilia; smeABC; smeDEF; Efflux pump; MLST
A homologue of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa, smeABC, was cloned from Stenotrophomonas maltophilia by using, as a probe, a PCR product amplified from this organism with primers based on the mexB sequence. The smeABC genes were hyperexpressed in a mutant strain displaying resistance to several antimicrobials, including aminoglycosides, β-lactams, and fluoroquinolones. Deletions in smeC but not smeB compromised this resistance, suggesting that SmeC contributed to the multidrug resistance of the mutant as part of another, as-yet-unidentified multidrug efflux system. Consistent with SmeC functioning independently of SmeAB, a promoter activity was identified upstream of smeC. Upstream of the smeABC genes, a putative two-gene operon, smeSR, encoding homologues of bacterial two-component regulatory systems was identified. The cloned smeR gene activated expression of a smeA-lacZ fusion, indicating that SmeR positively regulates expression of the smeABC genes. Consistent with this, the multidrug resistance of the SmeABC-hyperexpressing mutant was compromised by deletion of smeR. Intriguingly, SmeC expression in S. maltophilia paralleled a β-lactamase activity provided by a C-terminally truncated L2 enzyme, which was apparently responsible for the β-lactam resistance of the SmeABC-hyperexpressing mutant. This represents the first report of coregulation of an efflux resistance determinant and a β-lactamase.
KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded by smeU1 and smeU2 were found to belong to the family of short-chain dehydrogenases/reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to β-lactams and erythromycin was not affected. The expression of the smeU1-V-W-U2-X operon was regulated by the divergently transcribed LysR-type regulator gene smeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation of smeV and smeW completely abolished the activity of the SmeVWX pump, whereas inactivation of smeX alone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression.
The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.
We cloned a gene, bexA, that codes for a multidrug efflux transporter from the chromosomal DNA of Bacteroides thetaiotaomicron ATCC 29741 by using an Escherichia coli ΔacrAB ΔacrEF mutant as a host. Although the initial recombinant construct contained other open reading frames, the presence of bexA alone was sufficient to confer to the E. coli host elevated levels of resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Disruption of bexA in B. thetaiotaomicron made the strain more susceptible to norfloxacin, ciprofloxacin, and ethidium bromide, showing that this gene is expressed in this organism and functions as a multidrug efflux pump. The deduced BexA protein sequence was homologous to the protein sequence of Vibrio parahaemolyticus NorM, a multidrug efflux transporter, and thus, BexA belongs to the multidrug and toxic compound extrusion (MATE) family.
A Stenotrophomonas maltophilia mutant that coordinately hyper-expresses three resistance nodulation division-type efflux pump genes, smeZ, smeJ, and smeK, has been identified. SmeZ is responsible for elevating aminoglycoside MICs; SmeJ and SmeK are jointly responsible for elevating tetracycline, minocycline, and ciprofloxacin MICs and conferring levofloxacin resistance. One clinical isolate with this same phenotype was identified from a sample of six, and the isolate also coordinately hyper-expresses smeZ and smeJK, confirming the clinical relevance of our findings.
Screening of random fragments of Escherichia coli genomic DNA for their ability to increase the novobiocin resistance of a hypersusceptible ΔacrAB mutant resulted in the isolation of a plasmid containing baeR, which codes for the response regulator of the two-component regulatory system BaeSR. When induced for expression, baeR cloned in multicopy plasmid pTrc99A significantly increased the resistance of the ΔacrAB host strain to novobiocin (16-fold) and to deoxycholate (8-fold). Incubation of cells with novobiocin followed by a chromatographic assay for intracellular drug showed that overproduced BaeR decreased drastically the drug accumulation, presumably via increased active efflux. The genes baeSR are part of a putative operon, yegMNOB baeSR. Direct binding of BaeR to the yegM promoter was demonstrated in vitro by gel retardation assay. The gene yegB, which codes for a major facilitator superfamily transporter, was not necessary for increased resistance, but deletion of yegO or an in-frame deletion of yegN, both of which code for resistance-nodulation-cell division-type multidrug transporters, abolished the BaeR-induced increase in resistance. It is likely that both YegN and YegO produce a complex(es) with the membrane fusion protein family member YegM and pump out novobiocin and deoxycholate. We accordingly propose to rename yegMNOB as mdtABCD (mdt for multidrug transporter). Finally, the expression of two other genes, yicO and ygcL, was shown to be regulated by BaeR, but it is not known if they play any roles in resistance.
The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps in Pseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains of P. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.
The smeT-smeDEF region and the smeT gene, which encodes the smeDEF repressor, are highly polymorphic. Few changes in smeT might be associated with smeDEF overexpression. The results obtained with cellular extracts suggest that mutant SmeT proteins cannot bind to the operator and that other transcription factors besides SmeT are involved in the regulation of smeDEF expression.
The contributions of multicomponent-type multidrug efflux pumps to antimicrobial resistance and nodulation ability in Sinorhizobium meliloti were comprehensively analyzed. Computational searches identified genes in the S. meliloti strain 1021 genome encoding 1 pump from the ATP-binding cassette family, 3 pumps from the major facilitator superfamily, and 10 pumps from the resistance-nodulation-cell division family, and subsequently, these genes were deleted either individually or simultaneously. Antimicrobial susceptibility tests demonstrated that deletion of the smeAB pump genes resulted in increased susceptibility to a range of antibiotics, dyes, detergents, and plant-derived compounds and, further, that specific deletion of the smeCD or smeEF genes in a ΔsmeAB background caused a further increase in susceptibility to certain antibiotics. Competitive nodulation experiments revealed that the smeAB mutant was defective in competing with the wild-type strain for nodulation. The introduction of a plasmid carrying smeAB into the smeAB mutant restored antimicrobial resistance and nodulation competitiveness. These findings suggest that the SmeAB pump, which is a major multidrug efflux system of S. meliloti, plays an important role in nodulation competitiveness by mediating resistance toward antimicrobial compounds produced by the host plant.
The histone-like protein H-NS is a major component of the bacterial nucleoid and plays a crucial role in global gene regulation of enteric bacteria. It is known that the expression of a variety of genes is repressed by H-NS, and mutations in hns result in various phenotypes, but the role of H-NS in the drug resistance of Escherichia coli has not been known. Here we present data showing that H-NS contributes to multidrug resistance by regulating the expression of multidrug exporter genes. Deletion of the hns gene from the ΔacrAB mutant increased levels of resistance against antibiotics, antiseptics, dyes, and detergents. Decreased accumulation of ethidium bromide and rhodamine 6G in the hns mutant compared to that in the parental strain was observed, suggesting the increased expression of some drug exporter(s) in this mutant. The increased drug resistance and decreased drug accumulation caused by the hns deletion were completely suppressed by deletion of the multifunctional outer membrane channel gene tolC. At least eight drug exporter systems require TolC for their functions. Among these, increased expression of acrEF, mdtEF, and emrKY was observed in the Δhns strain by quantitative real-time reverse transcription-PCR analysis. The Δhns-mediated multidrug resistance pattern is quite similar to that caused by overproduction of the AcrEF exporter. Deletion of the acrEF gene greatly suppressed the level of Δhns-mediated multidrug resistance. However, this strain still retained resistance to some compounds. The remainder of the multidrug resistance pattern was similar to that conferred by overproduction of the MdtEF exporter. Double deletion of the mdtEF and acrEF genes completely suppressed Δhns-mediated multidrug resistance, indicating that Δhns-mediated multidrug resistance is due to derepression of the acrEF and mdtEF drug exporter genes.
Stenotrophomonas maltophilia is a multidrug-resistant organism increasingly isolated from the lungs of cystic fibrosis (CF) patients. One hundred twenty-five S. maltophilia isolates from 85 CF patients underwent planktonic and biofilm susceptibility testing against 9 different antibiotics, alone and in double antibiotic combinations. When S. maltophilia isolates were grown as a biofilm, 4 of the 10 most effective antibiotic combinations included high-dose levofloxacin and 7 of the 10 combinations included colistin at doses achievable by aerosolization.
Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log POW of the solvent, where POW is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of ΔacrAB and/or ΔtolC mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log POW = 5.5). The ΔtolC mutant was more sensitive to nonane than the ΔacrAB mutant. The solvent entered the E. coli cells rapidly. Entry of solvents with a log POW higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The ΔtolC mutant accumulated n-nonane or decane (log POW = 6.0) more abundantly than the parent or the ΔacrAB mutant. The AcrAB-TolC complex likely extrudes solvents with a log POW in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane.
Stenotrophomonas maltophilia is an environmental bacterium increasingly involved in nosocomial infections and resistant to most antibiotics. It is important to recognize and efficiently treat infections with this bacterium as soon as possible.
We present a case of Stenotrophomonas maltophilia prosthetic valve endocarditis secondary to an indwelling catheter infection. The patient was cured without surgery. We review other cases of S. maltophilia endocarditis from the literature and describe the peculiarities of this case.
S. maltophilia endocarditis is a rare disease that is often hospital-acquired and related to an indwelling catheter infection. The high lethality is likely related to the intrinsic resistance of nosocomial bloodstream infections to commonly prescribed antibiotics.
Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics, although the mechanisms of resistance are generally poorly understood. Multidrug resistant (MDR) strains were readily selected by plating a sensitive reference strain of the organism individually onto a variety of antibiotics, including tetracycline, chloramphenicol, ciprofloxacin, and norfloxacin. Tetracycline-selected MDR strains typically showed cross-resistance to erythromycin and fluoroquinolones and, in some instances, aminoglycosides. MDR mutants selected with the other agents generally displayed resistance to chloramphenicol and fluoroquinolones only, although two MDR strains (e.g., K1385) were also resistant to erythromycin and hypersusceptible to aminoglycosides. Many of the MDR strains expressed either moderate or high levels of a novel outer membrane protein (OMP) of ca. 50 kDa molecular mass, a phenotype typical of MDR strains of Pseudomonas aeruginosa hyperexpressing drug efflux systems. Indeed, the 50-kDa OMP of these S. maltophilia MDR strains reacted with antibody to OprM, the outer membrane component of the MexAB-OprM MDR efflux system of P. aeruginosa. Similarly, a ca. 110-kDa cytoplasmic membrane protein of these MDR strains also reacted with antibody to the MexB component of the P. aeruginosa pump. The outer and cytoplasmic membranes of several clinical S. maltophilia strains also reacted with the anti-OprM and anti-MexB antibodies. N-terminal amino acid sequencing of a cyanogen bromide-generated peptide of the 50-kDa OMP of MDR strain K1385, dubbed SmeM (Stenotrophomonas multidrug efflux), revealed it to be very similar to a number of outer membrane multidrug efflux components of P. aeruginosa and Pseudomonas putida. Deletion of the L1 and L2 β-lactamase genes confirmed that these enzymes were responsible for the bulk of the β-lactam resistance of K1385 and its parent. Still, overexpression of the MDR efflux mechanism in an L1- and L2-deficient derivative of K1385 did yield a modest increase in resistance to a few β-lactams. These data are consistent with the MDR efflux mechanism(s) playing a role in the multidrug resistance of S. maltophilia.
Stenotrophomonas maltophilia is an aerobic, glucose non- fermentative, gram negative bacillus, which is being increasingly recognized as a cause of serious infections such as bacteraemia, urinary tract infections, respiratory tract infections, skin and soft tissue infections, endocarditis, meningitis and ocular infections in hospitalized patients. The treatment of invasive S. maltophilia infections is difficult, as this pathogen shows high levels of intrinsic or acquired resistance to different antibiotics, thus reducing the options which are available for treatment. Meningitiscaused by S. maltophilia is rarely encountered and so its experience is also limited. We are describing here a case of a six months old, male child who developed meningitis caused by Stenotrophomonas maltophilia, after he underwent a neurosurgical procedure.
Stenotrophomonas maltophilia; Meningitis; Neurosurgery
The gram-negative bacterium Stenotrophomonas maltophilia is increasingly recognized as an important cause of nosocomial infection. Infection occurs principally, but not exclusively, in debilitated and immunosuppressed individuals. Management of S. maltophilia-associated infection is problematic because many strains of the bacterium manifest resistance to multiple antibiotics. These difficulties are compounded by methodological problems in in vitro susceptibility testing for which there are, as yet, no formal guidelines. Despite its acknowledged importance as a nosocomial pathogen, little is known of the epidemiology of S. maltophilia, and although it is considered an environmental bacterium, its sources and reservoirs are often not readily apparent. Molecular typing systems may contribute to our knowledge of the epidemiology of S. maltophilia infection, thus allowing the development of strategies to interrupt the transmission of the bacterium in the hospital setting. Even less is known of pathogenic mechanisms and putative virulence factors involved in the natural history of S. maltophilia infection and this, coupled with difficulties in distinguishing colonization from true infection, has fostered the view that the bacterium is essentially nonpathogenic. This article aims to review the current taxonomic status of S. maltophilia, and it discusses the laboratory identification of the bacterium. The epidemiology of the organism is considered with particular reference to nosocomial outbreaks, several of which have been investigated by molecular typing techniques. Risk factors for acquisition of the bacterium are also reviewed, and the ever-expanding spectrum of clinical syndromes associated with S. maltophilia is surveyed. Antimicrobial resistance mechanisms, pitfalls in in vitro susceptibility testing, and therapy of S. maltophilia infections are also discussed.
Cystic fibrosis (CF) patients are predisposed to chronic respiratory infection by nonfermentative gram-negative bacilli, including Stenotrophomonas maltophilia. S. maltophilia is highly resistant to most antibiotics, with the exception of sulfamethoxazole-trimethoprim (SXT). SXT-resistant S. maltophilia has been reported, but the mechanism of resistance is not well defined. Repeated findings of suspected small-colony-variant (SCV) S. maltophilia isolates from the sputa of five CF patients were confirmed by partial 16S rRNA gene sequencing. The SCV S. maltophilia isolates were the only S. maltophilia isolates in these cultures, and none were clonally related. DNA fingerprint analysis confirmed that once established, the SCV S. maltophilia strains persisted. Nutritional studies of SCV S. maltophilia have suggested auxotrophy in hemin, methionine, and thymidine associated with resistance to multiple antibiotics, including SXT. The phenotypic switch from wild-type to SCV S. maltophilia was reproducible in vitro by exposure to SXT, suggesting that prolonged exposure to antibiotics may select for both the SCV S. maltophilia phenotype and SXT resistance by interference with the dihydrofolate reductase pathway. Recovery of SCV S. maltophilia from the sputum of CF patients has implications for both laboratory testing and patient management.
The catechin epigallocatechin gallate, one of the main constituents of green tea, showed strong antibiotic activity against 18 isolates of Stenotrophomonas maltophilia (MIC range, 4 to 256 μg/ml). In elucidating its mechanism of action, we have shown that epigallocatechin gallate is an efficient inhibitor of S. maltophilia dihydrofolate reductase, a strategic enzyme that is considered an attractive target for the development of antibacterial agents. The inhibition of S. maltophilia dihydrofolate reductase by this tea compound was studied and compared with the mechanism of a nonclassical antifolate compound, trimethoprim. Investigation of dihydrofolate reductase was undertaken with both a trimethoprim-susceptible S. maltophilia isolate and an isolate with a high level of resistance. The enzymes were purified using ammonium sulfate precipitation, gel filtration, and methotrexate affinity chromatography. The two isolates showed similar levels of dihydrofolate reductase expression and similar substrate kinetics. However, the dihydrofolate reductase from the trimethoprim-resistant isolate demonstrated decreased susceptibility to inhibition by trimethoprim and epigallocatechin gallate. As with other antifolates, the action of epigallocatechin gallate was synergistic with that of sulfamethoxazole, a drug that blocks folic acid metabolism in bacteria, and the inhibition of bacterial growth was attenuated by including leucovorin in the growth medium. We conclude that the mechanism of action of epigallocatechin gallate on S. maltophilia is related to its antifolate activity.