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1.  In Vitro Properties of BAL30072, a Novel Siderophore Sulfactam with Activity against Multiresistant Gram-Negative Bacilli▿  
BAL30072 is a new monocyclic β-lactam antibiotic belonging to the sulfactams. Its spectrum of activity against significant Gram-negative pathogens with β-lactam-resistant phenotypes was evaluated and was compared with the activities of reference drugs, including aztreonam, ceftazidime, cefepime, meropenem, imipenem, and piperacillin-tazobactam. BAL30072 showed potent activity against multidrug-resistant (MDR) Pseudomonas aeruginosa and Acinetobacter sp. isolates, including many carbapenem-resistant strains. The MIC90s were 4 μg/ml for MDR Acinetobacter spp. and 8 μg/ml for MDR P. aeruginosa, whereas the MIC90 of meropenem for the same sets of isolates was >32 μg/ml. BAL30072 was bactericidal against both Acinetobacter spp. and P. aeruginosa, even against strains that produced metallo-β-lactamases that conferred resistance to all other β-lactams tested, including aztreonam. It was also active against many species of MDR isolates of the Enterobacteriaceae family, including isolates that had a class A carbapenemase or a metallo-β-lactamase. Unlike other monocyclic β-lactams, BAL30072 was found to trigger the spheroplasting and lysis of Escherichia coli rather than the formation of extensive filaments. The basis for this unusual property is its inhibition of the bifunctional penicillin-binding proteins PBP 1a and PBP 1b, in addition to its high affinity for PBP 3, which is the target of monobactams, such as aztreonam.
PMCID: PMC2876421  PMID: 20308379
2.  In vivo and in vitro activity of the siderophore monosulfactam BAL30072 against Acinetobacter baumannii 
New antibiotics that are active against multidrug-resistant (MDR) Acinetobacter baumannii are urgently needed. BAL30072, a siderophore monosulfactam antibiotic that rapidly penetrates the outer membrane of A. baumannii and has potent activity against most isolates, including those harbouring AmpC β-lactamases and metallo- (class B) or OXA- (class D) carbapenemases, is being developed to meet that need.
We assessed the in vitro activity of BAL30072, meropenem and the combination of BAL30072 and meropenem (2:1 and 1:1 ratios) by MIC and time–kill studies. Proof-of-principle in vivo efficacy was determined using a rat soft-tissue infection model. Five diverse strains with defined phenotypic and genetic profiles were tested (AB307-0294, AB8407, AB1697, AB3340 and AB0057).
In microdilution assays, combining BAL30072 with meropenem lowered meropenem MICs 2–8-fold. In time–kill studies, the BAL30072 and meropenem combinations resulted in bactericidal concentrations 2–8-fold lower than those of meropenem or BAL30072 alone. In the rat model, BAL30072 was active against four of five strains (AB307-0294, AB8407, AB1697 and AB3340), including meropenem-susceptible and -non-susceptible strains. AB0057 was the only strain resistant to BAL30072 in vivo and in vitro (MIC >64 mg/L). Meropenem was active in vivo against two of the five strains tested (AB307-0294 and AB3340). Both BAL30072 and BAL30072 with meropenem were equally effective in vivo.
These data support the continued evaluation of BAL30072 for use in the treatment of infections caused by MDR A. baumannii.
PMCID: PMC3058568  PMID: 21393224
MICs; time–kill assays; drug susceptibility testing; multidrug resistant
3.  Involvement of Fe Uptake Systems and AmpC β-Lactamase in Susceptibility to the Siderophore Monosulfactam BAL30072 in Pseudomonas aeruginosa 
BAL30072 is a monosulfactam conjugated with an iron-chelating dihydroxypyridone moiety. It is active against Gram-negative bacteria, including multidrug-resistant Pseudomonas aeruginosa. We selected mutants with decreased susceptibilities to BAL30072 in P. aeruginosa PAO1 under a variety of conditions. Under iron-deficient conditions, mutants with overexpression of AmpC β-lactamase predominated. These mutants were cross-resistant to aztreonam and ceftazidime. Similar mutants were obtained after selection at >16× the MIC in iron-sufficient conditions. At 4× to 8× the MIC, mutants with elevated MIC for BAL30072 but unchanged MICs for aztreonam or ciprofloxacin were selected. The expression of ampC and the major efflux pump genes were also unchanged. These BAL30072-specific mutants were characterized by transcriptome analysis, which revealed upregulation of the Fe-dicitrate operon, FecIRA. Whole-genome sequencing showed that this resulted from a single nucleotide change in the Fur-box of the fecI promoter. Overexpression of either the FecI ECF sigma factor or the FecA receptor increased BAL30072 MICs 8- to 16-fold. A fecI mutant and a fecA mutant of PAO1 were hypersusceptible to BAL30072 (MICs < 0.06 μg/ml). The most downregulated gene belonged to the pyochelin synthesis operon, although mutants in pyochelin receptor or synthesis genes had unchanged MICs. The piuC gene, coding for a Fe(II)-dependent dioxygenase located next to the piuA iron receptor gene, was also downregulated. The MICs of BAL30072 for piuC and piuA transposon mutants were increased 8- and 16-fold, respectively. We conclude that the upregulation of the Fe-dicitrate system impacts the expression of other TonB-dependent iron transporters and that PiuA and PiuC contribute to the susceptibility of P. aeruginosa PAO1 to BAL30072.
PMCID: PMC3632904  PMID: 23422914
4.  The In Vitro Antibiotic Susceptibility of Malaysian Isolates of Burkholderia pseudomallei 
Acute melioidosis may present as localised or septicaemic infections and can be fatal if left untreated. Burkholderia pseudomallei resistant to antibiotics used for the treatment of melioidosis had been reported. The aim of this study was to determine the in vitro antibiotic susceptibility patterns of Burkholderia pseudomallei isolated in Malaysia to a panel of antibiotics used for the treatment of melioidosis and also to potential alternative antibiotics such as tigecycline, ampicillin/sulbactam, and piperacillin/tazobactam. A total of 170 Burkholderia pseudomallei isolates were subjected to minimum inhibitory concentration determination using E-test method to eleven antibiotics. All isolates were sensitive to meropenem and piperacillin/tazobactam. For ceftazidime, imipenem, amoxicillin/clavulanic acid, and doxycycline resistance was observed in 1 isolate (0.6%) for each of the antibiotics. Trimethoprim/sulfamethoxazole resistance was observed in 17 (10%) isolates. For other antibiotics, ampicillin/sulbactam, chloramphenicol, tigecycline, and ciprofloxacin resistance were observed in 1 (0.6%), 6 (3.5%), 60 (35.3%) and 98 (57.7%) isolates respectively. One isolate B170/06 exhibited resistance to 4 antibiotics, namely, ciprofloxacin, chloramphenicol, trimethoprim/sulfamethoxazole, and tigecycline. In conclusion, the Malaysian isolates were highly susceptible to the current antibiotics used in the treatment of melioidosis in Malaysia. Multiple resistances to the antibiotics used in the maintenance therapy are the cause for a concern.
PMCID: PMC3803121  PMID: 24194761
5.  Workshop on Treatment of and Postexposure Prophylaxis for Burkholderia pseudomallei and B. mallei Infection, 2010 
The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.
PMCID: PMC3557896  PMID: 23171644
Burkholderia pseudomallei; melioidosis; Burkholderia mallei; glanders; drug therapy; postexposure prophylaxis; ceftazidime; carbapenems; trimethoprim/sulfamethoxazole; combination; amoxicillin/potassium clavulanate; clavulanic acid bacteria; antibiotic; antibacterial drugs; antimicrobial drugs; bacteria; Suggested citation for this article: Lipsitz R; Garges S; Aurigemma R; Baccam P; Blaney DD; Cheng AC; et al. Workshop on treatment of and postexposure prophylaxis for Burkholderia pseudomallei and B. mallei infection; 2010. Emerg Infect Dis [Internet]. 2012 Dec [date cited].
6.  In vitro susceptibilities of Pseudomonas pseudomallei to 27 antimicrobial agents. 
Antimicrobial Agents and Chemotherapy  1990;34(10):2027-2029.
Clinical isolates of Pseudomonas pseudomallei isolated in Thailand from 1981 to 1989 were tested for their in vitro susceptibilities to 27 antimicrobial agents, including older and newer quinolones, broad-spectrum cephems, carbapenems, monobactams, penicillins, tetracyclines, chloramphenicol, rifamycin, sulfamethoxazole, trimethoprim, and fosfomycin. Tosufloxacin, meropenem, CS-533, and minocycline were active against P. pseudomallei at levels comparable to or even greater than those of antimicrobial agents tested in previous studies, such as ciprofloxacin, ceftazidime, imipenem, carumonam, and piperacillin. Drug-resistant P. pseudomallei was found in only 1% of the isolates. The drug-resistant P. pseudomallei isolates displayed a unique pattern of susceptibility to the above-listed drugs.
PMCID: PMC171985  PMID: 2291671
7.  Mechanisms of antibiotic resistance in Burkholderia pseudomallei: implications for treatment of melioidosis 
Future microbiology  2012;7(12):1389-1399.
Burkholderia pseudomallei is the etiologic agent of melioidosis. This multifaceted disease is difficult to treat, resulting in high morbidity and mortality. Treatment of B. pseudomallei infections is lengthy and necessitates an intensive phase (parenteral ceftazidime, amoxicillin–clavulanic acid or meropenem) and an eradication phase (oral trimethoprim–sulfamethoxazole). The main resistance mechanisms affecting these antibiotics include enzymatic inactivation, target deletion and efflux from the cell, and are mediated by chromosomally encoded genes. Overproduction and mutations in the class A PenA β-lactamase cause ceftazidime and amoxicillin–clavulanic acid resistance. Deletion of the penicillin binding protein 3 results in ceftazidime resistance. BpeEF–OprC efflux pump expression causes trimethoprim and trimethoprim–sulfamethoxazole resistance. Although resistance is still relatively rare, therapeutic efficacies may be compromised by resistance emergence due to increased use of antibiotics in endemic regions. Novel agents and therapeutic strategies are being tested and, in some instances, show promise as anti-B. pseudomallei infectives.
PMCID: PMC3568953  PMID: 23231488
antibiotics; Burkholderia pseudomallei; melioidosis; resistance; therapy
8.  In Vitro Susceptibilities of Burkholderia mallei in Comparison to Those of Other Pathogenic Burkholderia spp. 
Antimicrobial Agents and Chemotherapy  1999;43(11):2773-2775.
The in vitro antimicrobial susceptibilities of isolates of Burkholderia mallei to 16 antibiotics were assessed and compared with the susceptibilities of Burkholderia pseudomallei and Burkholderia cepacia. The antibiotic susceptibility profile of B. mallei resembled that of B. pseudomallei more closely than that of B. cepacia, which corresponds to their similarities in terms of biochemistry, antigenicity, and pathogenicity. Ceftazidime, imipenem, doxycycline, and ciprofloxacin were active against both B. mallei and B. pseudomallei. Gentamicin was active against B. mallei but not against B. pseudomallei. Antibiotics clinically proven to be effective in the treatment of melioidosis may therefore be effective for treating glanders.
PMCID: PMC89557  PMID: 10543761
9.  Molecular Investigations of PenA-mediated β-lactam Resistance in Burkholderia pseudomallei 
Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium’s intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation β-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to this cephalosporin has been observed. The chromosomally encoded penA gene encodes a putative twin arginine translocase (TAT)-secreted β-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT-signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold) reduced the susceptibility to six of the nine β-lactams tested and ≥16-fold for ampicillin, amoxicillin, and carbenicillin. Overexpression of penA by single-copy, chromosomal expression of the gene under control of the inducible Ptac promoter, increased resistance levels for all β-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by ≥85- and 5- to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin and clavulanic acid. Susceptibility assays with PenA TAT-signal sequence and ΔtatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of penA expression.
PMCID: PMC3129521  PMID: 21747814
Burkholderia pseudomallei; melioidosis; antibiotic resistance; β-lactams; β-lactamase; TAT secretion
10.  Nitric oxide-dependent killing of aerobic, anaerobic and persistent Burkholderia pseudomallei 
Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe-4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.
PMCID: PMC3517295  PMID: 22521523
antibiotics; antimicrobials; melioidosis; reactive nitrogen species; therapy; [4Fe-4S] clusters
11.  Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection 
Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ), as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies, thereby improving patient outcomes for this serious disease.
PMCID: PMC3430440  PMID: 22977307
Burkholderia pseudomallei; ceftazidime; antibiotic resistance; melioidosis; β-lactamase; penA
12.  φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. mallei strain infectivity 
BMC Microbiology  2012;12:289.
Burkholderia pseudomallei and B. mallei are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237.
We have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains.
The broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.
PMCID: PMC3548686  PMID: 23217012
Bacteriophage; Burkholderia pseudomallei; B. mallei; P2; Prophage distribution; Phage-based diagnostics
13.  Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge 
Procedia in vaccinology  2010;2(1):10.1016/j.provac.2010.03.013.
Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.
PMCID: PMC3874274  PMID: 24379895
Burkholderia; B. mallei; B. pseudomallei; vaccine; subunit vaccination; intranasal infection
14.  Functional Characterization of Burkholderia pseudomallei Trimeric Autotransporters 
Infection and Immunity  2013;81(8):2788-2799.
Burkholderia pseudomallei is a tier 1 select agent and the causative agent of melioidosis, a severe and often fatal disease with symptoms ranging from acute pneumonia and septic shock to a chronic infection characterized by abscess formation in the lungs, liver, and spleen. Autotransporters (ATs) are exoproteins belonging to the type V secretion system family, with many playing roles in pathogenesis. The genome of B. pseudomallei strain 1026b encodes nine putative trimeric AT proteins, of which only four have been described. Using a bioinformatic approach, we annotated putative domains within each trimeric AT protein, excluding the well-studied BimA protein, and found short repeated sequences unique to Burkholderia species, as well as an unexpectedly large proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence to, invasion of, and plaque formation in A549 cells. The majority of the ATs (and/or the proteins encoded downstream) contributed to adherence to and efficient invasion of A549 cells. Using a BALB/c mouse model of infection, we determined the contributions of each AT to bacterial burdens in the lungs, liver, and spleen. At 48 h postinoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model.
PMCID: PMC3719557  PMID: 23716608
15.  Outcomes of Patients with Melioidosis Treated with Meropenem 
Melioidosis, an infection due to Burkholderia pseudomallei, is endemic in southeast Asia and northern Australia. We reviewed our experience with meropenem in the treatment of severe melioidosis in 63 patients over a 6-year period. Outcomes were similar to those of ceftazidime-treated patients (n = 153) despite a deliberate selection bias to more-unwell patients receiving meropenem. The mortality among meropenem-treated patients was 19%. One patient had a possible drug fever associated with the use of meropenem. We conclude that meropenem (1 g or 25 mg/kg every 8 h intravenously for ≥14 days) is an alternative to ceftazidime and imipenem in the treatment of melioidosis. The use of meropenem may be associated with improved outcomes in patients with severe sepsis associated with melioidosis.
PMCID: PMC400582  PMID: 15105132
16.  A Burkholderia pseudomallei ΔpurM Mutant Is Avirulent in Immunocompetent and Immunodeficient Animals: Candidate Strain for Exclusion from Select-Agent Lists▿  
Infection and Immunity  2010;78(7):3136-3143.
Burkholderia pseudomallei causes the disease melioidosis in humans and is classified as a category B select agent. Research utilizing this pathogen is highly regulated in the United States, and even basic studies must be conducted in biosafety level 3 (BSL-3) facilities. There is currently no attenuated B. pseudomallei strain available that is excluded from select-agent regulations and can be safely handled at BSL-2 facilities. To address this need, we created Bp82 and Bp190, which are ΔpurM derivatives of B. pseudomallei strains 1026b and K96243 that are deficient in adenine and thiamine biosynthesis but replication competent in vitro in rich medium. A series of animal challenge studies was conducted to ensure that these strains were fully attenuated. Whereas the parental strains 1026b and K96243 and the complemented mutants Bp410 and Bp454 were virulent in BALB/c mice following intranasal inoculation, the ΔpurM mutants Bp82 and Bp190 were avirulent even when they were administered at doses 4 logs higher than the doses used for the parental strains. Animals challenged with high doses of the ΔpurM mutants rapidly cleared the bacterium from tissues (lung, liver, and spleen) and remained free of culturable bacteria for the duration of the experiments (up to 60 days postinfection). Moreover, highly susceptible 129/SvEv mice and immune incompetent mice (IFN-γ−/−, SCID) were resistant to challenges with ΔpurM mutant Bp82. This strain was also avirulent in the Syrian hamster challenge model. We concluded that ΔpurM mutant Bp82 is fully attenuated and safe for use under BSL-2 laboratory conditions and thus is a candidate for exclusion from the select-agent list.
PMCID: PMC2897367  PMID: 20404077
17.  Genomic islands from five strains of Burkholderia pseudomallei 
BMC Genomics  2008;9:566.
Burkholderia pseudomallei is the etiologic agent of melioidosis, a significant cause of morbidity and mortality where this infection is endemic. Genomic differences among strains of B. pseudomallei are predicted to be one of the major causes of the diverse clinical manifestations observed among patients with melioidosis. The purpose of this study was to examine the role of genomic islands (GIs) as sources of genomic diversity in this species.
We found that genomic islands (GIs) vary greatly among B. pseudomallei strains. We identified 71 distinct GIs from the genome sequences of five reference strains of B. pseudomallei: K96243, 1710b, 1106a, MSHR668, and MSHR305. The genomic positions of these GIs are not random, as many of them are associated with tRNA gene loci. In particular, the 3' end sequences of tRNA genes are predicted to be involved in the integration of GIs. We propose the term "tRNA-mediated site-specific recombination" (tRNA-SSR) for this mechanism. In addition, we provide a GI nomenclature that is based upon integration hotspots identified here or previously described.
Our data suggest that acquisition of GIs is one of the major sources of genomic diversity within B. pseudomallei and the molecular mechanisms that facilitate horizontally-acquired GIs are common across multiple strains of B. pseudomallei. The differential presence of the 71 GIs across multiple strains demonstrates the importance of these mobile elements for shaping the genetic composition of individual strains and populations within this bacterial species.
PMCID: PMC2612704  PMID: 19038032
18.  Protection against Experimental Melioidosis following Immunization with Live Burkholderia thailandensis Expressing a manno-Heptose Capsule 
Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei. It is highly resistant to antibiotic treatment, and there is currently no licensed vaccine. Burkholderia thailandensis is a close relative of Burkholderia pseudomallei but is essentially avirulent in mammals. In this report, we detail the protective efficacy of immunization with live B. thailandensis E555, a strain which has been shown to express an antigenic capsule similar to that of B. pseudomallei. Immunization with E555 induced significant protection against a lethal intraperitoneal B. pseudomallei challenge in a mouse model of infection, with no mice succumbing to infection over the course of the study, even with challenges of up to 6,000 median lethal doses. By comparison, mice immunized with B. thailandensis not expressing a B. pseudomallei-like capsule had significantly decreased levels of protection. E555-immunized mice had significantly higher levels of IgG than mice immunized with noncapsulated B. thailandensis, and these antibody responses were primarily directed against the capsule.
PMCID: PMC3697456  PMID: 23677322
19.  Seronegative Bacteremic Melioidosis Caused by Burkholderia pseudomallei with Ambiguous Biochemical Profile: Clinical Importance of Accurate Identification by 16S rRNA Gene and groEL Gene Sequencing 
Journal of Clinical Microbiology  2003;41(8):3973-3977.
An aerobic gram-negative bacterium was isolated from the blood and sputum of an 84-year-old, chair-bound nursing home resident with acute bacteremic pneumonia. Although the phenotypic characteristics suggested that the bacterium could be Burkholderia pseudomallei, the Vitek 1 system (GNI+), which can successfully identify 99% of B. pseudomallei strains, showed that the bacterium was “unidentified.” Immunoglobulin G against the lipopolysaccharide (LPS) of B. pseudomallei, as detected by an LPS-based enzyme-linked immunosorbent assay with 95% sensitivity, was negative in both the acute-phase and convalescent-phase sera. Sequencing of the groEL gene showed that the isolate was B. pseudomallei. Proper identification of the bacterium in this study is crucial, since there would be a radical difference in the duration of antimicrobial therapy.
PMCID: PMC179777  PMID: 12904433
20.  Genomic Diversity of Burkholderia pseudomallei Clinical Isolates: Subtractive Hybridization Reveals a Burkholderia mallei-Specific Prophage in B. pseudomallei 1026b 
Journal of Bacteriology  2004;186(12):3938-3950.
Burkholderia pseudomallei is the etiologic agent of the disease melioidosis and is a category B biological threat agent. The genomic sequence of B. pseudomallei K96243 was recently determined, but little is known about the overall genetic diversity of this species. Suppression subtractive hybridization was employed to assess the genetic variability between two distinct clinical isolates of B. pseudomallei, 1026b and K96243. Numerous mobile genetic elements, including a temperate bacteriophage designated φ1026b, were identified among the 1026b-specific suppression subtractive hybridization products. Bacteriophage φ1026b was spontaneously produced by 1026b, and it had a restricted host range, infecting only Burkholderia mallei. It possessed a noncontractile tail, an isometric head, and a linear 54,865-bp genome. The mosaic nature of the φ1026b genome was revealed by comparison with bacteriophage φE125, a B. mallei-specific bacteriophage produced by Burkholderia thailandensis. The φ1026b genes for DNA packaging, tail morphogenesis, host lysis, integration, and DNA replication were nearly identical to the corresponding genes in φE125. On the other hand, φ1026b genes involved in head morphogenesis were similar to head morphogenesis genes encoded by Pseudomonas putida and Pseudomonas aeruginosa bacteriophages. Consistent with this observation, immunogold electron microscopy demonstrated that polyclonal antiserum against φE125 reacted with the tail of φ1026b but not with the head. The results presented here suggest that B. pseudomallei strains are genetically heterogeneous and that bacteriophages are major contributors to the genomic diversity of this species. The bacteriophage characterized in this study may be a useful diagnostic tool for differentiating B. pseudomallei and B. mallei, two closely related biological threat agents.
PMCID: PMC419931  PMID: 15175308
21.  Comparison of Diagnostic Laboratory Methods for Identification of Burkholderia pseudomallei 
Journal of Clinical Microbiology  2005;43(5):2201-2206.
Limited experience and a lack of validated diagnostic reagents make Burkholderia pseudomallei, the cause of melioidosis, difficult to recognize in the diagnostic microbiology laboratory. We compared three methods of confirming the identity of presumptive B. pseudomallei strains using a collection of Burkholderia species drawn from diverse geographic, clinical, and environmental sources. The 95 isolates studied included 71 B. pseudomallei and 3 B. thailandensis isolates. The API 20NE method identified only 37% of the B. pseudomallei isolates. The agglutinating antibody test identified 82% at first the attempt and 90% including results of a repeat test with previously negative isolates. Gas-liquid chromatography analysis of bacterial fatty acid methyl esters (GLC-FAME) identified 98% of the B. pseudomallei isolates. The agglutination test produced four false positive results, one B. cepacia, one B. multivorans, and two B. thailandensis. API produced three false positive results, one positive B. cepacia and two positive B. thailandensis. GLC-FAME analysis was positive for one B. cepacia isolate. On the basis of these results, the most robust B. pseudomallei discovery pathway combines the previously recommended isolate screening tests (Gram stain, oxidase test, gentamicin and polymyxin susceptibility) with monoclonal antibody agglutination on primary culture, followed by a repeat after 24 h incubation on agglutination-negative isolates and GLC-FAME analysis. Incorporation of PCR-based identification within this schema may improve percentages of recognition further but requires more detailed evaluation.
PMCID: PMC1153801  PMID: 15872242
22.  Polysaccharide Specific Monoclonal Antibodies Provide Passive Protection against Intranasal Challenge with Burkholderia pseudomallei 
PLoS ONE  2012;7(4):e35386.
Burkholderia pseudomallei is a Gram-negative bacillus that is the causative agent of melioidosis. The bacterium is inherently resistant to many antibiotics and mortality rates remain high in endemic areas. The lipopolysaccharide (LPS) and capsular polysaccharide (CPS) are two surface-associated antigens that contribute to pathogenesis. We previously developed two monoclonal antibodies (mAbs) specific to the CPS and LPS; the CPS mAb was shown to identify antigen in serum and urine from melioidosis patients. The goal of this study was to determine if passive immunization with CPS and LPS mAbs alone and in combination would protect mice from a lethal challenge with B. pseudomallei. Intranasal (i.n.) challenge experiments were performed with B. pseudomallei strains 1026b and K96423. Both mAbs provided significant protection when administered alone. A combination of mAbs was protective when low doses were administered. In addition, combination therapy provided a significant reduction in spleen colony forming units (cfu) compared to results when either the CPS or LPS mAbs were administered alone.
PMCID: PMC3328442  PMID: 22530013
23.  Isolation of Polymyxin B-Susceptible Mutants of Burkholderia pseudomallei and Molecular Characterization of Genetic Loci Involved in Polymyxin B Resistance 
Antimicrobial Agents and Chemotherapy  1999;43(11):2648-2656.
Burkholderia pseudomallei is a gram-negative bacterium that causes the disease known as melioidosis. This pathogen is endemic to Southeast Asia and northern Australia and is particularly problematic in northeastern Thailand. It has been previously reported that B. pseudomallei is resistant to the killing action of cationic antimicrobial peptides, including human neutrophil peptide, protamine sulfate, poly-l-lysine, magainins, and polymyxins. Recently, we have also found that the virulent clinical isolate B. pseudomallei 1026b is capable of replicating in media containing polymyxin B at concentrations of >100 mg/ml. In order to identify genetic loci that are associated with this particular resistance phenotype, we employed a Tn5-OT182 mutagenesis system in coordination with a replica plating screen to isolate polymyxin B-susceptible mutants. Of the 17,000 Tn5-OT182 mutants screened via this approach, five polymyxin B-susceptible mutants were obtained. Three of these mutants harbored Tn5-OT182 insertions within a genetic locus demonstrating strong homology to the lytB gene present in other gram-negative bacteria. Of the remaining two mutants, one contained a transposon insertion in a locus involved in lipopolysaccharide core biosynthesis (waaF), while the other contained an insertion in an open reading frame homologous to UDP-glucose dehydrogenase genes. Isogenic mutants were also constructed via allelic exchange and used in complementation analysis studies to further characterize the relative importance of each of the various genetic loci with respect to the polymyxin B resistance phenotype exhibited by B. pseudomallei 1026b.
PMCID: PMC89538  PMID: 10543742
24.  Characterization of Ceftazidime Resistance Mechanisms in Clinical Isolates of Burkholderia pseudomallei from Australia 
PLoS ONE  2012;7(2):e30789.
Burkholderia pseudomallei is a Gram-negative bacterium that causes the serious human disease, melioidosis. There is no vaccine against melioidosis and it can be fatal if not treated with a specific antibiotic regimen, which typically includes the third-generation cephalosporin, ceftazidime (CAZ). There have been several resistance mechanisms described for B. pseudomallei, of which the best described are amino acid changes that alter substrate specificity in the highly conserved class A β-lactamase, PenA. In the current study, we sequenced penA from isolates sequentially derived from two melioidosis patients with wild-type (1.5 µg/mL) and, subsequently, resistant (16 or ≥256 µg/mL) CAZ phenotypes. We identified two single-nucleotide polymorphisms (SNPs) that directly increased CAZ hydrolysis. One SNP caused an amino acid substitution (C69Y) near the active site of PenA, whereas a second novel SNP was found within the penA promoter region. In both instances, the CAZ resistance phenotype corresponded directly with the SNP genotype. Interestingly, these SNPs appeared after infection and under selection from CAZ chemotherapy. Through heterologous cloning and expression, and subsequent allelic exchange in the native bacterium, we confirmed the role of penA in generating both low-level and high-level CAZ resistance in these clinical isolates. Similar to previous studies, the amino acid substitution altered substrate specificity to other β-lactams, suggesting a potential fitness cost associated with this mutation, a finding that could be exploited to improve therapeutic outcomes in patients harboring CAZ resistant B. pseudomallei. Our study is the first to functionally characterize CAZ resistance in clinical isolates of B. pseudomallei and to provide proven and clinically relevant signatures for monitoring the development of antibiotic resistance in this important pathogen.
PMCID: PMC3283585  PMID: 22363490
25.  A Horizontal Gene Transfer Event Defines Two Distinct Groups within Burkholderia pseudomallei That Have Dissimilar Geographic Distributions▿ †  
Journal of Bacteriology  2007;189(24):9044-9049.
Burkholderia pseudomallei is the etiologic agent of melioidosis. Many disease manifestations are associated with melioidosis, and the mechanisms causing this variation are unknown; genomic differences among strains offer one explanation. We compared the genome sequences of two strains of B. pseudomallei: the original reference strain K96243 from Thailand and strain MSHR305 from Australia. We identified a variable homologous region between the two strains. This region was previously identified in comparisons of the genome of B. pseudomallei strain K96243 with the genome of strain E264 from the closely related B. thailandensis. In that comparison, K96243 was shown to possess a horizontally acquired Yersinia-like fimbrial (YLF) gene cluster. Here, we show that the homologous genomic region in B. pseudomallei strain 305 is similar to that previously identified in B. thailandensis strain E264. We have named this region in B. pseudomallei strain 305 the B. thailandensis-like flagellum and chemotaxis (BTFC) gene cluster. We screened for these different genomic components across additional genome sequences and 571 B. pseudomallei DNA extracts obtained from regions of endemicity. These alternate genomic states define two distinct groups within B. pseudomallei: all strains contained either the BTFC gene cluster (group BTFC) or the YLF gene cluster (group YLF). These two groups have distinct geographic distributions: group BTFC is dominant in Australia, and group YLF is dominant in Thailand and elsewhere. In addition, clinical isolates are more likely to belong to group YLF, whereas environmental isolates are more likely to belong to group BTFC. These groups should be further characterized in an animal model.
PMCID: PMC2168593  PMID: 17933898

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