Crystal structures of multidrug efflux transporters from Burkholderia pseudomallei suggest details of transport mechanism.

BpeB and BpeF are multidrug efflux transporters from Burkholderia pseudomallei that enable multidrug resistance. Here, we report the crystal structures of BpeB and BpeF at 2.94 Å and 3.0 Å resolution, respectively. BpeB was found as an asymmetric trimer, consistent with the widely-accepted functional rotation mechanism for this type of transporter. One of the monomers has a distinct structure that we interpret as an intermediate along this functional cycle. Additionally, a detergent molecule bound in a previously undescribed binding site provides insights into substrate translocation through the pathway. BpeF shares structural similarities with the crystal structure of OqxB from Klebsiella pneumoniae, where both are symmetric trimers composed of three "binding"-state monomers. The structures of BpeB and BpeF further our understanding of the functional mechanisms of transporters belonging to the HAE1-RND superfamily.

Exploring Cefiderocol Resistance Mechanisms in Burkholderia pseudomallei.

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.

Transmission of Antimicrobial Resistant Yersinia pestis During a Pneumonic Plague Outbreak.

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.

Expanding the Burkholderia pseudomallei Complex with the Addition of Two Novel Species: Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov.

Distinct Burkholderia strains were isolated from soil samples collected in tropical northern Australia (Northern Territory and the Torres Strait Islands, Queensland). Phylogenetic analysis of 16S rRNA and whole genome sequences revealed these strains were distinct from previously described Burkholderia species and assigned them to two novel clades within the B. pseudomallei complex (Bpc). Because average nucleotide identity and digital DNA-DNA hybridization calculations are consistent with these clades representing distinct species, we propose the names Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov. Strains assigned to B. mayonis sp. nov. include type strain BDU6T (=TSD-80; LMG 29941; ASM152374v2) and BDU8. Strains assigned to B. savannae sp. nov. include type strain MSMB266T (=TSD-82; LMG 29940; ASM152444v2), MSMB852, BDU18, and BDU19. Comparative genomics revealed unique coding regions for both putative species, including clusters of orthologous genes associated with phage. Type strains of both B. mayonis sp. nov. and B. savannae sp. nov. yielded biochemical profiles distinct from each other and from other species in the Bpc, and profiles also varied among strains within B. mayonis sp. nov. and B. savannae sp. nov. Matrix-assisted laser desorption ionization time-of-flight (MLST) analysis revealed a B. savannae sp. nov. cluster separate from other species, whereas B. mayonis sp. nov. strains did not form a distinct cluster. Neither B. mayonis sp. nov. nor B. savannae sp. nov. caused mortality in mice when delivered via the subcutaneous route. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in a total of eight species currently within the Bpc. IMPORTANCEBurkholderia species can be important sources of novel natural products, and new species are of interest to diverse scientific disciplines. Although many Burkholderia species are saprophytic, Burkholderia pseudomallei is the causative agent of the disease melioidosis. Understanding the genomics and virulence of the closest relatives to B. pseudomallei, i.e., the other species within the B. pseudomallei complex (Bpc), is important for identifying robust diagnostic targets specific to B. pseudomallei and for understanding the evolution of virulence in B. pseudomallei. Two proposed novel species, B. mayonis sp. nov. and B. savannae sp. nov., were isolated from soil samples collected from multiple locations in northern Australia. The two proposed species belong to the Bpc but are phylogenetically distinct from all other members of this complex. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in a total of eight species within this significant complex of bacteria that are available for future studies.

An in situ high-throughput screen identifies inhibitors of intracellular Burkholderia pseudomallei with therapeutic efficacy.

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 ∼220,000 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.

Loss of RND-Type Multidrug Efflux Pumps Triggers Iron Starvation and Lipid A Modifications in Pseudomonas aeruginosa.

Transporters belonging to the resistance-nodulation-division (RND) superfamily of proteins are invariably present in the genomes of Gram-negative bacteria and are largely responsible for the intrinsic antibiotic resistance of these organisms. The numbers of genes encoding RND transporters per genome vary from 1 to 16 and correlate with the environmental versatilities of bacterial species. Pseudomonas aeruginosa strain PAO1, a ubiquitous nosocomial pathogen, possesses 12 RND pumps, which are implicated in the development of clinical multidrug resistance and known to contribute to virulence, quorum sensing, and many other physiological functions. In this study, we analyzed how P. aeruginosa's physiology adapts to a lack of RND-mediated efflux activities. A combination of transcriptomics, metabolomics, genetic, and analytical approaches showed that the P. aeruginosa PΔ6 strain, lacking the six best-characterized RND pumps, activates a specific adaptation response that involves significant changes in the abundance and activities of several transport system, quorum sensing, iron acquisition, and lipid A modification pathways. Our results demonstrate that these cells accumulate large quantities of Pseudomonas quinolone signals (PQS), which triggers iron starvation and activation of siderophore biosynthesis and acquisition pathways. The accumulation of iron in turn activates lipid A modification and membrane protection pathways. A transcriptionally regulated RND pump, MuxABC-OpmB, contributes to these transformations by controlling the concentration of coumarins. Our results suggest that these changes reduce the permeability barrier of the outer membrane and are needed to protect the cell envelope of efflux-deficient P. aeruginosa.

Burkholderia ubonensis High-Level Tetracycline Resistance Is Due to Efflux Pump Synergy Involving a Novel TetA(64) Resistance Determinant.

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.

Conservation of Resistance-Nodulation-Cell Division Efflux Pump-Mediated Antibiotic Resistance in Burkholderia cepacia Complex and Burkholderia pseudomallei Complex Species.

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.

Efflux Pumps of Burkholderia thailandensis Control the Permeability Barrier of the Outer Membrane.

Burkholderia comprises species that are significant biothreat agents and common contaminants of pharmaceutical production facilities. Their extreme antibiotic resistance affects all classes of antibiotics, including polycationic polymyxins and aminoglycosides. The major underlying mechanism is the presence of two permeability barriers, the outer membrane with modified lipid A moieties and active drug efflux pumps. The two barriers are thought to be mechanistically independent and act synergistically to reduce the intracellular concentrations of antibiotics. In this study, we analyzed the interplay between active efflux pumps and the permeability barrier of the outer membrane in Burkholderia thailandensis We found that three efflux pumps, AmrAB-OprA, BpeEF-OprC, and BpeAB-OprB, of B. thailandensis are expressed under standard laboratory conditions and provide protection against multiple antibiotics, including polycationic polymyxins. Our results further suggest that the inactivation of AmrAB-OprA or BpeAB-OprB potentiates the antibacterial activities of antibiotics not only by reducing their efflux, but also by increasing their uptake into cells. Mass spectrometry analyses showed that in efflux-deficient B. thailandensis cells, lipid A species modified with 4-amino-4-deoxy-l-aminoarabinose are significantly less abundant than in the parent strain. Taken together, our results suggest that changes in the outer membrane permeability due to alterations in lipid A structure could be contributing factors in antibiotic hypersusceptibilities of B. thailandensis cells lacking AmrAB-OprA and BpeAB-OprB efflux pumps.

GC-072: A Novel Therapeutic Candidate for Oral Treatment of Melioidosis and Infections Caused by Select Biothreat Pathogens.

Burkholderia pseudomallei (B. pseudomallei), the etiological agent of melioidosis, is a Gram-negative bacterium with additional concern as a biothreat pathogen. The mortality rate from B. pseudomallei varies depending on the type of infection and extent of available health care, but in the case of septicemia left untreated it can range from 50 - 90%. Current therapy for melioidosis is biphasic, consisting of parenteral acute-phase treatment for two weeks or longer, followed by oral eradication-phase treatment lasting several months. An effective oral therapeutic for outpatient treatment of acute-phase melioidosis is needed. GC-072 is a potent, 4-oxoquinolizine antibiotic with selective inhibitory activity against bacterial topoisomerases. GC-072 has demonstrated in vitro potency against susceptible and drug-resistant strains of B. pseudomallei and is also active against Burkholderia mallei, Bacillus anthracis, Yersinia pestis, and Francisella tularensis GC-072 is bactericidal both extra- and intracellularly, with rapid killing noted within a few hours and reduced development of resistance compared to ceftazidime. GC-072, delivered intragastrically to mimic oral administration, promoted dose-dependent survival in mice using lethal inhalational models of B. pseudomallei infection following exposure to a 24 or 339 LD50 challenge with B. pseudomallei strain 1026b. Overall, GC-072 appears to be a strong candidate for first-line, oral treatment of melioidosis.

Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii.

The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of Acinetobacter baumannii In this study, mini-Tn7 vectors with zeocin and apramycin selection markers were created by cloning the ble and aac(3)-IV genes, respectively, enabling either inducible gene expression (pUC18T-mini-Tn7T-Zeo-LAC and pUC18T-mini-Tn7T-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-Tn7T-Zeo and pUC18T-mini-Tn7T-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn7 insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn7 vectors described above. Combinations of these novel mini-Tn7 plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of A. baumannii In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of A. baumannii via these apramycin and zeocin mini-Tn7 constructs to demonstrate their application.IMPORTANCEAcinetobacter baumannii is a high-priority pathogen for which research on mechanisms of resistance and virulence is a critical need. Commonly used antibiotic selection markers are not suitable for use in MDR and XDR isolates of A. baumannii due to the high antibiotic resistance of these isolates, which poses a barrier to the study of this pathogen. This study demonstrates the practical potential of using apramycin and zeocin mini-Tn7- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of A. baumannii displaying MDR and XDR phenotypes.

Lipid A Remodeling Is a Pathoadaptive Mechanism That Impacts Lipopolysaccharide Recognition and Intracellular Survival of Burkholderia pseudomallei.

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.

Spatial transcriptomes within the Pseudomonas aeruginosa biofilm architecture.

Bacterial cooperative associations and dynamics in biofilm microenvironments are of special interest in recent years. Knowledge of localized gene-expression and corresponding bacterial behaviors within the biofilm architecture at a global scale has been limited, due to a lack of robust technology to study limited number of cells in stratified layers of biofilms. With our recent pioneering developments in single bacterial cell transcriptomic analysis technology, we generated herein an unprecedented spatial transcriptome map of the mature in vitro Pseudomonas aeruginosa biofilm model, revealing contemporaneous yet altered bacterial behaviors at different layers within the biofilm architecture (i.e., surface, middle and interior of the biofilm). Many genes encoding unknown functions were highly expressed at the biofilm-solid interphase, exposing a critical gap in the knowledge of their activities that may be unique to this interior niche. Several genes of unknown functions are critical for biofilm formation. The in vivo importance of these unknown proteins was validated in invertebrate (fruit fly) and vertebrate (mouse) models. We envisage the future value of this report to the community, in aiding the further pathophysiological understanding of P. aeruginosa biofilms. Our approach will open doors to the study of bacterial functional genomics of different species in numerous settings.

Molecular determinants of Burkholderia pseudomallei BpeEF-OprC efflux pump expression.

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.

Thermoregulation of Biofilm Formation in Burkholderia pseudomallei Is Disrupted by Mutation of a Putative Diguanylate Cyclase.

Burkholderia pseudomallei, a tier 1 select agent and the etiological agent of melioidosis, transitions from soil and aquatic environments to infect a variety of vertebrate and invertebrate hosts. During the transition from an environmental saprophyte to a mammalian pathogen, B. pseudomallei encounters and responds to rapidly changing environmental conditions. Environmental sensing systems that control cellular levels of cyclic di-GMP promote pathogen survival in diverse environments. Cyclic di-GMP controls biofilm production, virulence factors, and motility in many bacteria. This study is an evaluation of cyclic di-GMP-associated genes that are predicted to metabolize and interact with cyclic di-GMP as identified from the annotated genome of B. pseudomallei 1026b. Mutants containing transposon disruptions in each of these genes were characterized for biofilm formation and motility at two temperatures that reflect conditions that the bacteria encounter in the environment and during the infection of a mammalian host. Mutants with transposon insertions in a known phosphodiesterase (cdpA) and a predicted hydrolase (Bp1026b_I2285) gene exhibited decreased motility regardless of temperature. In contrast, the phenotypes exhibited by mutants with transposon insertion mutations in a predicted diguanylate cyclase gene (Bp1026b_II2523) were strikingly influenced by temperature and were dependent on a conserved GG(D/E)EF motif. The transposon insertion mutant exhibited enhanced biofilm formation at 37°C but impaired biofilm formation at 30°C. These studies illustrate the importance of studying behaviors regulated by cyclic di-GMP under varied environmental conditions in order to better understand cyclic di-GMP signaling in bacterial pathogens.IMPORTANCE This report evaluates predicted cyclic di-GMP binding and metabolic proteins from Burkholderia pseudomallei 1026b, a tier 1 select agent and the etiologic agent of melioidosis. Transposon insertion mutants with disruptions in each of the genes encoding these predicted proteins were characterized in order to identify key components of the B. pseudomallei cyclic di-GMP-signaling network. A predicted hydrolase and a phosphodiesterase that modulate swimming motility were identified, in addition to a diguanylate cyclase that modulates biofilm formation and motility in response to temperature. These studies warrant further evaluation of the contribution of cyclic di-GMP to melioidosis in the context of pathogen acquisition from environmental reservoirs and subsequent colonization, dissemination, and persistence within the host.

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis.

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and ß-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A ß-lactamase and was previously implicated in resistance to ß-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

An avirulent Burkholderia pseudomallei ∆purM strain with atypical type B LPS: expansion of the toolkit for biosafe studies of melioidosis.

BACKGROUND: The work was undertaken to expand the tools available for researching Burkholderia pseudomallei (Bp), the etiological agent of the tropical disease melioidosis. Melioidosis has the potential to pose a severe threat to public health and safety. In the United States, Bp is listed as a Tier-1 select agent by the Centers for Disease Control and Prevention (CDC), thus requiring high levels of regulation and biosafety level 3 (BSL3) facilities for experimental manipulation of live organisms. An avirulent ∆purM derivative of strain 1026b (Bp82) has proven to be a valuable tool for biosafe research as a select-agent excluded strain, but the high level of genetic diversity between Bp strains necessitates an expansion of the biosafe toolset. RESULTS: The ∆purM mutation was recapitulated in the Bp 576a strain, a serotype B background. An important difference between strains 1026b and 576a is the lipopolysaccharide (LPS), a major virulence factor and protective antigen. Polyclonal sera from 1026b-challenged non-human primates showed no cross reactivity with strain 576a LPS and low reactivity with whole cell lysate. Strain 576a replicates to higher levels in mouse organs and induces more TNF-α in the lungs of BALB/c mice compared to 1026b. The newly created Bp 576a ∆purM strain, designated 576mn, was auxotrophic for adenine in minimal media, capable of wild-type growth in rich media with addition of adenine, and auxotrophy was abrogated with single-copy complementation. Bp 576mn was unable to replicate in human cells and was avirulent in BALB/c mice following high-dose intranasal inoculation, similar to Bp82. Organ loads indicated a significant reduction in bacterial replication. CONCLUSIONS: In this work, the new biosafe strain 576mn with atypical type B LPS was generated. This strain should prove a valuable addition to the toolkit for biosafe studies of Bp and development of therapeutic and preventative strategies aimed at combatting melioidosis. Strain 576mn is an ideal candidate for select-agent exclusion.

Antibiotic resistance in Burkholderia species.

The genus Burkholderia comprises metabolically diverse and adaptable Gram-negative bacteria, which thrive in often adversarial environments. A few members of the genus are prominent opportunistic pathogens. These include Burkholderia mallei and Burkholderia pseudomallei of the B. pseudomallei complex, which cause glanders and melioidosis, respectively. Burkholderia cenocepacia, Burkholderia multivorans, and Burkholderia vietnamiensis belong to the Burkholderia cepacia complex and affect mostly cystic fibrosis patients. Infections caused by these bacteria are difficult to treat because of significant antibiotic resistance. The first line of defense against antimicrobials in Burkholderia species is the outer membrane penetration barrier. Most Burkholderia contain a modified lipopolysaccharide that causes intrinsic polymyxin resistance. Contributing to reduced drug penetration are restrictive porin proteins. Efflux pumps of the resistance nodulation cell division family are major players in Burkholderia multidrug resistance. Third and fourth generation ß-lactam antibiotics are seminal for treatment of Burkholderia infections, but therapeutic efficacy is compromised by expression of several ß-lactamases and ceftazidime target mutations. Altered DNA gyrase and dihydrofolate reductase targets cause fluoroquinolone and trimethoprim resistance, respectively. Although antibiotic resistance hampers therapy of Burkholderia infections, the characterization of resistance mechanisms lags behind other non-enteric Gram-negative pathogens, especially ESKAPE bacteria such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa.

Exposing a ß-Lactamase "Twist": the Mechanistic Basis for the High Level of Ceftazidime Resistance in the C69F Variant of the Burkholderia pseudomallei PenI ß-Lactamase.

Around the world, Burkholderia spp. are emerging as pathogens highly resistant to ß-lactam antibiotics, especially ceftazidime. Clinical variants of Burkholderia pseudomallei possessing the class A ß-lactamase PenI with substitutions at positions C69 and P167 are known to demonstrate ceftazidime resistance. However, the biochemical basis for ceftazidime resistance in class A ß-lactamases in B. pseudomallei is largely undefined. Here, we performed site saturation mutagenesis of the C69 position and investigated the kinetic properties of the C69F variant of PenI from B. pseudomallei that results in a high level of ceftazidime resistance (2 to 64 mg/liter) when expressed in Escherichia coli. Surprisingly, quantitative immunoblotting showed that the steady-state protein levels of the C69F variant ß-lactamase were ∼4-fold lower than those of wild-type PenI (0.76 fg of protein/cell versus 4.1 fg of protein/cell, respectively). However, growth in the presence of ceftazidime increases the relative amount of the C69F variant to greater than wild-type PenI levels. The C69F variant exhibits a branched kinetic mechanism for ceftazidime hydrolysis, suggesting there are two different conformations of the enzyme. When incubated with an anti-PenI antibody, one conformation of the C69F variant rapidly hydrolyzes ceftazidime and most likely contributes to the higher levels of ceftazidime resistance observed in cell-based assays. Molecular dynamics simulations suggest that the electrostatic characteristics of the oxyanion hole are altered in the C69F variant. When ceftazidime was positioned in the active site, the C69F variant is predicted to form a greater number of hydrogen-bonding interactions than PenI with ceftazidime. In conclusion, we propose "a new twist" for enhanced ceftazidime resistance mediated by the C69F variant of the PenI ß-lactamase based on conformational changes in the C69F variant. Our findings explain the biochemical basis of ceftazidime resistance in B. pseudomallei, a pathogen of considerable importance, and suggest that the full repertoire of conformational states of a ß-lactamase profoundly affects ß-lactam resistance.

Membrane-Bound PenA ß-Lactamase of Burkholderia pseudomallei.

Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. ß-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA ß-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying ß-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of ß-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits ß-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose.