expression of resistance-nodulation-cell-division efflux pumps in commonly used burkholderia...

Transactions of the Royal Society of Tropical Medicine and Hygiene (2008) 102/S1, S145 S151

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Expression of resistance-nodulation-cell-divisionefflux pumps in commonly used Burkholderiapseudomallei strains and clinical isolates fromnorthern Australia

Ayush Kumara, 1, Mark Mayob, Lily A. Truncka, Allen C. Chengb,Bart J. Currieb, Herbert P. Schweizera, *aDepartment of Microbiology, Immunology, and Pathology, Rocky Mountain Regional Center of Excellence for Biodefense andEmerging Infectious Diseases Research, Colorado State University, Fort Collins, CO 80523-2025, USAbMenzies School of Health Research, Charles Darwin University and Royal Darwin Hospital, Darwin, NT, Australia

KEYWORDSMelioidosisBurkholderia

pseudomalleiEfflux pumpsAntibiotic resistanceResistance-

nodulation-celldivision

Polymerase chainreaction

Summary Burkholderia pseudomallei is the aetiological agent of melioidosis. Therapy for thisdisease is lengthy and limited to only a few antibiotics because of this bacterium’s intrinsicantibiotic resistance to many clinically useful antibiotics. These properties of B. pseudomalleimay partially be due to expression of efflux pumps of the resistance-nodulation-cell-division(RND) family. The patterns and magnitude of RND efflux pump expression in commonly usedstrains and clinical isolates of B. pseudomallei from the Royal Darwin Hospital, Darwin, Australia,were assessed in cells grown to late exponential phase using quantitative real-time PCR (qRT-PCR). Expression of the three previously identified RND efflux pumps AmrAB-OprA, BpeAB-OprBand BpeEF-OprC, as well as four other yet uncharacterized pumps, was found to be widespreadin the clinical isolates. In 45 of 50 isolates (90%), mRNA was detected for at least one of theseven RND pumps. Of these 45 isolates, 41 (82%) expressed multiple pumps with nine strainsexpressing all seven pumps tested. While these studies revealed no striking correlation betweenRND efflux pump expression and clinically significant antibiotic resistance, the data support thenotion that RND pumps probably play important roles in this bacterium’s physiology, defenceagainst toxic compounds, and perhaps virulence.© 2008 Published by Elsevier Ltd on behalf of Royal Society of Tropical Medicine and Hygiene.

1. IntroductionBurkholderia pseudomallei is the aetiological agent ofmelioidosis, a disease common to many of the tropical andsubtropical regions of the world.1,2 It is recognized as anemerging infectious disease and interest in this bacteriumand the related B. mallei has dramatically increasedfollowing their classification as category B Select Agents bythe U.S. Centers for Disease Control and Prevention.

* Corresponding author.Tel.: +1 970 491 3536; fax: +1 970 491 8708.

E-mail address: [email protected](H.P. Schweizer).

Burkholderia pseudomallei is intrinsically resistant tomany antibiotics, including aminoglycosides, macrolides,several penicillins, and first- and second-generationcephalosporins.3,4 Fluoroquinolones have only weak ac-tivity.5,6 Generally, B. pseudomallei is susceptible tochloramphenicol, the tetracyclines, trimethoprim sulpha-methoxazole (co-trimoxazole), third-generation cephalo-sporins, carbapenems and amoxicillin-clavulanate (co-amoxiclav). For optimal efficacy, prolonged therapy lasting12 20 weeks is required. Treatment is divided into intensive(intravenous) and eradication (oral) phases.1,7 Intensive-phase therapy involves ceftazidime or a carbapenemantibiotic which should be administered for at least 10 14 d.This is followed by at least 3 months of oral antibiotic

1 Present address: Faculty of Health Sciences, University of Ontario Institute of Technology, 2000 Simcoe Street N, Oshawa,ON L1H 7K4, Canada.

0035-9203/ $ see front matter © 2008 Published by Elsevier Ltd on behalf of Royal Society of Tropical Medicine and Hygiene.

S146 A. Kumar et al.

BPSL0307 BPSL0308 BPSL0309

BPSL0816BPSL0814 BPSL0815

bpeA bpeB oprB




amrA amrB oprA

BPSL2234 BPSL2235

BPSL2872BPSL2871

secD secF

BPSS0294BPSS0292 BPSS0293

bpeE bpeF oprC

BPSS1043 BPSS1042 BPSS1041

czcC czcB czcA


bpeG bpeH oprD

Chromosome 2

Chromosome 1

bpeR

BPSL0813

BPSL1269

BPSL1569

amrR

BPSL1805

BPSS0291BPSS0290

bpeT

BPSS1117



bpeA bpeB oprB




amrA amrB oprA

BPSL2234 BPSL2235

BPSL2872BPSL2871

secD secF


bpeE bpeF oprC

BPSS1043 BPSS1042 BPSS1041

czcC czcB czcA


bpeG bpeH oprD

Chromosome 2

Chromosome 1

bpeR

BPSL0813

BPSL1269

BPSL1569

amrR

BPSL1805

BPSS0291BPSS0290

bpeT

BPSS1117

Fig. 1. Resistance-nodulation-cell-division (RND) efflux systems encoded by the Burkholderia pseudomallei K96243 genome. Membrane fusion protein,RND transporter and outer membrane channel protein-encoding genes are indicated by yellow, blue and green arrows, respectively. Orange arrowsindicate known or probable regulatory genes. Note that the bpEF-oprC operon is preceded by BPSS0291 (grey arrow) encoding a putative lipase. Actualgene names are given for characterized or highly probable gene products.

therapy involving either co-trimoxazole (with or withoutdoxycycline) or co-amoxiclav in patients with intolerancefor co-trimoxazole. We were interested in investigating thepotential role of energy-dependent efflux in the intrinsicantibiotic resistance of B. pseudomallei.

Antibiotic efflux is now recognized as the majordeterminant of multidrug resistance (MDR) phenotype invarious bacterial pathogens. In Gram-negative bacteria,efflux pumps belonging to the resistance-nodulation-cell-division (RND) superfamily are most commonly responsiblefor the MDR phenotype. RND pumps have been shown to beactive in various clinically important organisms, includingPseudomonas aeruginosa, Escherichia coli, Acinetobacterbaumannii and Salmonella spp.8 These pumps functionby forming a tripartite complex with a periplasmicprotein belonging to the membrane fusion protein (MFP)family and an outer membrane protein of the outermembrane factor (OMF) family. RND pumps exhibit theability to bind various structurally unrelated substratesand pump them out directly into the external mediausing the proton gradient as the energy source. Whileunderstudied, evidence obtained with a limited numberof clinical isolates suggests that efflux pumps play amajor role in B. pseudomallei’s inherent antibiotic resis-tance: (i) treatment-emergent chloramphenicol-resistantstrains often demonstrate cross-resistance to tetracyclines,

sulphamethoxazole, trimethoprim and ciprofloxacin3,9; and(ii) intrinsic aminoglycoside, macrolide, chloramphenicoland trimethoprim resistance is clearly attributable to thepresence of several characterized efflux systems, AmrAB-OprA,10 BpeAB-OprB11 and BpeEF-OprC.12 Efflux pumpmutants become exquisitely susceptible to the respectivesubstrates.

The genome sequence of B. pseudomallei K9624313

reveals the presence of at least 10 operons that mayencode for RND efflux pump components; seven onchromosome 1 and three on chromosome 2 (Figure 1).Although annotated differently, these pumps are conservedin other B. pseudomallei strains. As has previously beenshown for P. aeruginosa,14 all of these operons encodethe MFP and RND transporter components. Most operons,with the exception of BPSL2234-BPSL2235, also encodean OMP component. Since all RND efflux pumps aretripartite, i.e. require the MFP, RND transporter andOMP for function, the absence of an OMP-encodinggene implies that this efflux pump either utilizes anOMP encoded by one of the other RND operons, oran unlinked OMP of the export OMP family, similar toE. coli TolC15 and P. aeruginosa OpmH.16,17 Finally, mostoperons also contain linked regulatory genes that probablyencode local transcriptional regulators. The absence oflocal regulators does not mean that these genes are

RND pumps in Burkholderia pseudomallei strains and clinical isolates from northern Australia S147

constitutively expressed, but rather that their expressionis probably governed by global transcriptional regulatorymechanism(s) (reviewed by Kumar and Schweizer8 and Liand Nikaido18).

Of the 10 RND export systems, three are unlikelycandidates for drug efflux systems. SecD-SecF is probably acomponent of a general protein export (secretion) system,CzcC-CzcB-CzcA is most likely a metal-ion efflux system,while BPSL2234-BPSL2235 appears to be a cation-effluxpump (based on sequence homology, data not shown). Todate, three RND drug efflux pumps have been characterized,namely, AmrAB-OprA from strain 1026b,10 BpeAB-OprBfrom strain KHW,11 and BpeEF-OprC from strain 1026bof B. pseudomallei.12 AmrAB-OprA and BpeAB-OprB areaminoglycoside and macrolide pumps, while BpeEF-OprCwas shown to efflux trimethoprim and chloramphenicol in asurrogate P. aeruginosa strain. Two other pumps, BPSS1118-BPSS1119-BPSS1120 (bpeGH-oprD) and BPSL1568-BPSL1567-BPSL1566, are currently being characterized in ourlaboratory (Department of Microbiology, Immunology, andPathology, Rocky Mountain Regional Center of Excellencefor Biodefense and Emerging Infectious Diseases Research,Colorado State University, Fort Collins, CO, USA).

In this study we investigated the expression of sevendifferent RND pump genes in a collection of commonlyused laboratory strains and 50 different clinical isolatesof B. pseudomallei from tropical northern Australia usingquantitative real-time PCR (qRT-PCR).

2. Materials and methods2.1. Bacterial strains and culture media

Strains 1026b and DD503,10 1710a&b, K96243, Pasteur 6608and S13 were obtained from Dr Mark Schell, Universityof Georgia, USA, and Dr Donald Woods, University ofCalgary, Canada. Fifty clinical isolates of B. pseudomalleiwere selected from the B. pseudomallei culture collectionat the Menzies School of Health Research at the RoyalDarwin Hospital, Darwin, Australia. These isolates includedserial isolates from patients with treatment failure orchronic infection, with or without the emergence ofreduced susceptibility to one of the commonly usedantimicrobials (including doxycycline, meropenem andceftazidime). Clinical isolates of B. pseudomallei werecultured on chocolate agar (Oxoid Australia, Adelaide,South Australia), while Luria Bertani (LB) (EM Sciences,Gibbstown, NJ, USA) broth was used to cultivate cells inliquid medium. All cultures were incubated at 37ºC.

2.2. RNA extraction and cDNA synthesis

RNA extractions were performed using the RNeasy kit(Qiagen-Australia, Doncaster, VIC, Australia). One ml oflate log phase (A600 nm ≈ 0.8) culture was harvested and thepellet frozen at 80ºC for 15 min to facilitate cell lysis.RNA extraction was carried out following the manufacturer’sinstructions. Synthesis of the cDNA was performed usingthe Superscript First Strand Synthesis Supermix (Invitrogen-Australia, Mount Waverley, VIC, Australia) following themanufacturer’s instructions.

Table 1 List of primers used for quantitative real-time PCR

Target gene Primer name Sequence (5′ → 3′)

23S rRNA Bp23S_F gta gac ccg aaa cca ggt ga

Bp23S_R cac ccc tat cca cag ctc at

amrB amrB_RT_Rev gtc agc acg ttg atc gag aa

amrB_RT_For cgc tct gat gtt cct ctt ca

bpeB bpeB-F1_RT ggc ctc gac aac ttc ctg ta

bpeB-R1_RT ttc ttc tgc acc tga acc tg

bpeF bpeF-F1_RT tcc gag tat ccg gaa gtc gt

bpeF-R1_RT gtc ctc gac acc gtt gat ct

bpeH bpeH-F1_RT cat tca acg aga tgg tcg tg

bpeH-R1_RT cgc gct cga gtt gta gtt ct

BPSL0309 BPSL0309F gtg ggc tgg gtc tac gaa ta

BPSL0309R agc tcg aac ttc agg aac ca

BPSL1267 BPSL1267F gaa cca gct gtt cct gat cc

BPSL1267R cgg atg gat gta gct ctc gt

BPSL1567 BPSL1567F aat aca cgc gct cga tct tc

BPSL1567R ctc ggg cag cat gtg ata g

2.3. Quantitative real-time PCR

Quantitative RT-PCR was performed using the SYBRGreenER™ qRT-PCR master mix (Invitrogen, Carlsbad, CA,USA). A list of primers used in the study is given in Table 1.Primers were designed using the OligoPerfect primer de-signing tool from Invitrogen (http://www.invitrogen.com).23S rRNA was used as the housekeeping control, whileB. pseudomallei 1026b was used as the control strain. Allprimer pairs were subjected to melt-curve analysis to ruleout secondary products and primer dimer formation priorto use. cDNA samples were diluted 1:10 in dH2O beforeanalysis.

2.4. Antibiotic susceptibility tests

Minimal inhibitory concentrations (MICs) were determinedin Mueller Hinton broth from Becton Dickinson (FranklinLakes, New Jersey, USA) by the two-fold broth microdilutionmethod19 or by the E-test method (AB Biodisk, Solna,Sweden).

3. Results3.1. Expression of efflux pumps in commonly used strains

To assess efflux pump gene expression in the commonly usedstrains 1026b, 1106b, 1710a, 1710b, DD503, K96243, Pasteur6608 and S13, RNA was isolated from late log phase culturesand expression of AmrAB-OprA, BpeAB-OprB, BpeEF-OprCand BpeGH-OprD assessed using qRT-PCR and amrB-, bpeB-,bpeF- and bpeH-specific primers. Data obtained from qRT-PCR were analysed by normalizing with the 23S rRNAhousekeeping control and using B. pseudomallei 1026b asthe control strain. To score the expression of a pumpas positive, a Ct-value of 30 was set as the cut-off. Aspresented in Figure 2, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC and BpeGH-OprD were expressed in all strains tested,albeit to various degrees.


Fig. 2 Relative resistance-nodulation-cell-division efflux pump geneexpression in common Burkholderia pseudomallei laboratory strains. Datawere normalized using the 23S rRNA gene as the housekeeping control.Relative quantification was performed using B. pseudomallei 1026b as thecontrol strain. Note that DD503 is an isogenetic D(amrAB-oprA) derivative of1026b and thus shows no expression of amrB. Error bars represent SD.

3.2. Expression of RND efflux pumps in clinical isolates

Using the same criteria described above for laboratorystrains, expression of AmrAB-OprA, BpeAB-OprB, BpeEF-OprC and BpeGH-OprD, plus three other putative RNDpumps (BPSL0307-BPSL0308-BPSL0309, BPSL1268-BPSL1267-BPSL1266 and BPSL1568-BPSL1567-BPSL1566) was assessedin clinical isolates archived in the Royal Darwin Hospital.We only analysed the expression of seven of the 10 putativeRND export systems found in K96243 (Figure 1) becauseSecD-SecF and CzcC-CzcB-CzcA are unlikely candidates fordrug efflux systems as they are likely to be involvedin protein and heavy-metal export, respectively. A thirdsystem encoded by BPSL2234-BPSL2235 was not includedbecause, based on sequence homology, it appears to bea cation efflux pump (data not shown). The results aresummarised in Table 2.

In 45 of 50 isolates (90%), mRNA was detected forat least one of the seven RND pumps examined. Of these45 isolates, nine were found to express all seven pumpstested, five expressed six pumps, six expressed five pumps,seven expressed four pumps, seven expressed three pumps,another seven expressed two pumps, while four strainsexpressed only one of the seven pumps. Although nine (18%)of the isolates tested were found to express all seven of thepumps tested, only five (10%) of the strains tested did notshow mRNA for any of the pumps.

3.3. Expression of individual efflux pumps

To assess the possible significance of efflux pump expressionin clinical isolates, the prevalence of expression of indi-vidual, characterized and uncharacterized efflux systemswas more closely scrutinized. The BpeAB-OprB system wasexpressed in 43 (86%) of the analysed isolates. The AmrAB-OprA pump was found to be expressed in 23 isolates (46%),and 21 of these isolates (91% of the amrB positive strains)also expressed bpeB. Overall, 25 isolates (50%) were shownto be positive for bpeF mRNA encoding the RND componentof the BpeEF-OprC pump. The BpeGH-OprD pump wasfound to be expressed in 23 (46%) isolates. The BPSL0309,BPSL1267 and BPSL1567 encoding components of efflux

pumps of unknown function were found to be expressedin 13 (26%), 26 (52%), and 33 (66%) isolates, respectively.

3.4. Comparative efflux operon expression

For the amrB, bpeB, bpeH, and BPSL1567 genes, datawere analysed by comparing the expression in clinicalisolates with that of B. pseudomallei 1026b (Figure 3). Thedata indicate that 18 different clinical isolates expressedamrB at levels that are either similar to or higher thanthose seen in B. pseudomallei 1026b (Figure 3A), while15 clinical isolates showed expression levels of bpeB thatare either comparable to or higher than those observed inB. pseudomallei 1026b (Figure 3B). Sixteen and 20 isolatesexpressed bpeH (Figure 3C) and BPSL1567 (Figure 3D),respectively, at levels that were higher than those seen in1026b. It should be noted that the expression of bpeH andBPSL1567 in B. pseudomallei 1026b was found to be lowerthan that of amrB and bpeB. We could not perform relativequantification analyses for bpeF, BPSL0309, and BPSL1267as the control strain used in our study (1026b) did not showany significant expression of these genes.

3.5. Correlation between efflux pump expression andantibiotic resistance

Antibiotic susceptibility tests were performed using drugsfor the treatment of melioidosis with the common strainsof B. pseudomallei. The expression of amrAB-oprA, bpeAB-oprB, bpeEF-oprC and bpeGH-oprD (Figure 2) revealed noobvious correlations between the expression of the effluxsystems encoded by these operons and drug resistance.Most strains exhibited MIC levels below the clinicalbreakpoints for the tested antibiotics, with the exceptionof strains 1710a and 1710b, which showed intermediateceftazidime resistance/susceptibility. A small number ofthe clinical isolates tested included serial isolates frompatients with treatment failure or chronic infection, with orwithout the emergence of reduced susceptibility to one ofthe commonly used antimicrobials (including doxycycline,meropenem and ceftazidime). With this small sample size,we did not observe a significant correlation betweenthe expression of the individual pumps and antibioticsusceptibility phenotype or a consistent increase in pumpexpression in serial isolates from patients with chronicinfection (data not shown).

4. DiscussionThis is the first comprehensive study of RND efflux pumpexpression in B. pseudomallei. Because many of the effluxoperons studied possess putative regulatory proteins, theexperimental approach used here will only reveal genes thatare transcribed constitutively due to regulatory mutationsor the experimental conditions employed. Despite thesepossible limitations, the data nonetheless convincinglyshow that RND efflux system expression is very prevalentin commonly used B. pseudomallei laboratory strainsand clinical isolates. Expression of the three previouslyidentified RND efflux pumps, AmrAB-OprA, BpeAB-OprB andBpeEF-OprC, as well as four other yet uncharacterizedpumps, was found to be widespread in clinical and othercommonly used isolates. In 45 of the 50 isolates (90%),


Table 2 Expression of resistance-nodulation-cell-division pumps in clinical Burkholderia pseudomallei isolates

Isolate Isolate ID Isolation site amrB bpeB bpeF bpeH BPSL0309 BPSL1267 BPSL1567 No. of pumps

1 58 NA + + + 3

2 87 Blood + 1

3 95 NA + + + + 4

5 118 Blood + + + + + + 6

6 223 Sputum + + + + 4

7 304 Urine + + + + + + + 7

8 335 Wound + + + + + + + 7

9 342 Prostatic abscess + + + + + + 6

10 378 Prostatic abscess + + + + + + + 7

11 443 Sputum + + + + + + 6

12 459 NA + + + + + 5

13 516 Sputum + + + + + + 6

14 524 Sputum + + + + + + + 7

15 527 Blood + + + + + + + 7

16 672 Blood + + + + + + + 7

17 686 Blood + + + + + + 6

18 737 NA + + + + 4

19 759 Sputum + + + + + + + 7

20 783 NA + + + + + 5

21 788 Urine + + 2

23 870 NA + + + + + + + 7

24 936 Urine + + + 3

25 938 Lung 0

26 1044 Lymph node 0

27 1048 Throat + + + + + + + 7

28 1068 NA + 1

29 1092 Blood + 2

30 1202 Urine 0

31 1223 Blood + + 2

32 1288 Throat + 1

34 1300 Faeces 0

35 1378 Blood + + + 3

36 1390 NA + + + 3

37 1391 NA + + 2

38 1428 Blood + 1

39 1814 NA + + + 3

41 1943 NA + + + + + 5

42 2260 Blood + + + + + 4

43 367b Bone + + + + 2

47 799 Joint + + 0

48 888 Blood 4

49 1218 Sputum + + + + 5

50 1287 Nose + + + + + 3

51 1418 Sputum + + + 3

52 1459 Sputum + + + 4

61 238 Blood + + + + 5

62 305 Brain + + + + + 5

63 346 Sputum + + + + + 4

64 465A Blood + + + + 2

65 668 Blood + + 2

Total 23 43 25 23 13 26 33

NA: not available.


Fig. 3. Relative quantification of resistance-nodulation-cell-division pump gene expression in Burkholderia pseudomallei clinical isolates. Data werenormalized using the 23S rRNA gene as the housekeeping control. Relative quantification of the indicated genes was performed using reference strainB. pseudomallei 1026b (R) as the comparator. 1 → 65 indicates the isolates listed in Table 2. Error bars represent SD.

mRNA was detected for at least one of the seven RNDpumps. Of these 45 isolates, 41 (82%) expressed multiplepumps with nine strains expressing all seven pumps tested.Of the commonly used strains, S13 over-expressed AmrAB-OprA, BpeAB-OprB, BpeEF-OprC and BpeGH-OprD, althoughthe reason(s) for this are currently unclear. Because thephysiological role(s) of virtually all of these pumps remaina mystery, strains for laboratory studies should be chosenwisely because over-expression of RND pumps may haveundesired consequences and may make comparisons withother strains difficult.

Closer analyses of the expression patterns of individualpumps revealed some interesting trends. First, the highprevalence of the expression of BpeAB-OprB, a pumprequired for quorum sensing and virulence,20,21 in clinicalisolates, is supportive of the previously postulated roleof BpeAB-OprB in the virulence of B. pseudomallei.Second, AmrAB-OprA and BpeAB-OprB were simultaneouslyexpressed in a high percentage of strains. Co-expressionof two pumps with similar substrate profiles in thesame isolates suggests (a) physiological role(s) other thanantibiotic efflux for these systems. This notion is supportedby the finding that a loss of AmrAB-OprA expression instrain DD503, an isogenetic D(amrAB-oprA) derivative of1026b, is accompanied by an increased expression of BpeAB-OprB (Figure 2). However, since the data obtained in thisstudy only reflect mRNA levels for different pumps, theyshould be interpreted with caution in lieu of any additionalexperimentation. Third, the high prevalence of expressionof the BpeEF-OprC and BpeGH-OprD pumps, as well asthe BPSL1267- and BPSL1567-encoded pump components inclinical isolates, may be indicative of a significant role(s) ofthese efflux systems in the physiology of B. pseudomallei.

We did not observe a significant correlation betweenthe expression of individual pumps and ceftazidime,

doxycycline and meropenem susceptibility phenotypes inthe small number of antibiotic-resistant isolates presentin the sample population. This finding may imply thatexposure to the respective drugs during therapy may notsignificantly exert selective pressure leading to expressionof the pumps observed in these isolates. Alternatively, andperhaps more likely, this may simply mean that the drugsstudied here (doxycycline, meropenem and ceftazidime)are not substrates for the pumps expressed in the individualisolates. However, because the purpose of this study wasnot to examine a possible correlation between antibioticresistance and efflux pump expression, such interpretationsare merely speculations at this time and the validation ofthese notions, if any, awaits further experimentation.

RND pumps are now recognized as major players inthe MDR of many Gram-negative bacteria, and mountingevidence suggests that the energy-dependent efflux ofantibiotics should be an important consideration whiledesigning new antimicrobials.22 Our efforts to characterizeRND efflux pumps in this organism may contribute to thedevelopment of more effective chemotherapies by aidingin the discovery of new drugs that are poor substrates ofthese pumps or efflux pump inhibitors,23 which may beused in combination with existing antibiotics. The findingin this study that RND pumps are widely expressed inB. pseudomallei clinical isolates supports the notion thatRND pumps probably play important roles in this bacterium’sphysiology, defence against toxic compounds and perhapsvirulence.

Authors’ contributions: AK, HPS, BJC and ACC designed thestudy protocol; AK and MM isolated RNA samples; AK andLAT performed the qRT-PCR experiments, and analysis andinterpretation of these data; AK, HPS, BJC and ACC drafted


the manuscript. All authors read and approved the finalmanuscript. HPS is guarantor of the paper.

Acknowledgements: We acknowledge the gift of strainsby Dr Mark Schell, University of Georgia and Dr DonaldWoods, University of Calgary. We thank Dr Gary Lum andthe Microbiology Laboratory staff at Royal Darwin Hospitalfor providing the RDH strains.

Funding: This study was supported by NIH grant AI065357and a Project grant from the Australian National Health andMedical Research Council. AC is supported by an AustralianNational Health and Medical Research Council PostdoctoralFellowship.

Conflicts of interest: None declared.

Ethics approval: Not required.

References1. Cheng AC, Currie BJ. Melioidosis: epidemiology, pathophys-

iology, and management. Clin Microbiol Rev 2005;18:383416.

2. Wiersinga WJ, van der Poll T, White NJ, Day NP, Peacock SJ.Melioidosis: insights into the pathogenicity of Burkholderiapseudomallei. Nat Rev Microbiol 2006;4:272 82.

3. Dance DA, Wuthiekanun V, Chaowagul W, White NJ. Theantimicrobial susceptibility of Pseudomonas pseudomallei.Emergence of resistance in vitro and during treatment.J Antimicrob Chemother 1989;24:295 309.

4. Jenney AW, Lum G, Fisher DA, Currie BJ. Antibioticsusceptibility of Burkholderia pseudomallei from tropicalnorthern Australia and implications for therapy ofmelioidosis. Int J Antimicrob Agents 2001;17:109 13.

5. Ashdown LR, Currie BJ. Melioidosis: when in doubt leavethe quinolone alone! Med J Aust 1992;157:427 8.

6. Chaowagul W, Suputtamongkul Y, Smith MD, White NJ. Oralfluoroquinolones for maintenance treatment of melioidosis.Trans R Soc Trop Med Hyg 1997;91:599 601.

7. Wuthiekanun V, Peacock SJ. Management of melioidosis.Expert Rev Anti Infect Ther 2006;4:445 55.

8. Kumar A, Schweizer HP. Bacterial resistance to antibiotics:active efflux and reduced uptake. Adv Drug Deliv Rev 2005;57:1486 513.

9. Piliouras P, Ulett GC, Ashurst-Smith C, Hirst RG, Norton RE.A comparison of antibiotic susceptibility testing methodsof cotrimoxazole with Burkholderia pseudomallei. Int JAntimicrob Agents 2002;19:427 9.

10. Moore RA, DeShazer D, Reckseidler S, Weissman A,Woods DE. Efflux-mediated aminoglycoside and macrolide

resistance in Burkholderia pseudomallei. AntimicrobAgents Chemother 1999;43:465 70.

11. Chan YY, Tan TMC, Ong YM, Chua KL. BpeAB-OprB,a multidrug efflux pump in Burkholderia pseudomallei.Antimicrob Agents Chemother 2004;48:1128 35.

12. Kumar A, Chua K-L, Schweizer HP. Method for regulatedexpression of single-copy efflux pump genes in a surrogatePseudomonas aeruginosa strain: identification of theBpeEF-OprC chloramphenicol and trimethoprim effluxpump of Burkholderia pseudomallei 1026b. AntimicrobAgents Chemother 2006;50:3460 3.

13. Holden MTG, Titball RW, Peacock SJ, Cerdeno-Tarraga AM,Atkins TP, Crossman LC, et al. Genomic plasticity of thecausative agent of melioidosis, Burkholderia pseudomallei.Proc Natl Acad Sci USA 2004;101:14240 5.

14. Stover CK, Pham X-Q, Erwin AL, Mizoguchi SD, Warrener P,Hickey MJ, et al. Complete genome sequence ofPseudomonas aeruginosa, an opportunistic pathogen.Nature 2000;406:959 64.

15. Fralick JA. Evidence that TolC is required for functioning ofthe Mar/AcrAB efflux pump of Escherichia coli. J Bacteriol1996;178:5803 5.

16. Chuanchuen R, Murata T, Gotoh N, Schweizer HP.Substrate-dependent utilization of OprM or OpmH by thePseudomonas aeruginosa MexJK efflux pump. AntimicrobAgents Chemother 2005;49:2133 6.

17. Jo JTH, Brinkman FS, Hancock REW. Aminoglycosideefflux in Pseudomonas aeruginosa: involvement of novelouter membrane proteins. Antimicrob Agents Chemother2003;47:1101 11.

18. Li XZ, Nikaido H. Efflux-mediated drug resistance inbacteria. Drugs 2004;64:159 204.

19. Clinical and Laboratory Standards Institute. M7-A7,Methods for dilution antimicrobial susceptibility tests forbacteria that grow aerobically; approved standard. 7th ed.PA: Wayne; 2006.

20. Chan YY, Chua KL. The Burkholderia pseudomallei BpeAB-OprB efflux pump: expression and impact on quorumsensing and virulence. J Bacteriol 2005;187:4707 19.

21. Chan YY, Bian HS, Tan TM, Mattmann ME, Geske GD,Igarashi J, et al. Control of quorum sensing bya Burkholderia pseudomallei multidrug efflux pump.J Bacteriol 2007;189:4320 4.

22. Li X, Zolli-Juran M, Cechetto JD, Daigle DM, Wright GD,Brown ED. Multicopy suppressors for novel antibacterialcompounds reveal targets and drug efflux susceptibility.Chem Biol 2004;11:1423 30.

23. Lomovskaya O, Bostian KA. Practical applications andfeasibility of efflux pump inhibitors in the clinic a visionfor applied use. Biochem Pharmacol 2006;71:910 8.

expression of resistance-nodulation-cell-division efflux pumps in commonly used burkholderia...

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