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.
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.
MICs; time–kill assays; drug susceptibility testing; multidrug resistant
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.
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]. http://dx.doi.org/10.3201/eid1812.120638
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.
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.
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.
Burkholderia pseudomallei; ceftazidime; antibiotic resistance; melioidosis; β-lactamase; penA
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.
Bacteriophage; Burkholderia pseudomallei; B. mallei; P2; Prophage distribution; Phage-based diagnostics
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.
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.
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.
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.
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.
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.
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.
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.
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.
Although melioidosis in endemic regions is usually caused by a diverse range of Burkholderia pseudomallei strains, clonal outbreaks from contaminated potable water have been described. Furthermore B. pseudomallei is classified as a CDC Group B bioterrorism agent. Ribotyping, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) have been used to identify genetically related B. pseudomallei isolates, but they are time consuming and technically challenging for many laboratories.
We have adapted repetitive sequence typing using a BOX A1R primer for typing B. pseudomallei and compared BOX-PCR fingerprinting results on a wide range of well-characterized B. pseudomallei isolates with MLST and PFGE performed on the same isolates.
BOX-PCR typing compared favourably with MLST and PFGE performed on the same isolates, both discriminating between the majority of multilocus sequence types and showing relatedness between epidemiologically linked isolates from various outbreak clusters.
Our results suggest that BOX-PCR can be used to exclude a clonal outbreak of melioidosis within 10 hours of receiving the bacterial strains.
Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism.
Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms) for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc-diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2s) enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/ml). The cell viability was measured using tetrazolium salts (XTT) based cytotoxic assay.
The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Ceftazidime (30 μg/disc). Among those tested, phospholipase A2 enzymes (crotoxin B and daboiatoxin) showed the most potent antibacterial activity against Gram-negative (TES) bacteria. Naturally occurring venom peptides and phospholipase A2 proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05–10 mg/mL) of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL.
This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei.
Burkholderia pseudomallei is the causative agent of melioidosis. In northeast Thailand, this gram-negative bacterium is a major cause of mortality from septicemia. The definitive diagnosis of this disease is made by bacterial culture. In this study, we produced a monoclonal antibody (MAb) specific to the 30-kDa protein of B. pseudomallei by in vivo and in vitro immunization of BALB/c mice with a crude culture filtrate antigen. The MAb could directly agglutinate with all 243 clinical isolates of B. pseudomallei but not with other gram-negative bacteria, except for one strain of Burkholderia mallei. However, the MAb cross-reacted with the gram-positive Bacillus sp. and Streptococcus pyogenes. B. pseudomallei in brain heart infusion broth (BHIB) subcultured from a BacT/Alert automated blood culture system could be identified by simple agglutination with this MAb assay. The sensitivity and specificity of direct agglutination compared to the “gold standard,” the culture method, were 94.12 and 98.25%, respectively. However, the MAb adsorbed to polystyrene beads or latex particles directly identified the bacterium in blood culture specimens and in BHIB subcultured from a BacT/Alert automated blood culture system. The sensitivity of the latex agglutination test was 100% for both blood culture and BHIB specimens. The specificity was 85.96 and 96.49% for the blood culture and BHIB specimens, respectively. The specificity could be increased if the nonspecific materials in the blood culture broths were eradicated by centrifugation at low speeds. Thus, a combination of blood culture and the agglutination method could be used for the rapid diagnosis of melioidosis in the routine bacteriological laboratory. This method could speed up detection of the bacterium in blood culture by at least 2 days, compared to the conventional bacterial culture method. In addition, the MAb is stable at room temperature for 2 weeks and at 4, −20, and −70°C for at least 1 year. The latex reagent was stable for at least 6 months at 4°C.
Lipopolysaccharide (LPS) is one of the most important virulence and antigenic components of Burkholderia pseudomallei, the causative agent of melioidosis. LPS diversity in B. pseudomallei has been described as typical, atypical or rough, based upon banding patterns on SDS-PAGE. Here, we studied the genetic and molecular basis of these phenotypic differences. Bioinformatics was used to determine the diversity of genes known or predicted to be involved in biosynthesis of the O-antigenic moiety of LPS in B. pseudomallei and its near-relative species. Multiplex-PCR assays were developed to target diversity of the O-antigen biosynthesis gene patterns or LPS genotypes in B. pseudomallei populations. We found that the typical LPS genotype (LPS genotype A) was highly prevalent in strains from Thailand and other countries in Southeast Asia, whereas the atypical LPS genotype (LPS genotype B) was most often detected in Australian strains (∼13.8%). In addition, we report a novel LPS ladder pattern, a derivative of the atypical LPS phenotype, associated with an uncommon O-antigen biosynthesis gene cluster that is found in only a small B. pseudomallei sub-population. This new LPS group was designated as genotype B2. We also report natural mutations in the O-antigen biosynthesis genes that potentially cause the rough LPS phenotype. We postulate that the diversity of LPS may correlate with differential immunopathogenicity and virulence among B. pseudomallei strains.
Burkholderia pseudomallei is an environmental Gram-negative bacterium and the cause of melioidosis, an often life-threatening disease affecting people in Southeast Asia and northern Australia. Melioidosis is usually contracted by bacterial inoculation, ingestion or inhalation. Effective vaccines for melioidosis are currently unavailable. This organism contains a large genome, which varies greatly among strains due to a high frequency of genetic recombination. We report here on diversity of lipopolysaccharides (LPS) in this species, a major component of the bacterial outer membrane and a known immunogenic virulence factor. We developed LPS genotyping techniques to study frequency of two major LPS types, known as typical and atypical LPS, in B. pseudomallei strains collected from two endemic regions: Southeast Asia and Northern Australia. LPS genotype variation differed among B. pseudomallei populations. During the investigation, we discovered a new LPS genotype in a sub-population group of B. pseudomallei in Australia. We postulate that such differences are likely to be associated with variable immunopathogenicity and clinical presentation in the human host.
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.
Burkholderia pseudomallei; melioidosis; antibiotic resistance; β-lactams; β-lactamase; TAT secretion
From January 1982 to May 2000, 17 infections caused by Burkholderia pseudomallei were diagnosed in 15 patients in Taiwan; almost all the infections were diagnosed from 1994 to May 2000. Of the 15 patients, 9 (60%) had underlying diseases, and 10 (67%) had bacteremic pneumonia. Thirteen (76%) episodes of infection were considered indigenous. Four patients died of melioidosis. Seventeen B. pseudomallei isolates, recovered from eight patients from November 1996 to May 2000, were analyzed to determine their in vitro susceptibilities to 14 antimicrobial agents, cellular fatty acid and biochemical reaction profiles, and randomly amplified polymorphic DNA patterns. Eight strains (highly related isolates) were identified. All isolates were arabinose non-assimilators and were susceptible to amoxicillin-clavulanate, piperacillin-tazobactam, imipenem, and meropenem. No spread of the strain was documented.
Burkholderia pseudomallei is the causative agent of melioidosis, a disease increasingly recognized as an important cause of morbidity and mortality in many regions of the world. B. pseudomallei is a facultative intracellular pathogen capable of invading eukaryotic cells. We used Tn5-OT182 mutagenesis to generate mutants deficient in the ability to invade a human type II pneumocyte cell line (A549 cells). One of these mutants, AJ1D8, exhibited approximately 10% of the ability of the parental strain, 1026b, to invade A549 cells. There was no difference in the abilities of 1026b and AJ1D8 to resist killing by RAW macrophages or the human defensin HNP-1. The nucleotide sequence flanking the Tn5-OT182 integration in AJ1D8 was determined, and two open reading frames were identified. The predicted proteins shared considerable homology with two-component regulatory systems involved in the regulation of heavy-metal resistance in other organisms. AJ1D8 was 16-fold more sensitive to Cd2+ and twofold more sensitive to Zn2+ than was 1026b but was not sensitive to any of the other heavy metals examined. The B. pseudomallei two-component regulatory system, termed irlRS, complemented the invasion-deficient and heavy-metal-sensitive phenotype of AJ1D8 in trans. There was no significant difference between the virulence of AJ1D8 and that of 1026b in infant diabetic rats and Syrian hamsters, suggesting that the irlRS locus is probably not a virulence determinant in these animal models of acute B. pseudomallei infection.
Burkholderia pseudomallei is a facultative intracellular gram-negative bacterium that can survive and multiply inside macrophages. One of the mechanisms by which B. pseudomallei escapes macrophage killing is by interfering with the expression of inducible nitric oxide synthase (iNOS). However, the bacterial components that modulate antimicrobial activity of the macrophage have not been fully elucidated. In the present study, we demonstrated that B. pseudomallei strain SRM117, a lipopolysaccharide (LPS) mutant that lacks the O-antigenic polysaccharide moiety, was more susceptible to macrophage killing during the early phase of infection than the parental wild-type strain (1026b). Unlike the wild type, the LPS mutant could readily stimulate Y701-STAT-1 phosphorylation (pY701-STAT-1) and interferon-regulatory factor 1 (IRF-1) expression, both of which are essential transcription factors of iNOS. Neutralizing antibody against beta interferon was able to inhibit the phosphorylation of Y701-STAT-1 and the expression of IRF-1 and iNOS, all of which resulted in an increased rate of intracellular replication. These data suggest that the O-antigenic polysaccharide moiety of B. pseudomallei modulates the host cell response, which in turn controls the intracellular fate of B. pseudomallei inside macrophages.
Burkholderia (Pseudomonas) pseudomallei is the causative agent of melioidosis, a bacterial infection of considerable morbidity in areas of endemicity of Southeast Asia and northern Australia. Clinical isolates of B. pseudomallei have been demonstrated to produce a lipopolysaccharide (LPS) containing two separate and chemically distinct antigenic O polysaccharides against which infected patients produced antibodies. A putative capsular polysaccharide (CPS) has also been reported and is thought to be antigenically conserved based on results of serological studies with clinical B. pseudomallei isolates. In the present study, the CPS isolated from B. pseudomallei 304b from northeastern Thailand was found to have an [alpha]D of +99 degrees (water), was composed of D-galactose (D-Gal), 3-deoxy-D-manno-2-octulosonic acid (KDO), and O-acetyl 3:1:1), and was a linear unbranched polymer of repeating tetrasaccharide units having the following structure: -3)-2-O-Ac-beta-D-Galp-(1-4)-alpha-D-Galp-(1-3)-beta-D -Galp-(1-5)-beta-D-KDOp-(2-. Sera from 13 of 15 patients with different clinical manifestations of melioidosis but not normal controls recognize the CPS, which suggests that it is immunogenic and raises the possibility that it may have a role as a vaccine candidate and/or diagnostic agent.