Yi-Wei Huang

AmpDI Is Involved in Expression of the Chromosomal L1 and L2 β-Lactamases of Stenotrophomonas maltophilia

ABSTRACT Two ampD homologues, ampDI and ampDII, of Stenotrophomonas maltophilia have been cloned and analyzed. Comparative genomic analysis revealed that the genomic context of the ampDII genes is quite different, whereas that of the ampDI genes is more conserved in S. maltophilia strains. The ampD system of S. maltophilia is distinct from that of the Enterobacteriaceae and Pseudomonas aeruginosa in three respects. (i) AmpDI of S. maltophilia is not encoded in an ampDE operon, in contrast to what happens in the Enterobacteriaceae and P. aeruginosa. (ii) The AmpD systems of the Enterobacteriaceae and P. aeruginosa are generally involved in the regulation of ampR-linked ampC gene expression, while AmpDI of S. maltophilia is responsible for the regulation of two intrinsic β-lactamase genes, of which the L2 gene, but not the L1 gene, is linked to ampR. (iii) S. maltophilia exhibits a one-step L1 and L2 gene derepression model involving ampDI, distinct from the two- or three-step derepression of the Enterobacteriaceae and P. aeruginosa. Moreover, the ampDI and ampDII genes are constitutively expressed and not regulated by the inducer and AmpR protein, and the expression of ampDII is weaker than that of ampDI. Finally, AmpDII is not associated with the derepression of β-lactamases, and its role in S. maltophilia remains unclear.

The SmeYZ Efflux Pump of Stenotrophomonas maltophilia Contributes to Drug Resistance, Virulence-Related Characteristics, and Virulence in Mice

ABSTRACT The resistance-nodulation-division (RND)-type efflux pump is one of the causes of the multidrug resistance of Stenotrophomonas maltophilia. The roles of the RND-type efflux pump in physiological functions and virulence, in addition to antibiotic extrusion, have attracted much attention. In this study, the contributions of the constitutively expressed SmeYZ efflux pump to drug resistance, virulence-related characteristics, and virulence were evaluated. S. maltophilia KJ is a clinical isolate of multidrug resistance. The smeYZ isogenic deletion mutant, KJΔYZ, was constructed by a gene replacement strategy. The antimicrobial susceptibility, virulence-related physiological characteristics, susceptibility to human serum and neutrophils, and in vivo virulence between KJ and KJΔYZ were comparatively assessed. The SmeYZ efflux pump contributed resistance to aminoglycosides and trimethoprim-sulfamethoxazole. Inactivation of smeYZ resulted in attenuation of oxidative stress susceptibility, swimming, flagella formation, biofilm formation, and secreted protease activity. Furthermore, loss of SmeYZ increased susceptibility to human serum and neutrophils and decreased in vivo virulence in a murine model. These findings suggest the possibility of attenuation of the resistance and virulence of S. maltophilia with inhibitors of the SmeYZ efflux pump.

An Inducible Fusaric Acid Tripartite Efflux Pump Contributes to the Fusaric Acid Resistance in Stenotrophomonas maltophilia

Background Fusaric acid (5-butylpicolinic acid), a mycotoxin, is noxious to some microorganisms. Stenotrophomonas maltophilia displays an intrinsic resistance to fusaric acid. This study aims to elucidate the mechanism responsible for the intrinsic fusaric acid resistance in S. maltophilia. Methodology A putative fusaric acid resistance-involved regulon fuaR-fuaABC was identified by the survey of the whole genome sequence of S. maltophilia K279a. The fuaABC operon was verified by reverse transcriptase-PCR. The contribution of the fuaABC operon to the antimicrobial resistance was evaluated by comparing the antimicrobials susceptibility between the wild-type strain and fuaABC knock-out mutant. The regulatory role of fuaR in the expression of the fuaABC operon was assessed by promoter transcription fusion assay. Results The fuaABC operon was inducibly expressed by fusaric acid and the inducibility was fuaR dependent. FuaR functioned as a repressor of the fuaABC operon in absence of a fusaric acid inducer and as an activator in its presence. Overexpression of the fuaABC operon contributed to the fusaric acid resistance. Significance A novel tripartite fusaric acid efflux pump, FuaABC, was identified in this study. Distinct from the formally classification, the FuaABC may constitute a new type of subfamily of the tripartite efflux pump.

SmeOP-TolCsm Efflux Pump Contributes to the Multidrug Resistance of Stenotrophomonas maltophilia

ABSTRACT A five-gene cluster, tolCSm-pcm-smeRo-smeO-smeP, of Stenotrophomonas maltophilia was characterized. The presence of smeOP and smeRo-pcm-tolCSm operons was verified by reverse transcription (RT)-PCR. Both operons were negatively regulated by the TetR-type transcriptional regulator SmeRo, as demonstrated by quantitative RT-PCR and a promoter-fusion assay. SmeO and SmeP were associated with TolCSm (the TolC protein of S. maltophilia) for the assembly of a resistance-nodulation-cell-division (RND)-type pump. The compounds extruded by SmeOP-TolCSm mainly included nalidixic acid, doxycycline, amikacin, gentamicin, erythromycin, leucomycin, carbonyl cyanide 3-chlorophenylhydrazone, crystal violet, sodium dodecyl sulfate, and tetrachlorosalicylanilide.

Inactivation of mrcA gene derepresses the basal-level expression of L1 and L2 β-lactamases in Stenotrophomonas maltophilia

OBJECTIVES To characterize the relationship between inactivation of the mrcA gene and β-lactamase expression and β-lactams resistance in Stenotrophomonas maltophilia KJ and to investigate the involvement of ampR, ampN-ampG, ampD(I) and creBC in this. METHODS The mrcA deletion mutant KJΔmrcA was constructed to investigate the role of this putative penicillin-binding protein 1a (PBP1a) in β-lactamase expression and β-lactam resistance. The ΔampR, ΔampNG, ΔampDI and ΔcreBC alleles were introduced into KJΔmrcA, and KJΔDIΔBC and KJΔDIΔmrcAΔBC were also constructed for comparison. All the mutants and their corresponding parent strains were assayed for β-lactamase activities and MICs of β-lactams. RESULTS Inactivation of mrcA caused basal L1/L2 β-lactamase production to increase by ∼100-fold, but made little difference to cefuroxime-induced β-lactamase activity and the MICs of β-lactams. The ΔmrcA-derived basal β-lactamase hyperproduction was ampR and ampN-ampG dependent. Simultaneous inactivation of ampD(I) and mrcA did not augment β-lactamase production over and above that seen in an ampD(I) mutant alone. Furthermore, we could find no evidence for a role of the creBC two-component regulatory system in β-lactamase hyperproduction in a ΔampD(I) or ΔmrcA background. CONCLUSIONS Inactivation of mrcA, predicted to encode PBP1a, causes basal L1/L2 β-lactamase hyperproduction in S. maltophilia.

AmpN-AmpG Operon Is Essential for Expression of L1 and L2 β-Lactamases in Stenotrophomonas maltophilia

ABSTRACT AmpG is an inner membrane permease which transports products of murein sacculus degradation from the periplasm into the cytosol in Gram-negative bacteria. This process is linked to induction of the chromosomal ampC beta-lactamase gene in some members of the Enterobacteriaceae and in Pseudomonas aeruginosa. In this study, the ampG homologue of Stenotrophomonas maltophilia KJ was analyzed. The ampG homologue and its upstream ampN gene form an operon and are cotranscribed under the control of the promoter PampN. Expression from PampN was found to be independent of β-lactam exposure and ampN and ampG products. A ΔampN allele exerted a polar effect on the expression of ampG and resulted in a phenotype of null β-lactamase inducibility. Complementation assays elucidated that an intact ampN-ampG operon is essential for β-lactamase induction. Consistent with ampG of Escherichia coli, the ampN-ampG operon of S. maltophilia did not exhibit a gene dosage effect on β-lactamase expression. The AmpG permease of E. coli could complement the β-lactamase inducibility of ampN or ampG mutants of S. maltophilia, indicating that both species have the same precursor of activator ligand(s) for β-lactamase induction.

A Linkage between SmeIJK Efflux Pump, Cell Envelope Integrity, and σE-Mediated Envelope Stress Response in Stenotrophomonas maltophilia

Resistance nodulation division (RND) efflux pumps, such as the SmeIJK pump of Stenotrophomonas maltophilia, are known to contribute to the multidrug resistance in Gram-negative bacteria. However, some RND pumps are constitutively expressed even though no antimicrobial stresses occur, implying that there should be some physical implications for these RND pumps. In this study, the role of SmeIJK in antimicrobials resistance, envelope integrity, and σE-mediated envelope stress response (ESR) of S. maltophilia was assessed. SmeIJK was involved in the intrinsic resistance of S. maltophilia KJ to aminoglycosides and leucomycin. Compared with the wild-type KJ, the smeIJK deletion mutant exhibited growth retardation in the MH medium, an increased sensitivity to membrane-damaging agents (MDAs), as well as activation of an σE-mediated ESR. Moreover, the expression of smeIJK was further induced by sub-lethal concentrations of MDAs or surfactants in an σE-dependent manner. These data collectively suggested an alternative physiological role of smeIJK in cell envelope integrity maintenance and σE-mediated ESR beyond the efflux of antibiotics. Because of the necessity of the physiological role of SmeIJK in protecting S. maltophilia from the envelope stress, smeIJK is constitutively expressed, which, in turn, contributes the intrinsic resistance to aminoglycoside and leucomycin. This is the first demonstration of the linkage among RND-type efflux pump, cell envelope integrity, and σE-mediated ESR in S. maltophilia.

NagZ-Dependent and NagZ-Independent Mechanisms for β-Lactamase Expression in Stenotrophomonas maltophilia

ABSTRACT β-N-Acetylglucosaminidase (NagZ), encoded by the nagZ gene, is a critical enzyme for basal-level ampC derepression (ampC expression in the absence of β-lactam challenge) in ampD and dacB mutants of Pseudomonas aeruginosa. Three mutants with a phenotype of basal-level L1 and L2 β-lactamase derepression in Stenotrophomonas maltophilia have been reported, including KJΔDI (ampDI mutant), KJΔmrcA (mrcA mutant), and KJΔDIΔmrcA (ampDI and mrcA double mutant). In this study, nagZ of S. maltophilia was characterized, and its roles in basal-level β-lactamase derepression, induced β-lactamase activities, and β-lactam resistance of KJΔDI, KJΔmrcA, and KJΔDIΔmrcA were evaluated. Expression of the nagZ gene was constitutive and not regulated by AmpR, AmpDI, AmpN, AmpG, PBP1a, and NagZ. Introduction of ΔnagZ into KJΔDI nearly abolished basal-level derepressed β-lactamase activity; conversely, introduction of ΔnagZ into KJΔmrcA did not affect it. At least two activator ligands (ALs) are thus considered responsible for β-lactamase expression in the S. maltophilia system, specifically, the NagZ-dependent (AL1) and NagZ-independent (AL2) ligands responsible for the basal-level derepressed β-lactamase activities of KJΔDI and KJΔmrcA, respectively. The contributions of AL1 and AL2 to the induced β-lactamase activities may vary with the types of β-lactams. nagZ inactivation did not affect aztreonam-, cefoxitin-, and carbenicillin-induced β-lactamase activities, but it attenuated cefuroxime- and piperacillin-induced β-lactamase activities. Introduction of ΔnagZ into KJ, KJΔDI, KJΔmrcA, and KJΔDIΔmrcA did not significantly change the MICs of the β-lactams tested except that the MICs of cefuroxime and piperacillin moderately decreased in strains KJΔZ and KJΔDIΔZ (nagZ mutants).

MacABCsm, an ABC-type tripartite efflux pump of Stenotrophomonas maltophilia involved in drug resistance, oxidative and envelope stress tolerances and biofilm formation

OBJECTIVES To characterize a five gene cluster, macRS-macABCsm, in Stenotrophomonas maltophilia. METHODS The presence of macABCsm operon was verified by RT-PCR. The substrate spectrum of the MacABCsm efflux pump was investigated by mutant construction and susceptibility testing. The physiological role of MacABCsm was assessed by comparing the growth of wild-type and macABCsm mutant under different stresses. To examine the regulatory role of the two-component regulatory system (TCS) macRS in the expression of macABCsm operon, mutant construction, quantitative RT-PCR and susceptibility testing were employed. RESULTS macAsm, macBsm and macCsm genes formed a three-membered operon. The MacABCsm efflux pump extruded macrolides, aminoglycosides and polymyxins and contributed to oxidative and envelope stress tolerances and biofilm formation. Inactivation of macRS TCS hardly influenced the expression of macABCsm operon and the antimicrobial susceptibility. CONCLUSIONS The MacABCsm pump has physiological roles in protecting S. maltophilia from the attack of oxidative and envelope stresses and in biofilm formation, which may be the reason why it can be constitutively expressed in the absence of antibiotics and is highly conserved in S. maltophilia isolates isolated from different environmental niches. However, the constitutive expression of macABCsm contributes to the intrinsic resistance of S. maltophilia to macrolides, aminoglycosides and polymyxins.

Role of the pcm-tolCsm operon in the multidrug resistance of Stenotrophomonas maltophilia

OBJECTIVES To elucidate the role of the pcm-tolCsm operon in the multidrug resistance of Stenotrophomonas maltophilia. METHODS The presence of the pcm-tolCsm operon was verified by RT-PCR. The phylogenetic relationship between the outer membrane proteins known to be involved in functional tripartite efflux in Escherichia coli, Pseudomonas aeruginosa and S. maltophilia was analysed. The contribution of TolCsm to resistance to a variety of compounds was investigated by susceptibility testing of the ΔtolCsm mutant. The role of pcm in the expression and function of tolCsm was assessed by quantitative real-time PCR and complementation assay. RESULTS The pcm and tolCsm genes formed an operon. TolCsm of S. maltophilia, OpmH of P. aeruginosa and TolC of E. coli formed a distinguishing phylogenetic TolC-like clade. TolCsm deletion increased the susceptibility of S. maltophilia KJ2 to several antimicrobial agents (aminoglycoside, macrolide, β-lactam, chloramphenicol, nalidixic acid, doxycycline and trimethoprim/sulfamethoxazole) and chemical compounds (acriflavine, carbonyl cyanide 3-chlorophenylhydrazone, crystal violet, fusaric acid, menadione, Paraquat, plumbagin, SDS and tetrachlorosalicylanilide). The in-frame deletion of pcm caused a polar effect on the expression of tolCsm, which compromised the resistance to amikacin and gentamicin. Nevertheless, the presence of the PCM protein made an insignificant contribution to the function of TolCsm in the resistance to amikacin and gentamicin. CONCLUSIONS The pcm-tolCsm operon makes a significant contribution to the multidrug resistance of S. maltophilia.