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1.  In vivo and in vitro activity of the siderophore monosulfactam BAL30072 against Acinetobacter baumannii 
Objectives
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.
Methods
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).
Results
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.
Conclusions
These data support the continued evaluation of BAL30072 for use in the treatment of infections caused by MDR A. baumannii.
doi:10.1093/jac/dkr013
PMCID: PMC3058568  PMID: 21393224
MICs; time–kill assays; drug susceptibility testing; multidrug resistant
2.  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.
doi:10.1128/AAC.02474-12
PMCID: PMC3632904  PMID: 23422914
3.  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.
doi:10.1128/AAC.01525-09
PMCID: PMC2876421  PMID: 20308379
4.  Urinary Concentrations and Antibacterial Activity of BAL30072, a Novel Siderophore Monosulfactam, against Uropathogens after Intravenous Administration in Healthy Subjects 
This annex study to a phase 1 study aimed to correlate urinary concentrations and bactericidal titers (UBTs) of BAL30072, a novel siderophore monosulfactam, in healthy subjects in order to evaluate which dosage of BAL30072 should be investigated in a clinical study on complicated urinary tract infection (UTI). Three cohorts of a total of 19 healthy male subjects were included in the add-on study and received the following BAL30072 dosages. The 1st cohort received 1 g once a day (q.d.) intravenously (i.v.) (1 h) on day 1 and 1 g thrice daily (t.i.d.) on day 2, the 2nd cohort received 2 g q.d. i.v. (1 h) on day 1 and 2 g t.i.d. on day 2, and the 3rd cohort received 1 g q.d. i.v. (4-h infusion) on day 8. Urine was collected up to 24 h after drug administration. UBTs were determined for seven Escherichia coli isolates (three wild type [WT], CTX-M-15, TEM-3, TEM-5, NDM-1), two Klebsiella pneumoniae isolates (WT, KPC), one Proteus mirabilis isolate (WT), and two Pseudomonas aeruginosa isolates (WT, VIM-1 plus AmpC). Urine drug concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The median urinary excretions of BAL30072 ranged between 38% and 46% (3 cohorts). The median UBTs after i.v. administration of 1 or 2 g q.d. and after 1 or 2 g t.i.d. showed positive UBTs for 24 h after the lowest dosage (1 g q.d.) for 5 of 7 of the Enterobacteriaceae strains and after the higher dosage of 2 g administered i.v. t.i.d. for all strains tested. After i.v. infusion of 1 g over 4 h, positive UBTs were demonstrated for three E. coli strains for up to 12 h, for the K. pneumoniae (KPC) strain for up to 8 h, and for the P. aeruginosa (VIM-1 plus AmpC) strain for up to only 4 h. The minimal bactericidal concentrations (MBCs) of the E. coli (NDM-1) strain and the K. pneumoniae (WT) strain correlated well between broth and urine but did not correlate well for the two P. aeruginosa strains. BAL30072 exhibits positive UBTs for 24 h even after a dosage of 1 g administered i.v. q.d. for 5 of 7 Enterobacteriaceae strains and after 2 g administered i.v. t.i.d. for all strains except one P. aeruginosa strain (50% of the time). In general, the UBTs correlated well with the MICs of the Enterobacteriaceae but were lower for P. aeruginosa. The clinical efficacy with a dosage regimen of BAL30072 of 2 g administered i.v. t.i.d. should be evaluated in the treatment of complicated UTI.
doi:10.1128/AAC.02425-15
PMCID: PMC4879366  PMID: 26976871
5.  A kinetic analysis of the inhibition of FOX-4 β-lactamase, a plasmid-mediated AmpC cephalosporinase, by monocyclic β-lactams and carbapenems 
Objectives
Class C β-lactamases are prevalent among Enterobacteriaceae; however, these enzymes are resistant to inactivation by commercially available β-lactamase inhibitors. In order to find novel scaffolds to inhibit class C β-lactamases, the comparative efficacy of monocyclic β-lactam antibiotics (aztreonam and the siderophore monosulfactam BAL30072), the bridged monobactam β-lactamase inhibitor BAL29880, and carbapenems (imipenem, meropenem, doripenem and ertapenem) were tested in kinetic assays against FOX-4, a plasmid-mediated class C β-lactamase (pmAmpC).
Methods
The FOX-4 β-lactamase was purified. Steady-state kinetics, electrospray ionization mass spectrometry (ESI-MS) and ultraviolet difference (UVD) spectroscopy were conducted using the β-lactam scaffolds described.
Results
The Ki values for the monocyclic β-lactams against FOX-4 β-lactamase were 0.04 ± 0.01 μM (aztreonam) and 0.66 ± 0.03 μM (BAL30072), and the Ki value for the bridged monobactam BAL29880 was 8.9 ± 0.5 μM. For carbapenems, the Ki values ranged from 0.27 ± 0.05 μM (ertapenem) to 2.3 ± 0.3 μM (imipenem). ESI-MS demonstrated the formation of stable covalent adducts when the monocyclic β-lactams and carbapenems were reacted with FOX-4 β-lactamase. UVD spectroscopy suggested the appearance of different chromophoric intermediates.
Conclusions
Monocyclic β-lactam and carbapenem antibiotics are effective mechanism-based inhibitors of FOX-4 β-lactamase, a clinically important pmAmpC, and provide stimulus for the development of new inhibitors to inactivate plasmidic and chromosomal class C β-lactamases.
doi:10.1093/jac/dkt434
PMCID: PMC3922156  PMID: 24235094
bridged monobactams; monosulfactam; β-lactamase inhibitors; carbapenems
6.  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.
doi:10.2147/IDR.S35529
PMCID: PMC3430440  PMID: 22977307
Burkholderia pseudomallei; ceftazidime; antibiotic resistance; melioidosis; β-lactamase; penA
7.  Burkholderia pseudomallei Known Siderophores and Hemin Uptake Are Dispensable for Lethal Murine Melioidosis 
Burkholderia pseudomallei is a mostly saprophytic bacterium, but can infect humans where it causes the difficult-to-manage disease melioidosis. Even with proper diagnosis and prompt therapeutic interventions mortality rates still range from >20% in Northern Australia to over 40% in Thailand. Surprisingly little is yet known about how B. pseudomallei infects, invades and survives within its hosts, and virtually nothing is known about the contribution of critical nutrients such as iron to the bacterium's pathogenesis. It was previously assumed that B. pseudomallei used iron-acquisition systems commonly found in other bacteria, for example siderophores. However, our previous discovery of a clinical isolate carrying a large chromosomal deletion missing the entire malleobactin gene cluster encoding the bacterium's major high-affinity siderophore while still being fully virulent in a murine melioidosis model suggested that other iron-acquisition systems might make contributions to virulence. Here, we deleted the major siderophore malleobactin (mba) and pyochelin (pch) gene clusters in strain 1710b and revealed a residual siderophore activity which was unrelated to other known Burkholderia siderophores such as cepabactin and cepaciachelin, and not due to increased secretion of chelators such as citrate. Deletion of the two hemin uptake loci, hmu and hem, showed that Hmu is required for utilization of hemin and hemoglobin and that Hem cannot complement a Hmu deficiency. Prolonged incubation of a hmu hem mutant in hemoglobin-containing minimal medium yielded variants able to utilize hemoglobin and hemin suggesting alternate pathways for utilization of these two host iron sources. Lactoferrin utilization was dependent on malleobactin, but not pyochelin synthesis and/or uptake. A mba pch hmu hem quadruple mutant could use ferritin as an iron source and upon intranasal infection was lethal in an acute murine melioidosis model. These data suggest that B. pseudomallei may employ a novel ferritin-iron acquisition pathway as a means to sustain in vivo growth.
Author Summary
Burkholderia pseudomallei is the etiologic agent of melioidosis, a multifaceted deadly and difficult to treat disease of equatorial regions of the world. Disease manifestations range from acute infections to long term chronic infections. The factors by which this bacterium causes disease are not yet well understood. Studies thus far focused on elucidation of the roles of traditional virulence factors such as secreted proteins and exopolysaccharides, but virtually nothing is known about the roles of nutrient acquisition systems in B. pseudomallei's survival in its mammalian hosts. One nutrient that is essential for bacterial metabolism and pathogenicity is iron. As free iron is not readily available in nature, bacteria developed numerous mechanisms for iron acquisition from abiotic and biotic sources. These mechanisms include siderophores and hemin/hemoglobin utilization systems, and it is therefore not too surprising that mutants defective in these systems are often impaired in virulence. In this study we show that defined B. pseudomallei mutants defective in siderophore and hemin/hemoglobin utilization systems remain fully lethal in a murine melioidosis model and present evidence for in vitro ferritin-iron acquisition which may be one or perhaps the main means by which this pathogen sustains in vivo growth.
doi:10.1371/journal.pntd.0001715
PMCID: PMC3383733  PMID: 22745846
8.  Antimicrobial Action of the Cyclic Peptide Bactenecin on Burkholderia pseudomallei Correlates with Efficient Membrane Permeabilization 
Burkholderia pseudomallei is a category B agent that causes Melioidosis, an acute and chronic disease with septicemia. The current treatment regimen is a heavy dose of antibiotics such as ceftazidime (CAZ); however, the risk of a relapse is possible. Peptide antibiotics are an alternative to classical antibiotics as they exhibit rapid action and are less likely to result in the development of resistance. The aim of this study was to determine the bactericidal activity against B. pseudomallei and examine the membrane disrupting abilities of the potent antimicrobial peptides: bactenecin, RTA3, BMAP-18 and CA-MA. All peptides exhibited >97% bactericidal activity at 20 µM, with bactenecin having slightly higher activity. Long term time-kill assays revealed a complete inhibition of cell growth at 50 µM bactenecin and CA-MA. All peptides inhibited biofilm formation comparable to CAZ, but exhibited faster kinetics (within 1 h). Bactenecin exhibited stronger binding to LPS and induced perturbation of the inner membrane of live cells. Interaction of bactenecin with model membranes resulted in changes in membrane fluidity and permeability, leading to leakage of dye across the membrane at levels two-fold greater than that of other peptides. Modeling of peptide binding on the membrane showed stable and deep insertion of bactenecin into the membrane (up to 9 Å). We propose that bactenecin is able to form dimers or large β-sheet structures in a concentration dependent manner and subsequently rapidly permeabilize the membrane, leading to cytosolic leakage and cell death in a shorter period of time compared to CAZ. Bactenecin might be considered as a potent antimicrobial agent for use against B. pseudomallei.
Author Summary
Burkholderia pseudomallei is a category B agent that causes Melioidosis, an acute and chronic disease with septicemia. The current treatment regimen is a heavy dose of antibiotics such as ceftazidime (CAZ), however, the risk of a relapse is possible. In this study we demonstrate that bactenecin, CA-MA, RTA3 and BMAP-18 are able to inhibit the growth and biofilm formation of B. pseudomallei. The strong bactericidal activity of bactenecin is attributed to its greater ability to permeabilize the membrane. Computational modeling of these peptide-membrane interactions provide support for a model in which bactenecin is able to penetrate the membrane most effectively due to its cyclical structure. The peptide, bactenecin has the potential to act as a highly effective alternative to CAZ, or as a combination therapy with CAZ in the treatment of melioidosis. Furthermore, understanding the mechanism of bactenecin may help us better design more effective peptides therapeutics of choice for melioidosis.
doi:10.1371/journal.pntd.0002267
PMCID: PMC3681726  PMID: 23785532
9.  Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis) 
PLoS Medicine  2007;4(7):e248.
Background
Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis.
Methods and Findings
Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury.
Conclusions
Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.
Willem Wiersinga and colleagues find up-regulation of multiple Toll-like receptors (TLRs) in peripheral blood cells of patients with melioidosis. However, only TLR2 had an effect on the immune response in a mouse model.
Editors' Summary
Background.
Melioidosis is a severe tropical infection caused by the bacterium Burkholderia pseudomallei. This soil-dwelling pathogen (disease-causing organism) enters the body through cuts, by swallowed contaminated water, or by inhaled contaminated dust. Here, it can cause a severe lung infection or spread into the blood stream and around the body, where it causes widespread inflammation (sepsis) and organ failure. Untreated septic melioidosis is usually fatal. Even with antibiotic therapy, half the people who develop it in Thailand (a hot spot for melioidosis) die. B. pseudomallei is a “gram-negative” bacterium. That is, it is surrounded by a membrane that stops it taking up a stain used to detect bacteria. This membrane contains a molecule called lipopolysaccharide (LPS). Proteins on immune system cells called Toll-like receptors (TLRs), of which there are many, recognize LPS and other surface molecules common to different pathogens and tell the cells to make cytokines. These cytokines stimulate the immune system to kill the pathogen but also cause inflammation, the underlying problem in septic melioidosis and other forms of sepsis. In other words, TLRs are two-edged swords—they provide an essential first-line defense against pathogens, but cause life-threatening inflammation if overstimulated.
Why Was This Study Done?
It isn't known which TLRs are involved in melioidosis. TLR4 normally detects LPS, but the surface of B. pseudomallei also carries molecules that interact with TLR2. Understanding how B. pseudomallei interacts with TLRs might suggest new, more effective ways to treat septic melioidosis. Better remedies for this disease are badly needed because, as well as the infections it causes in the community, the US Centers for Disease Control and Prevention has identified B. pseudomallei as a potential bioterrorism agent. In this study, the researchers have characterized the expression and function of TLRs in septic melioidosis using human, in vitro (test tube), and animal approaches.
What Did the Researchers Do and Find?
The researchers isolated monocytes and granulocytes (immune system cells involved in first-line defenses against pathogens) from patients with melioidosis and from healthy people. The patients' cells made more TLR1, TLR2, TLR4, and CD14 (a protein that enhances the activation of immune system cells by LPS) than those of the healthy controls and more of the mRNAs encoding several other TLRs. Next, the researchers tested the ability of heat-killed B. pseudomallei to induce the release of TNFα (a cytokine produced in response to TLR signaling) from macrophages (immune system cells that swallow up pathogens) isolated from wild-type mice and from mice lacking TLR2 or TLR4. Macrophages isolated from wild-type mice made more TNFα than those from TLR2- or TLR4-deficient mice. In addition, a human kidney cell line engineered to express CD14/TLR2 or CD14/TLR4 but not the parent cell line released IL8 (another cytokine) when stimulated with heat-killed B. pseudomallei. Other experiments in these human cell lines showed that LPS purified from B. pseudomallei signals through TLR2 but not through TLR4. Finally, the researchers tested the ability of TLR2- and TLR4-deficient mice to survive after infection with live B. pseudomallei. Compared with TLR4-deficient or wild-type mice, the TLR2-deficient mice had a strong survival advantage, a lower bacterial load, reduced lung inflammation, and less organ damage.
What Do These Findings Mean?
These findings show that people with melioidosis have increased expression of several TLRs, any one of which might cause the sepsis associated with B. pseudomallei infection. The in vitro findings indicate that TLR2 and TLR4 both contribute to the responsiveness of immune cells to B. pseudomallei in test tubes, but that only TLR2 detects the LPS of this bacterium. This unexpected result—TLR4 normally responds to LPS—might indicate that there is something unique about the LPS of B. pseudomallei. Finally, the survival of TLR2-deficient mice after infection with B. pseudomallei suggests that TLR2-mediated dysregulation of the immune system in response to invasive B. pseudomallei might cause septic melioidosis. Although these results need confirming in people, they suggest that inhibition of TLR2 in combination with antibiotic therapy might improve outcomes for people with melioidosis.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040248.
Information is available from the US Centers for Disease Control and Prevention on melioidosis (in English and Spanish)
The UK Health Protection Agency provides information for the public and health professionals on melioidosis
Wikipedia has pages on melioidosis and on Toll-like receptors (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The MedlinePlus encyclopedia contains a page on sepsis (in English and Spanish)
doi:10.1371/journal.pmed.0040248
PMCID: PMC1950213  PMID: 17676990
10.  A Burkholderia pseudomallei Outer Membrane Vesicle Vaccine Provides Protection against Lethal Sepsis 
The environmental Gram-negative encapsulated bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a disease associated with high morbidity and mortality rates in areas of Southeast Asia and northern Australia in which the disease is endemic. B. pseudomallei is also classified as a tier I select agent due to the high level of lethality of the bacterium and its innate resistance to antibiotics, as well as the lack of an effective vaccine. Gram-negative bacteria, including B. pseudomallei, secrete outer membrane vesicles (OMVs) which are enriched with multiple protein, lipid, and polysaccharide antigens. Previously, we demonstrated that immunization with multivalent B. pseudomallei-derived OMVs protects highly susceptible BALB/c mice against an otherwise lethal aerosol challenge. In this work, we evaluated the protective efficacy of OMV immunization against intraperitoneal challenge with a heterologous strain because systemic infection with phenotypically diverse environmental B. pseudomallei strains poses another hazard and a challenge to vaccine development. We demonstrated that B. pseudomallei OMVs derived from strain 1026b afforded significant protection against septicemic infection with B. pseudomallei strain K96243. OMV immunization induced robust OMV-, lipopolysaccharide-, and capsular polysaccharide-specific serum IgG (IgG1, IgG2a, and IgG3) and IgM antibody responses. OMV-immune serum promoted bacterial killing in vitro, and passive transfer of B. pseudomallei OMV immune sera protected naive mice against a subsequent challenge. These results indicate that OMV immunization provides antibody-mediated protection against acute, rapidly lethal sepsis in mice. B. pseudomallei-derived OMVs may represent an efficacious multivalent vaccine strategy against melioidosis.
doi:10.1128/CVI.00119-14
PMCID: PMC4018892  PMID: 24671550
11.  Antimicrobial Susceptibility and Genetic Characterisation of Burkholderia pseudomallei Isolated from Malaysian Patients 
The Scientific World Journal  2014;2014:132971.
Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics. Ceftazidime (CAZ), the synthetic β-lactam, is normally used as the first-line antibiotic therapy for treatment of melioidosis. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, leading to mortality if therapy is not switched to a different antibiotic(s) in a timely manner. In this study, susceptibilities of 81 B. pseudomallei isolates to nine different antimicrobial agents were determined using the disk diffusion method, broth microdilution test and Etest. Highest percentage of susceptibility was demonstrated to CAZ, amoxicillin/clavulanic acid, meropenem, imipenem, and trimethoprim/sulfamethoxazole. Although these drugs demonstrated the highest percentage of susceptibility in B. pseudomallei, the overall results underline the importance of the emergence of resistance in this organism. PCR results showed that, of the 81 B. pseudomallei, six multidrug resistant (MDR) isolates carried bpeB, amrB, and BPSS1119 and penA genes. Genotyping of the isolates using random amplified polymorphic DNA analysis showed six different PCR fingerprinting patterns generated from the six MDR isolates clusters (A) and eight PCR fingerprinting patterns generated for the remaining 75 non-MDR isolates clusters (B).
doi:10.1155/2014/132971
PMCID: PMC4213392  PMID: 25379514
12.  Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei 
Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-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 β-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 β-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 β-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 β-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.
doi:10.1128/AAC.02444-15
PMCID: PMC4775993  PMID: 26711764
13.  Two-Phase Bactericidal Mechanism of Silver Nanoparticles against Burkholderia pseudomallei 
PLoS ONE  2016;11(12):e0168098.
Silver nanoparticles (AgNPs) have a strong antimicrobial activity against a variety of pathogenic bacteria. The killing mechanism of AgNPs involves direct physical membrane destruction and subsequent molecular damage from both AgNPs and released Ag+. Burkholderia pseudomallei is the causative agent of melioidosis, an endemic infectious disease primarily found in northern Australia and Southeast Asia. B. pseudomallei is intrinsically resistant to most common antibiotics. In this study, the antimicrobial activity and mechanism of AgNPs (10–20 nm) against B. pseudomallei were investigated. The MIC and MBC for nine B. pseudomallei strains ranged from 32–48 μg/mL and 96–128 μg/mL, respectively. Concentrations of AgNPs less than 256 μg/mL were not toxic to human red blood cells. AgNPs exhibited a two-phase mechanism: cell death induction and ROS induction. The first phase was a rapid killing step within 5 min, causing the direct damage of the cytoplasmic membrane of the bacterial cells, as observed by a time-kill assay and fluorescence microscopy. During the period of 5–30 min, the cell surface charge was rapidly neutralized from -8.73 and -7.74 to 2.85 and 2.94 mV in two isolates of B. pseudomallei, as revealed by zeta potential measurement. Energy-dispersive X-ray (EDX) spectroscopy showed the silver element deposited on the bacterial membrane, and TEM micrographs of the AgNP-treated B. pseudomallei cells showed severe membrane damage and cytosolic leakage at 1/5 MIC and cell bursting at MBC. During the killing effect the released Ag+ from AgNPs was only 3.9% from the starting AgNPs concentration as observed with ICP-OES experiment. In the second phase, the ROS induction occurred 1–4 hr after the AgNP treatment. Altogether, we provide direct kinetic evidence of the AgNPs killing mechanism, by which cell death is separable from the ROS induction and AgNPs mainly contributes in the killing action. AgNPs may be considered a potential candidate to develop a novel alternative agent for melioidosis treatment with fast action.
doi:10.1371/journal.pone.0168098
PMCID: PMC5158019  PMID: 27977746
14.  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.
doi:10.1371/journal.pone.0030789
PMCID: PMC3283585  PMID: 22363490
15.  Comparative In Vitro Activities of Meropenem, Imipenem, Temocillin, Piperacillin, and Ceftazidime in Combination with Tobramycin, Rifampin, or Ciprofloxacin against Burkholderia cepacia Isolates from Patients with Cystic Fibrosis 
We evaluated the activities of meropenem, imipenem, temocillin, piperacillin, and ceftazidime by determination of the MICs for 66 genotypically characterized Burkholderia cepacia isolates obtained from the sputum of cystic fibrosis patients. In vitro synergy assays, as performed by the time-kill methodology, of two- and three-drug combinations of the β-lactams with tobramycin, rifampin, and/or ciprofloxacin were also performed with 10 strains susceptible, intermediate, or resistant to fluoroquinolones. On the basis of the MICs, meropenem and temocillin were the most active β-lactam agents, with MICs at which 90% of isolates are inhibited of 8 and 32 μg/ml, respectively. The addition of ciprofloxacin significantly enhanced the killing activities of piperacillin, imipenem, and meropenem against the 10 strains tested (P < 0.05). The best killing activity was obtained with the combination of meropenem and ciprofloxacin, with bactericidal activity of 3.31 ± 0.36 log10 CFU/ml (P < 0.05). Compared to the activity of the two-drug β-lactam–ciprofloxacin combination, the addition of rifampin or tobramycin did not significantly increase the killing activity (P > 0.05). The three-drug combinations (with or without ciprofloxacin) significantly enhanced the killing activities of piperacillin, imipenem, and meropenem relative to the activities of the β-lactams used alone (P < 0.05). The combination β-lactam–ciprofloxacin–tobramycin was the combination with the most consistently synergistic effect.
PMCID: PMC89053  PMID: 9925508
16.  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.
doi:10.3389/fmicb.2011.00139
PMCID: PMC3129521  PMID: 21747814
Burkholderia pseudomallei; melioidosis; antibiotic resistance; β-lactams; β-lactamase; TAT secretion
17.  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.
doi:10.2217/fmb.12.116
PMCID: PMC3568953  PMID: 23231488
antibiotics; Burkholderia pseudomallei; melioidosis; resistance; therapy
18.  Nitric Oxide from IFNγ-Primed Macrophages Modulates the Antimicrobial Activity of β-Lactams against the Intracellular Pathogens Burkholderia pseudomallei and Nontyphoidal Salmonella 
Our investigations show that nonlethal concentrations of nitric oxide (NO) abrogate the antibiotic activity of β-lactam antibiotics against Burkholderia pseudomallei, Escherichia coli and nontyphoidal Salmonella enterica serovar Typhimurium. NO protects B. pseudomallei already exposed to β-lactams, suggesting that this diatomic radical tolerizes bacteria against the antimicrobial activity of this important class of antibiotics. The concentrations of NO that elicit antibiotic tolerance repress consumption of oxygen (O2), while stimulating hydrogen peroxide (H2O2) synthesis. Transposon insertions in genes encoding cytochrome c oxidase-related functions and molybdenum assimilation confer B. pseudomallei a selective advantage against the antimicrobial activity of the β-lactam antibiotic imipenem. Cumulatively, these data support a model by which NO induces antibiotic tolerance through the inhibition of the electron transport chain, rather than by potentiating antioxidant defenses as previously proposed. Accordingly, pharmacological inhibition of terminal oxidases and nitrate reductases tolerizes aerobic and anaerobic bacteria to β-lactams. The degree of NO-induced β-lactam antibiotic tolerance seems to be inversely proportional to the proton motive force (PMF), and thus the dissipation of ΔH+ and ΔΨ electrochemical gradients of the PMF prevents β-lactam-mediated killing. According to this model, NO generated by IFNγ-primed macrophages protects intracellular Salmonella against imipenem. On the other hand, sublethal concentrations of imipenem potentiate the killing of B. pseudomallei by NO generated enzymatically from IFNγ-primed macrophages. Our investigations indicate that NO modulates the antimicrobial activity of β-lactam antibiotics.
Author Summary
β-lactam drugs that inhibit peptidoglycan biosynthesis are often used in the treatment of bacterial infections, including melioidosis. Independent of their antibiotic activity, we have noted that submicromolar concentrations of β-lactams potentiate the killing of intracellular B. pseudomallei supported by NO generated by IFNγ-primed macrophages. The production of NO can nonetheless be a double-edged sword, as indicated by our observations that sublethal concentrations of nitric oxide (NO), a diatomic radical produced by phylogenetically diverse organisms to regulate neurotransmission, vascular tone and host defense, tolerize B. pseudomallei, nontyphoidal Salmonella and E. coli against the antimicrobial activity of β-lactams. Accordingly, NO produced in the inflammatory response of macrophages protects nontyphoidal Salmonella against β-lactam antibiotics. NO mediates bacterial tolerance to β-lactam antibiotics by inhibiting the electrochemical gradient supported by terminal cytochrome oxidases of the respiratory chain, rather than by decreasing oxidative stress as previously thought.
doi:10.1371/journal.pntd.0003079
PMCID: PMC4133387  PMID: 25121731
19.  Pseudomonas pseudomallei, a common pathogen in Thailand that is resistant to the bactericidal effects of many antibiotics. 
The purpose of this investigation was to identify newer antimicrobial agents that may be useful in the therapy of melioidosis. The in vitro susceptibilities of 199 clinical isolates of Pseudomonas pseudomallei to 22 antibiotics were determined by standard disk diffusion, and those to 13 antibiotics were determined by agar dilution. Over 90% of the isolates were susceptible to imipenem, piperacillin-tazobactam, piperacillin, ceftazidime, ticarcillin-clavulanate, ampicillin-sulbactam, and carumonam by both methods. Standard disk diffusion yielded unacceptably high false-susceptibility results with aztreonam, ciprofloxacin, and temafloxacin. Piperacillin, ceftazidime, imipenem, and ciprofloxacin were not bactericidal for three selected P. pseudomallei strains as determined by time-kill curve methods. Furthermore, addition of ciprofloxacin to piperacillin, ceftazidime, or imipenem did not enhance bactericidal activity. One hundred ninety-four strains showed weak beta-lactamase production that did not increase upon incubation with cefoxitin. These findings suggest that several newer antimicrobial agents may prove useful in the treatment of melioidosis. However, results of susceptibility studies involving P. pseudomallei and newer agents must be interpreted with caution.
PMCID: PMC245036  PMID: 2039198
20.  Post-Exposure Therapeutic Efficacy of COX-2 Inhibition against Burkholderia pseudomallei 
Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus and the etiologic agent of melioidosis, a severe disease in Southeast Asia and Northern Australia. Like other multidrug-resistant pathogens, the inherent antibiotic resistance of B. pseudomallei impedes treatment and highlights the need for alternative therapeutic strategies that can circumvent antimicrobial resistance mechanisms. In this work, we demonstrate that host prostaglandin E2 (PGE2) production plays a regulatory role in the pathogenesis of B. pseudomallei. PGE2 promotes B. pseudomallei intracellular survival within macrophages and bacterial virulence in a mouse model of pneumonic melioidosis. PGE2-mediated immunosuppression of macrophage bactericidal effector functions is associated with increased arginase 2 (Arg2) expression and decreased nitric oxide (NO) production. Treatment with a commercially-available COX-2 inhibitor suppresses the growth of B. pseudomallei in macrophages and affords significant protection against rapidly lethal pneumonic melioidosis when administered post-exposure to B. pseudomallei-infected mice. COX-2 inhibition may represent a novel immunotherapeutic strategy to control infection with B. pseudomallei and other intracellular pathogens.
Author Summary
Burkholderia pseudomallei is the etiologic agent of melioidosis, a severe disease endemic in Southeast Asia and Northern Australia. B. pseudomallei is also classified as a Tier 1 select agent due to the threat of malicious use of the organism. Treatment of melioidosis is complicated by the inherent multidrug resistance of B. pseudomallei, leading to high case fatality rates or disease relapse. New therapeutic strategies are urgently needed to improve patient survival and to protect against a deliberate release of B. pseudomallei. Immunotherapeutics that can enhance the host immune response and delay disease progression represent a significant area of research interest. A number of immunomodulatory agents delivered locally to the lung prior to B. pseudomallei infection have afforded significant protection against pulmonary disease in animal models of melioidosis; however, their protective capacity significantly wanes upon post-exposure administration. In this work, we identify the PGE2 pathway as an immunotherapeutic target in pulmonary melioidosis and show that post-exposure COX-2 inhibition provides significant protection against lethal B. pseudomallei lung infection in mice. Further research examining FDA-approved COX-2 inhibitors as post-exposure prophylaxis for B. pseudomallei is warranted, as this may represent a safe, affordable, and efficacious immunotherapeutic strategy.
doi:10.1371/journal.pntd.0002212
PMCID: PMC3649956  PMID: 23675544
21.  Antimicrobial activity of Tachyplesin 1 against Burkholderia pseudomallei: an in vitro and in silico approach 
PeerJ  2016;4:e2468.
Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many conventional antibiotics. Therefore, alternative antimicrobial agents such as antimicrobial peptides (AMPs) are extensively studied to combat this issue. Our study aims to identify and understand the mode of action of the potential AMP(s) that are effective against B. pseudomallei in both planktonic and biofilm state as well as to predict the possible binding targets on using in vitro and in silico approaches. In the in vitro study, 11 AMPs were tested against 100 B. pseudomallei isolates for planktonic cell susceptibility, where LL-37, and PG1, demonstrated 100.0% susceptibility and TP1 demonstrated 83% susceptibility. Since the B. pseudomallei activity was reported on LL-37 and PG1, TP1 was selected for further investigation. TP1 inhibited B. pseudomallei cells at 61.69 μM, and membrane blebbing was observed using scanning electron microscopy. Moreover, TP1 inhibited B. pseudomallei cell growth, reaching bactericidal endpoint within 2 h post exposure as compared to ceftazidime (CAZ) (8 h). Furthermore, TP1 was shown to suppress the growth of B. pseudomallei cells in biofilm state at concentrations above 221 μM. However, TP1 was cytotoxic to the mammalian cell lines tested. In the in silico study, molecular docking revealed that TP1 demonstrated a strong interaction to the common peptide or inhibitor binding targets for lipopolysaccharide of Escherichia coli, as well as autolysin, pneumolysin, and pneumococcal surface protein A (PspA) of Streptococcus pneumoniae. Homology modelled B. pseudomallei PspA protein (YDP) also showed a favourable binding with a strong electrostatic contribution and nine hydrogen bonds. In conclusion, TP1 demonstrated a good potential as an anti-B. pseudomallei agent.
doi:10.7717/peerj.2468
PMCID: PMC5088614  PMID: 27812400
Burkholderia pseudomallei; Melioidosis; Antimicrobial peptides; Molecular docking; Tachyplesin 1
22.  Distinct roles for nitric oxide in resistant C57BL/6 and susceptible BALB/c mice to control Burkholderia pseudomallei infection 
BMC Immunology  2011;12:20.
Background
Burkholderia pseudomallei is the causative agent of melioidosis, an emerging bacterial infectious disease in tropical and subtropical areas. We recently showed that NADPH oxidase but not nitric oxide (NO) contributes to resistance in innately resistant C57BL/6 mice in a B. pseudomallei respiratory infection model. However, the function of NO for resistance was shown to differ among distinct strains of mice and proved also to be stage dependent in various infection models. The present study therefore aimed to examine the role of NO in a systemic infection model of melioidosis and to test whether the function of NO differs among innately resistant C57BL/6 and susceptible BALB/c mice after B. pseudomallei infection.
Results
C57BL/6 iNOS-/- mice that were intravenously infected with B. pseudomallei survived several weeks, whereas most of the wild type animals succumbed during this period. The bacterial burden in liver and spleen was significantly higher in wild type animals compared to iNOS-/- mice 13 days after challenge. In contrast, BALB/c mice that were treated with amminoguanidine to inhibit NO expression in vivo showed significantly enhanced mortality rates and higher bacterial loads in liver and spleen compared to control animals. The bactericidal function of IFN-γ stimulated C57BL/6 iNOS-/- macrophages were not altered after B. pseudomallei infection, but BALB/c macrophages exhibited reduced killing activity against the pathogen when NO was inhibited.
Conclusion
Our present data indicate a dual role of NO among resistant and susceptible mouse strains after B. pseudomallei infection. NO mediated mechanisms are an essential component to control the infection in susceptible BALB/c mice. In contrast, NO production in B. pseudomallei infected C57BL/6 mice rather harmed the host likely due to its detrimental effects.
doi:10.1186/1471-2172-12-20
PMCID: PMC3072354  PMID: 21410970
23.  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.
doi:10.1016/j.niox.2012.04.001
PMCID: PMC3517295  PMID: 22521523
antibiotics; antimicrobials; melioidosis; reactive nitrogen species; therapy; [4Fe-4S] clusters
24.  In vitro antimicrobial activity of natural toxins and animal venoms tested against Burkholderia pseudomallei 
Background
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.
Methods
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2334-6-100
PMCID: PMC1569838  PMID: 16784542
25.  Analyses of the Distribution Patterns of Burkholderia pseudomallei and Associated Phages in Soil Samples in Thailand Suggest That Phage Presence Reduces the Frequency of Bacterial Isolation 
PLoS Neglected Tropical Diseases  2016;10(9):e0005005.
Background
Burkholderia pseudomallei is a soil saprophytic bacterium that causes melioidosis. The infection occurs through cutaneous inoculation, inhalation or ingestion. Bacteriophages (phages) in the same ecosystem may significantly impact the biology of this bacterium in the environment, and in their culturability in the laboratory.
Methods/Principal Findings
The soil samples were analysed for the presence of bacteria using culture methods, and for phages using plaque assays on B. pseudomallei strain 1106a lawns. Of the 86 soil samples collected from northeastern Thailand, B. pseudomallei was cultured from 23 (26.7%) samples; no phage capable of infecting B. pseudomallei was detected in these samples. In contrast, phages capable of infecting B. pseudomallei, but no bacteria, were present in 10 (11.6%) samples. B. pseudomallei and their phages were co-isolated from only 3 (3.5%) of soil samples. Since phage capable of infecting B. pseudomallei could not have appeared in the samples without the prior presence of bacteria, or exposure to bacteria nearby, our data suggest that all phage-positive/bacteria-negative samples have had B. pseudomallei in or in a close proximity to them. Taken together, these findings indicate that the presence of phages may influence the success of B. pseudomallei isolation. Transmission electron microscopy revealed that the isolated phages are podoviruses. The temperate phages residing in soil-isolated strains of B. pseudomallei that were resistant to the dominant soil borne phages could be induced by mitomycin C. These induced-temperate phages were closely related, but not identical, to the more dominant soil-isolated phage type.
Conclusion/Significance
The presence of podoviruses capable of infecting B. pseudomallei may affect the success of the pathogen isolation from the soil. The currently used culture-based methods of B. pseudomallei isolation appear to under-estimate the bacterial abundance. The detection of phage capable of infecting B. pseudomallei from environmental samples could be a useful preliminary test to indicate the likely presence of B. pseudomallei in environmental samples.
Author Summary
Burkholderia pseudomallei is a motile, Gram-negative bacterium that causes melioidosis. The disease is endemic in Southeast Asia and Northern Australia and can be fatal. In the zones of endemicity, B. pseudomallei are commonly found in soils, and the bacteria can move to the surface during the rainy season. In Thailand, rice farmers rarely wear protective footwear, and thus they are exposed to a risk of infection with B. pseudomallei by cutaneous inoculation. Biological factors such as bacteriophages (phages), viruses of bacteria, present in the same ecosystem as B. pseudomallei may affect the population dynamics of this bacterium in the environment. In order to study this, we have investigated the distribution patterns of B. pseudomallei and associated phages in nature and the impact of these phages on the bacterial culturability. This is the first study demonstrating that the presence of phages capable of infecting B. pseudomallei may affect the success of the pathogen isolation from the soil. Currently culture-based methods of B. pseudomallei appear to under-estimate the bacterial abundance.
doi:10.1371/journal.pntd.0005005
PMCID: PMC5036839  PMID: 27668750

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