RESUMEN
Cefiderocol is a siderophore cephalosporin designed mainly for treatment of infections caused by ß-lactam and multidrug-resistant Gram-negative bacteria. Burkholderia pseudomallei clinical isolates are usually highly cefiderocol susceptible, with in vitro resistance found in a few isolates. Resistance in clinical B. pseudomallei isolates from Australia is caused by a hitherto uncharacterized mechanism. We show that, like in other Gram-negatives, the PiuA outer membrane receptor plays a major role in cefiderocol nonsusceptibility in isolates from Malaysia.
Asunto(s)
Antibacterianos , Burkholderia pseudomallei , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Bacterias Gramnegativas , Cefalosporinas/farmacología , Cefalosporinas/uso terapéutico , Farmacorresistencia Bacteriana Múltiple/genética , Pruebas de Sensibilidad Microbiana , CefiderocolRESUMEN
Burkholderia pseudomallei is a soil- and water-dwelling Gram-negative bacterium that causes melioidosis in humans and animals. Amoxicillin-clavulanic acid (AMC) susceptibility has been hailed as an integral part of the screening algorithm for identification of B. pseudomallei, but the molecular basis for the inherent AMC susceptibility of this bacterium remains undefined. This study showed that B. pseudomallei (and the closely-related B. mallei) wild-type strains are the only Burkholderia spp. that contain a 70STSK73 PenA Ambler motif. This motif was present in >99.5% of 1820 analysed B. pseudomallei strains and 100% of 83 analysed B. mallei strains, and is proposed as the likely cause for their inherent AMC sensitivity. The authors developed a polymerase chain reaction (PCR) assay that specifically amplifies the penA70ST(S/F)K73-containing region from B. pseudomallei and B. mallei, but not from the remaining B. pseudomallei complex species or the 70STFK73 region from the closely-related penB of B. cepacia complex species. The abundance and purity of the 193-bp PCR fragment from putative B. pseudomallei isolates from clinical and environmental samples is likely sufficient for reliable confirmation of the presence of B. pseudomallei. The PCR assay is designed to be especially suited for use in resource-constrained areas. While not further explored in this study, the assay may allow diagnosis of putative B. mallei in culture isolates from animal and human samples.
Asunto(s)
Burkholderia mallei , Burkholderia pseudomallei , Melioidosis , Animales , Humanos , Burkholderia mallei/genética , Burkholderia pseudomallei/genética , Melioidosis/diagnóstico , Melioidosis/microbiología , Combinación Amoxicilina-Clavulanato de Potasio/farmacología , beta-Lactamasas , Dominio Catalítico , Reacción en Cadena de la PolimerasaRESUMEN
In vitro activities of delafloxacin and ciprofloxacin were evaluated against Burkholderia pseudomallei mutants expressing or lacking defined resistance-nodulation-cell division (RND) efflux pumps using CLSI methodology at pHs of 5.8 and 7.2. Delafloxacin MIC values were as much as 8-fold lower at pH 5.8 than those at pH 7.2, while ciprofloxacin MICs increased as much as 8-fold. The data from this study suggest that compared to ciprofloxacin, delafloxacin may have improved efflux avoidance, notably at acidic pH. In contrast to ciprofloxacin, delafloxacin may thus retain its therapeutic potential, even in BpeEF-OprC efflux-pump-expressing B. pseudomallei strains that compromise the use of fluoroquinolones, such as ciprofloxacin. IMPORTANCE Resistance-nodulation-cell division (RND) efflux pumps play a major role in intrinsic and acquired antibiotic resistance in Burkholderia pseudomallei, and these pumps are its only known multidrug resistance determinants. Fluoroquinolones have performed poorly in clinical settings and are currently not recommended for treatment of B. pseudomallei infections. While the reasons for the poor clinical performance of this pathogen remain unclear, efflux may be partially responsible since fluoroquinolones like ciprofloxacin are prone to efflux by RND pumps, notably BpeEF-OprC. In vitro efficacy testing using a panel of efflux-proficient and efflux-deficient strains allows identification of fluoroquinolones that compared to ciprofloxacin are less prone to efflux.
Asunto(s)
Burkholderia pseudomallei , Burkholderia pseudomallei/genética , Farmacorresistencia Bacteriana Múltiple , Antibacterianos/farmacología , Fluoroquinolonas/farmacología , Pruebas de Sensibilidad Microbiana , Ciprofloxacina/farmacologíaRESUMEN
BACKGROUND: Pneumonic plague (PP), caused by Yersinia pestis, is the most feared clinical form of plague due to its rapid lethality and potential to cause outbreaks. PP outbreaks are now rare due to antimicrobial therapy. METHODS: A PP outbreak in Madagascar involving transmission of a Y. pestis strain resistant to streptomycin, the current recommended first-line treatment in Madagascar, was retrospectively characterized using epidemiology, clinical diagnostics, molecular characterization, and animal studies. RESULTS: The outbreak occurred in February 2013 in the Faratsiho district of Madagascar and involved 22 cases, including 3 untreated fatalities. The 19 other cases participated in funeral practices for the fatal cases and fully recovered after combination antimicrobial therapy: intramuscular streptomycin followed by oral co-trimoxazole. The Y. pestis strain that circulated during this outbreak is resistant to streptomycin resulting from a spontaneous point mutation in the 30S ribosomal protein S12 (rpsL) gene. This same mutation causes streptomycin resistance in 2 unrelated Y. pestis strains, one isolated from a fatal PP case in a different region of Madagascar in 1987 and another isolated from a fatal PP case in China in 1996, documenting this mutation has occurred independently at least 3 times in Y. pestis. Laboratory experiments revealed this mutation has no detectable impact on fitness or virulence, and revertants to wild-type are rare in other species containing it, suggesting Y. pestis strains containing it could persist in the environment. CONCLUSIONS: Unique antimicrobial resistant (AMR) strains of Y. pestis continue to arise in Madagascar and can be transmitted during PP outbreaks.
Asunto(s)
Peste , Yersinia pestis , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Brotes de Enfermedades , Peste/tratamiento farmacológico , Peste/epidemiología , Estudios Retrospectivos , Yersinia pestis/genéticaRESUMEN
Burkholderia cepacia complex (Bcc) and Burkholderia pseudomallei complex (Bpc) species include pathogens that are typically multidrug resistant. Dominant intrinsic and acquired multidrug resistance mechanisms are efflux mediated by pumps of the resistance-nodulation-cell division (RND) family. From comparative bioinformatic and, in many instances, functional studies, we infer that RND pump-based resistance mechanisms are conserved in Burkholderia. We propose to use these findings as a foundation for adoption of a uniform RND efflux pump nomenclature.
Asunto(s)
Complejo Burkholderia cepacia , Burkholderia pseudomallei , Antibacterianos/farmacología , Complejo Burkholderia cepacia/genética , Burkholderia pseudomallei/genética , División Celular , Farmacorresistencia Microbiana , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad MicrobianaRESUMEN
Burkholderia ubonensis, a nonpathogenic soil bacterium belonging to the Burkholderia cepacia complex (Bcc), is highly resistant to some clinically significant antibiotics. The concern is that B. ubonensis may serve as a resistance reservoir for Bcc or B. pseudomallei complex (Bpc) organisms that are opportunistic human pathogens. Using a B. ubonensis strain highly resistant to tetracycline (MIC, ≥256 µg/ml), we identified and characterized tetA(64) that encodes a novel tetracycline-specific efflux pump of the major facilitator superfamily. TetA(64) and associated TetR(64) regulator expression are induced by tetracyclines. Although TetA(64) is the primary tetracycline and doxycycline resistance determinant, maximum tetracycline and doxycycline resistance requires synergy between TetA(64) and the nonspecific AmrAB-OprA resistance nodulation cell division efflux pump. TetA(64) does not efflux minocycline, tigecycline, and eravacycline. Comprehensive screening of genome sequences showed that TetA(64) is unequally distributed in the Bcc and absent from the Bpc. It is present in some major cystic fibrosis pathogens, like Burkholderia cenocepacia, but absent from others like Burkholderia multivorans The tetR(64)-tetA(64) genes are located in a region of chromosome 1 that is highly conserved in Burkholderia sp. Because there is no evidence for transposition, the tetR(64)-tetA(64) genes may have been acquired by homologous recombination after horizontal gene transfer. Although Burkholderia species contain a resident multicomponent efflux pump that allows them to respond to tetracyclines up to a certain concentration, the acquisition of the single-component TetA(64) by some species likely provides the synergy that these bacteria need to defend against high tetracycline concentrations in niche environments.
Asunto(s)
Complejo Burkholderia cepacia , Tetraciclina , Antibacterianos/farmacología , Burkholderia , Complejo Burkholderia cepacia/genética , Humanos , Tetraciclina/farmacología , Resistencia a la Tetraciclina/genéticaRESUMEN
Burkholderia pseudomallei, the founding member of the B. pseudomallei complex (Bpc), is a biothreat agent and causes melioidosis, a disease whose treatment mainly relies on ceftazidime and meropenem. The concern is that B. pseudomallei could enhance its drug resistance repertoire by the acquisition of DNA from resistant near-neighbor species. Burkholderia ubonensis, a member of the B. cepacia complex (Bcc), is commonly coisolated from environments where B. pseudomallei is present. Unlike B. pseudomallei, in which significant primary carbapenem resistance is rare, it is not uncommon in B. ubonensis, but the underlying mechanisms are unknown. We established that carbapenem resistance in B. ubonensis is due to an inducible class A PenB ß-lactamase, as has been shown for other Bcc bacteria. Inducibility is not sufficient for high-level resistance but also requires other determinants, such as a PenB that is more robust than that present in susceptible isolates, as well as other resistance factors. Curiously and diagnostic for the two complexes, both Bpc and Bcc bacteria contain distinct annotated PenA class A ß-lactamases. However, the protein from Bcc bacteria is missing its essential active-site serine and, therefore, is not a ß-lactamase. Regulated expression of a transcriptional penB'-lacZ (ß-galactosidase) fusion in the B. pseudomallei surrogate B. thailandensis confirms that although Bpc bacteria lack an inducible ß-lactamase, they contain the components required for responding to aberrant peptidoglycan synthesis resulting from ß-lactam challenge. Understanding the diversity of antimicrobial resistance in Burkholderia species is informative about how the challenges arising from potential resistance transfer between them can be met.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a tropical disease that is highly fatal if not properly treated. Our data show that, in contrast to B. pseudomallei, B. ubonensis ß-lactam resistance is fundamentally different because intrinsic resistance is mediated by an inducible class A ß-lactamase. This includes resistance to carbapenems. Our work demonstrates that studies with near-neighbor species are informative about the diversity of antimicrobial resistance in Burkholderia and can also provide clues about the potential of resistance transfer between bacteria inhabiting the same environment. Knowledge about potential adverse challenges resulting from the horizontal transfer of resistance genes between members of the two complexes enables the design of effective countermeasures.
Asunto(s)
Antibacterianos/farmacología , Complejo Burkholderia cepacia/enzimología , Burkholderia pseudomallei/enzimología , Burkholderia/efectos de los fármacos , Meropenem/farmacología , Resistencia betalactámica/genética , beta-Lactamasas/genética , Burkholderia/enzimología , Complejo Burkholderia cepacia/genética , Burkholderia pseudomallei/genética , Humanos , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/clasificaciónRESUMEN
Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Tier-1 Select Agents that cause melioidosis and glanders, respectively. These are highly lethal human infections with limited therapeutic options. Intercellular spread is a hallmark of Burkholderia pathogenesis, and its prominent ties to virulence make it an attractive therapeutic target. We developed a high-throughput cell-based phenotypic assay and screened â¼220,000 small molecules for their ability to disrupt intercellular spread by Burkholderia thailandensis, a closely related BSL-2 surrogate. We identified 268 hits, and cross-species validation found 32 hits that also disrupt intercellular spread by Bp and/or Bm Among these were a fluoroquinolone analog, which we named burkfloxacin (BFX), which potently inhibits growth of intracellular Burkholderia, and flucytosine (5-FC), an FDA-approved antifungal drug. We found that 5-FC blocks the intracellular life cycle at the point of type VI secretion system 5 (T6SS-5)-mediated cell-cell spread. Bacterial conversion of 5-FC to 5-fluorouracil and subsequently to fluorouridine monophosphate is required for potent and selective activity against intracellular Burkholderia In a murine model of fulminant respiratory melioidosis, treatment with BFX or 5-FC was significantly more effective than ceftazidime, the current antibiotic of choice, for improving survival and decreasing bacterial counts in major organs. Our results demonstrate the utility of cell-based phenotypic screening for Select Agent drug discovery and warrant the advancement of BFX and 5-FC as candidate therapeutics for melioidosis in humans.
Asunto(s)
Burkholderia pseudomallei/efectos de los fármacos , Ciprofloxacina/farmacología , Reposicionamiento de Medicamentos , Flucitosina/farmacología , Melioidosis/tratamiento farmacológico , Animales , Burkholderia pseudomallei/patogenicidad , Ciprofloxacina/análogos & derivados , Ciprofloxacina/uso terapéutico , Citoplasma/efectos de los fármacos , Citoplasma/microbiología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Flucitosina/uso terapéutico , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Melioidosis/microbiología , Ratones , Pruebas de Sensibilidad Microbiana , Resultado del Tratamiento , VirulenciaRESUMEN
Ceftazidime (CAZ) is the antibiotic of choice for the treatment of Burkholderia pseudomallei infection (melioidosis). The chromosomally-encoded PenA ß-lactamase possesses weak cephalosporinase activity. The wild-type penA gene confers clinically significant CAZ resistance only when overexpressed due to a promoter mutation, transcriptional antitermination or by gene duplication and amplification (GDA). Here we characterise a reversible 33-kb GDA event involving wild-type penA in a CAZ-resistant B. pseudomallei clinical isolate from Thailand. We show that duplication arises from exchanges between short (<10 bp) chromosomal sequences, which in this example consist of 4-bp repeats flanked by 3-bp inverted repeats. GDA involving ß-lactamases may be a common CAZ resistance mechanism in B. pseudomallei.
Asunto(s)
Antibacterianos/farmacología , Burkholderia pseudomallei/efectos de los fármacos , Ceftazidima/farmacología , Farmacorresistencia Bacteriana , Amplificación de Genes , Duplicación de Gen , beta-Lactamasas/genética , Burkholderia pseudomallei/enzimología , Burkholderia pseudomallei/genética , ADN Bacteriano/genética , Humanos , Melioidosis/microbiología , TailandiaRESUMEN
AIM: To develop a probe-based triplex quantitative real-time PCR assay to simultaneously detect the upregulation of the efflux pumps AmrAB-OprA, BpeAB-OprB and BpeEF-OprC in Burkholderia pseudomallei strains exhibiting increased minimum inhibitory concentrations toward meropenem, doxycycline or trimethoprim-sulfamethoxazole. METHODS: The triplex assay was developed and subsequently tested on RNA isolated from eight clinical and eight laboratory-generated B. pseudomallei mutants harboring efflux pump regulator mutations. RESULTS: The triplex assay accurately detected efflux pump upregulation in all clinical and laboratory mutants, which corresponded with decreased antibiotic susceptibility or antibiotic resistance. CONCLUSION: Rapid detection of antibiotic resistance provides clinicians with a tool to identify potential treatment failure in near real time, enabling informed alteration of treatment during an infection and improved patient outcomes.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Burkholderia pseudomallei/aislamiento & purificación , Farmacorresistencia Bacteriana , Melioidosis/microbiología , Proteínas de Transporte de Membrana/genética , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/efectos de los fármacos , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/metabolismo , Humanos , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , MutaciónRESUMEN
Burkholderia pseudomallei causes the severe disease melioidosis. The bacterium subverts the host immune system and replicates inside cells, and host mortality results primarily from sepsis-related complications. Lipopolysaccharide (LPS) is a major virulence factor and mediator of sepsis that many pathogens capable of intracellular growth modify to reduce their immunological "footprint." The binding strength of B. pseudomallei LPS for human LPS binding protein (hLBP) was measured using surface plasmon resonance. The structures of lipid A isolated from B. pseudomallei under different temperatures were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and the gene expression of two lipid A remodeling genes, lpxO and pagL, was investigated. The LPS was characterized for its ability to trigger tumor necrosis factor alpha (TNF-α) release and to activate caspase-11-triggered pyroptosis by introduction of LPS into the cytosol. Lipid A from long-term chronic-infection isolates was isolated and characterized by MALDI-TOF MS and also by the ability to trigger caspase-11-mediated cell death. Lipid A from B. pseudomallei 1026b lpxO and pagL mutants were characterized by positive- and negative-mode MALDI-TOF MS to ultimately identify their role in lipid A structural modifications. Replication of lpxO and pagL mutants and their complements within macrophages showed that lipid A remodeling can effect growth in host cells and activation of caspase-11-mediated cytotoxicity.
Asunto(s)
Burkholderia pseudomallei/metabolismo , Burkholderia pseudomallei/patogenicidad , Lípido A/metabolismo , Lipopolisacáridos/metabolismo , Melioidosis/microbiología , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/metabolismo , Animales , Apoptosis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/crecimiento & desarrollo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Lípido A/química , Melioidosis/genética , Melioidosis/metabolismo , Melioidosis/fisiopatología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Viabilidad Microbiana , Unión Proteica , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Burkholderia pseudomallei, the cause of melioidosis, is intrinsically resistant to many antibiotics. Acquired multidrug resistance, including resistance to doxycycline and co-trimoxazole used for melioidosis eradication phase therapy, is mainly attributed to constitutive expression of the BpeEF-OprC efflux pump. Constitutive expression of this pump is caused by mutations affecting two highly similar LysR-type transcriptional regulators (LTTR), BpeT and BpeS, but their interaction with the regulatory region governing BpeEF-OprC expression has not yet been studied. The bpeE-bpeF-oprC genes are distally located in the llpE-bpeE-bpeF-oprC operon. The llpE gene encodes a putative lipase/esterase of unknown function. We show that in a bpeT mutant llpE is constitutively co-transcribed with bpeE-bpeF-oprC. As expected from previous studies with B. cenocepacia, deletion of llpE does not affect antibiotic efflux. Using transcriptional bpeE'-lacZ fusions, we demonstrate that the 188 bp bpeT-llpE intergenic region located between bpeT and the llpE-bpeE-bpeF-oprC operon contains regulatory elements needed for control of bpeT and llpE-bpeE-bpeF-oprC operon expression. By native polyacrylamide gel electrophoresis and electrophoretic mobility shift assays with purified recombinant BpeT and BpeS proteins, we show BpeT and BpeS form oligomers that share a 14 bp binding site overlapping the essential region required for llpE-bpeE-bpeF-oprC expression. The binding site contains the conserved T-N11-A LTTR box motif involved in binding of LysR proteins, which in concert with two other possible LTTR boxes may mediate BpeT and BpeS regulation of BpeEF-OprC expression. These studies form the basis for further investigation of BpeEF-OprC expression and regulation at the molecular level by yet unknown external stimuli.
Asunto(s)
Burkholderia pseudomallei/enzimología , Burkholderia pseudomallei/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Antibacterianos/metabolismo , Sitios de Unión , Transporte Biológico Activo , ADN Bacteriano , Farmacorresistencia Bacteriana , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Eliminación de Gen , Operón , Unión Proteica , Multimerización de Proteína , Transcripción GenéticaRESUMEN
Therapy of Burkholderia pseudomallei acute infections is largely limited to a few ß-lactam antibiotics such as ceftazidime or meropenem. Although relatively rare, resistance emergence during therapy leads to treatment failures with high mortality rates. In the absence of acquired external resistance determinants in B. pseudomallei emergence of ß-lactam resistance is invariably caused by mutational modification of genomically encoded factors. These include the deletion of the ceftazidime target penicillin-binding protein 3 or amino acid changes in the Class A PenA ß-lactamase that expand its substrate spectrum, as well as penA gene duplication and amplification or its overexpression via transcriptional up-regulation. Evidence is presented that penA is co-transcribed with the upstream nlpD1 gene, that the transcriptional terminator for nlpD1 serves as a penA attenuator and that generation of a new promoter immediately upstream of the terminator/attenuator by a conserved G to A transition leads to anti-termination and thus constitutive PenA expression and extended ß-lactam resistance. Further evidence obtained with the extensively ß-lactam resistant clinical isolate Bp1651 shows that in addition to PenA overexpression and structural mutations other adaptive mechanisms contribute to intrinsic and acquired B. pseudomallei ß-lactam resistance.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Burkholderia pseudomallei/genética , Lipoproteínas/genética , Melioidosis/tratamiento farmacológico , Resistencia betalactámica/genética , beta-Lactamasas/genética , Antibacterianos/uso terapéutico , Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/efectos de los fármacos , Ceftazidima/farmacología , Ceftazidima/uso terapéutico , Duplicación de Gen/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos/efectos de los fármacos , Genes Bacterianos/genética , Interacciones Huésped-Patógeno/genética , Humanos , Lipoproteínas/metabolismo , Melioidosis/microbiología , Meropenem/uso terapéutico , Pruebas de Sensibilidad Microbiana , Mutación/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Procesamiento Postranscripcional del ARN , ARN Bacteriano/genética , Regulación hacia Arriba/efectos de los fármacos , Resistencia betalactámica/efectos de los fármacos , beta-Lactamasas/metabolismoRESUMEN
Burkholderia pseudomallei, the causative agent of melioidosis, is an important public health threat due to limited therapeutic options for treatment. Efforts to improve therapeutics for B. pseudomallei infections are dependent on the need to understand the role of B. pseudomallei biofilm formation and its contribution to antibiotic tolerance and persistence as these are bacterial traits that prevent effective therapy. In order to reveal the genes that regulate and/or contribute to B. pseudomallei 1026b biofilm formation, we screened a sequence defined two-allele transposon library and identified 118 transposon insertion mutants that were deficient in biofilm formation. These mutants include transposon insertions in genes predicted to encode flagella, fimbriae, transcriptional regulators, polysaccharides, and hypothetical proteins. Polysaccharides are key constituents of biofilms and B. pseudomallei has the capacity to produce a diversity of polysaccharides, thus there is a critical need to link these biosynthetic genes with the polysaccharides they produce to better understand their biological role during infection. An allelic exchange deletion mutant of the entire B. pseudomallei biofilm-associated exopolysaccharide biosynthetic cluster was decreased in biofilm formation and produced a smooth colony morphology suggestive of the loss of exopolysaccharide production. Conversely, deletion of the previously defined capsule I polysaccharide biosynthesis gene cluster increased biofilm formation. Bioinformatics analyses combined with immunoblot analysis and glycosyl composition studies of the partially purified exopolysaccharide indicate that the biofilm-associated exopolysaccharide is neither cepacian nor the previously described acidic exopolysaccharide. The biofilm-associated exopolysaccharide described here is also specific to the B. pseudomallei complex of bacteria. Since this novel exopolysaccharide biosynthesis cluster is retained in B. mallei, it is predicted to have a role in colonization and infection of the host. These findings will facilitate further advances in understanding the pathogenesis of B. pseudomallei and improve diagnostics and therapeutic treatment strategies.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Burkholderia pseudomallei/genética , Burkholderia pseudomallei/fisiología , Genoma Bacteriano , Polisacáridos Bacterianos/genética , Burkholderia cenocepacia/genética , Hibridación Genómica Comparativa , Regulación Bacteriana de la Expresión Génica , Familia de Multigenes , MutaciónRESUMEN
The Pseudomonas aeruginosa PAO1 thiol peroxidase homolog (Tpx) belongs to a family of enzymes implicated in the removal of toxic peroxides. We have shown the expression of tpx to be highly inducible with redox cycling/superoxide generators and diamide and weakly inducible with organic hydroperoxides and hydrogen peroxide (H(2)O(2)). The PAO1 tpx pattern is unlike the patterns for other peroxide-scavenging genes in P. aeruginosa. Analysis of the tpx promoter reveals the presence of a putative IscR binding site located near the promoter. The tpx expression profiles in PAO1 and the iscR mutant, together with results from gel mobility shift assays showing that purified IscR specifically binds the tpx promoter, support the role of IscR as a transcriptional repressor of tpx that also regulates the oxidant-inducible expression of the gene. Recombinant Tpx has been purified and biochemically characterized. The enzyme catalyzes thioredoxin-dependent peroxidation and can utilize organic hydroperoxides and H(2)O(2) as substrates. The Δtpx mutant demonstrates differential sensitivity to H(2)O(2) only at moderate concentrations (0.5 mM) and not at high (20 mM) concentrations, suggesting a novel protective role of tpx against H(2)O(2) in P. aeruginosa. Altogether, P. aeruginosa tpx is a novel member of the IscR regulon and plays a primary role in protecting the bacteria from submillimolar concentrations of H(2)O(2).
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/toxicidad , Peroxidasa/metabolismo , Pseudomonas aeruginosa/enzimología , Proteínas Represoras/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Sitios de Unión , ADN Bacteriano/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Eliminación de Gen , Perfilación de la Expresión Génica , Peroxidasa/genética , Peroxidasa/aislamiento & purificación , Regiones Promotoras Genéticas , Unión Proteica , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , RegulónRESUMEN
BACKGROUND: Burkholderia gladioli pathovar cocovenenans (BGC) is responsible for sporadic food-poisoning outbreaks with high morbidity and mortality in Asian countries. Little is known about the regulation of virulence factor and toxin production in BGC, and studies in this bacterium have been hampered by lack of genetic tools. FINDINGS: Establishment of a comprehensive antibiotic susceptibility profile showed that BGC strain ATCC33664 is susceptible to a number of antibiotics including aminoglycosides, carbapenems, fluoroquinolones, tetracyclines and trimethoprim. In this study, we established that gentamicin, kanamycin and trimethoprim are good selection markers for use in BGC. Using a 10 min method for preparation of electrocompetent cells, the bacterium could be transformed by electroporation at high frequencies with replicative plasmids containing the pRO1600-derived origin of replication. These plasmids exhibited a copy number of > 100 in BGC. When co-conjugated with a transposase expressing helper plasmid, mini-Tn7 vectors inserted site- and orientation-specifically at a single glmS-associated insertion site in the BGC genome. Lastly, a Himar1 transposon was used for random transposon mutagenesis of BGC. CONCLUSIONS: A series of genetic tools previously developed for other Gram-negative bacteria was adapted for use in BGC. These tools now facilitate genetic studies of this pathogen and allow establishment of toxin biosynthetic pathways and their genetic regulation.
RESUMEN
A novel five-domain Kazal-type serine proteinase inhibitor, SPIPm2, identified from the hemocyte cDNA library of black tiger shrimp Penaeus monodon was successfully expressed in the Escherichia coli expression system. The expressed recombinant SPIPm2 (rSPIPm2) as inclusion bodies was solubilized with a sodium carbonate buffer, pH10, and purified by gel filtration chromatography. The molecular mass of rSPIPm2 was determined using MALDI-TOF mass spectrometry to be 29.065 kDa. The inhibitory activities of rSPIPm2 were tested against trypsin, alpha-chymotrypsin, subtilisin and elastase. The inhibitor exhibited potent inhibitory activities against subtilisin and elastase, weak inhibitory activity against trypsin, and did not inhibit chymotrypsin. Tight-binding inhibition assay suggested that the molar ratios of SPIPm2 to subtilisin and elastase were 1:2 and 1:1, respectively. The inhibition against subtilisin and elastase was a competitive type with inhibition constants (Ki) of 0.52 and 3.27 nM, respectively. The inhibitory activity of SPIPm2 against subtilisin implies that, in shrimp, it may function as a defense component against proteinases from pathogenic bacteria but the elastase inhibitory function is not known.