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Investigation of aminoglycoside resistance inducing conditions and a putative AmrAB-OprM efflux system… Jassem, Agatha N; Forbes, Connor M; Speert, David P Jan 6, 2014

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SHORT REPORT Open AccessInvestigation of aminoglycoside resistanceinducing conditions and a putative AmrAB-OprMefflux system in Burkholderia vietnamiensisAgatha N Jassem1,3*, Connor M Forbes2,3 and David P Speert1,2,3AbstractBackground: Burkholderia cepacia complex (BCC) bacteria are highly virulent, typically multidrug-resistant,opportunistic pathogens in cystic fibrosis (CF) patients and other immunocompromised individuals. B. vietnamiensisis more often susceptible to aminoglycosides than other BCC species, and strains acquire aminoglycoside resistanceduring chronic CF infection and under tobramycin and azithromycin exposure in vitro, apparently from gain ofantimicrobial efflux as determined through pump inhibition. The aims of the present study were to determine ifoxidative stress could also induce aminoglycoside resistance and provide further observations in support of a rolefor antimicrobial efflux in aminoglycoside resistance in B. vietnamiensis.Findings: Here we identified hydrogen peroxide as an additional aminoglycoside resistance inducing agent inB. vietnamiensis. After antibiotic and hydrogen peroxide exposure, isolates accumulated significantly less [3H]gentamicin than the susceptible isolate from which they were derived. Strains that acquired aminoglycosideresistance during infection and after exposure to tobramycin or azithromycin overexpressed a putative resistance-nodulation-division (RND) transporter gene, amrB. Missense mutations in the repressor of amrB, amrR, were identifiedin isolates that acquired resistance during infection, and not in those generated in vitro.Conclusions: These data identify oxidative stress as an inducer of aminoglycoside resistance in B. vietnamiensis andfurther suggest that active efflux via a RND efflux system impairs aminoglycoside accumulation in clinical B. vietnamiensisstrains that have acquired aminoglycoside resistance, and in those exposed to tobramycin and azithromycin, but nothydrogen peroxide, in vitro. Furthermore, the repressor AmrR is likely just one regulator of the putative AmrAB-OprM effluxsystem in B. vietnamiensis.Keywords: Burkholderia vietnamiensis, Aminoglycoside, Azithromycin, Hydrogen peroxide, Efflux, AmrB, AmrRFindingsMembers of the Burkholderia cepacia complex (BCC)can cause severe respiratory infections in individualswith cystic fibrosis (CF) [1]. Furthermore, many strainsare highly and intrinsically resistant to various antimicro-bials, including aminoglycosides [2], ribosome-targetingantibiotics important in the treatment of CF respiratorydisease [3].B. cenocepacia studies suggest that resistance-nodulation-division (RND) efflux systems are involved in BCC resist-ance to aminoglycosides [4-6]. The MexXY-OprM RNDpump is the predominant determinant of aminoglycosideresistance in CF isolates of Pseudomonas aeruginosa [7],and aminoglycoside susceptibility in B. pseudomalleiresults from loss of AmrAB-OprA [8]. At subinhibitoryconcentrations, ribosome-targeting antibiotics and oxida-tive stress induce mexXY expression [9,10]. mexXY isunder the control of the MexZ repressor [11], and mexZmutations are common in pan-aminoglycoside resistantisolates [12].We previously reported that B. vietnamiensis isolates areoften aminoglycoside-susceptible and strains acquire resist-ance during chronic CF infection and under tobramycin* Correspondence: agatha.jassem@gmail.com1Department of Pathology and Laboratory Medicine, University of BritishColumbia, 950 W 28th Avenue, Vancouver, British Columbia V5Z 4H4, Canada3Centre for Understanding and Preventing Infection in Children, Child &Family Research Institute, University of British Columbia, 950 W 28th Avenue,Vancouver, British Columbia V5Z 4H4, CanadaFull list of author information is available at the end of the article© 2014 Jassem et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedicationwaiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwisestated.Jassem et al. Annals of Clinical Microbiology and Antimicrobials 2014, 13:2http://www.ann-clinmicrob.com/content/13/1/2and azithromycin pressure in vitro [13]. Decreased accessof aminoglycosides to their target resulted from apparentgain of antimicrobial efflux via a RND pump, the latter de-termined with an inhibitor [13].B. vietnamiensis develops aminoglycoside resistanceunder hydrogen peroxide pressure in vitroAminoglycoside resistance can be induced in susceptibleCF isolates of B. vietnamiensis following serial exposureto tobramycin (Table 1: C8395TE, D0072TE) or a singleexposure to subinhibitory concentrations of azithromycin[13]. To characterize resistance inducing antimicrobialpressures further, after serial passage in cation-adjustedMueller-Hinton broth (CAMHB) containing azithromy-cin, meropenem, ceftazidime, and co-trimoxazole at doub-ling concentrations as described previously [13] the drugsusceptibility of C8395 was evaluated. Triplicate minimuminhibitory concentrations (MICs) were determined usingbroth microdilution methods [14], and their stabilityconfirmed after 20 passages on antibiotic-free media.P. aeruginosa and non-Enterobacteriaceae breakpointswere used in the absence of B. cepacia breakpoints.Only serial exposure of C8395 to azithromycin resultedin notable (≥4 fold) increases in aminoglycoside MICs(Table 1). By previously described methods [9], but withselective agar containing tobramycin at 2.5 times theMIC, serial exposure of C8395 to hydrogen peroxideat half the MIC resulted in a 16-fold stable increasein aminoglycoside MIC for C8395PE (Table 1). Otheracquired resistance was also observed: after passagewith all antimicrobials the MICs of the respectiveagents against C8395 increased greatly (Table 1:C8395AE, C8395ME, C8395CE, and C8395SE), andsome other cross-resistance, most notably betweenthe β-lactams antibiotics, was also seen.Hydrogen peroxide is, therefore, an additional inducerof aminoglycoside resistance in B. vietnamiensis in vitro, aparticularly important finding because CF airways are richin reactive oxygen species [15]. Moreover, B. vietnamiensiscan acquire resistance after exposure to other antimicro-bials used in treating BCC-infected CF patients, namelymeropenem, ceftazidime, and co-trimoxazole [16].The aminoglycoside-resistant derived isolates C8395TEand C8395PE accumulated 2.65 and 3.50 times less [3H]gentamicin than C8395, respectively (P = 0.0118, one-wayANOVA) (data not shown). Accumulation was deter-mined in triplicate in Luria-Bertani (LB) medium as previ-ously used to show the late, aminoglycoside-resistantisolate D0774 accumulates less gentamicin than C8395[13]. There were no significant differences in theCFU/ml between C8395 and the comparison isolatesat starting time (data not shown). Decreased access ofaminoglycosides to their intracellular target is, therefore,responsible for the observed in vitro antibiotic andoxidative stress-induced resistance.Analysis of putative efflux system genes in clinical andin vitro stress exposed B. vietnamiensis isolatesOf the 11 putative RND transporters that the sequencedenvironmental B. vietnamiensis isolate G4 (accessionTable 1 Antimicrobial susceptibilities of B. vietnamiensis after serial exposure to antibiotics or hydrogen peroxideIsolatea MIC (μg/ml)bAMK GEN KAN TOB AZM MEM CAZ SXT CIPClinical CFC8395 (3/11/1998, Bv1) 2 4 2 2 32 1 4 2/10 1D0774 (25/7/2003, Bv1) >128 128 128 128 >2048 128 128 64/320 >32D0072 (15/03/2002, Bv3) 2 4 1 2 32 0.5 2 2/10 1D2910 (31/03/2008, Bv3) 128 32 64 32 >32 2 4 1/5 16In vitro exposedC8395TE (TOB) >128 >128 128 >128 64 1 4 4/20 4C8395AE (AZM) 32 16 16 16 2048 2 16 8/40 4C8395ME (MEM) 16 8 8 8 32 16 64 4/20 16C8395CE (CAZ) 8 8 4 4 32 8 16 2/10 16C8395SE (SXT) 8 8 2 2 32 0.5 4 >64/320 8C8395PE (peroxide) 32 64 32 32 32 4 16 4/20 4C8395PC (control) 8 8 4 8 32 1 4 1/5 1D0072TE (TOB) 32 32 16 16 >32 1 2 2/10 1aPatient identification numbers and bacterial isolation dates are noted in brackets. Abbreviations: TE, TOB exposed; AE, AZM exposed; ME, MEM exposed; CE, CAZexposed; SE, SXT exposed; PE, hydrogen peroxide exposed; PC, passage control.bAminoglycoside and azithromycin MICs for C8395 and D0774, and tobramycin and azithromycin MICs for D0072, D2910, C8395TE, and D0072TE were previouslypublished to some extent [13] and are shown here for comparison. MICs represent susceptibility after 3 passages on antibiotic-free media. Abbreviations: AMK,amikacin; GEN, gentamicin; KAN, kanamycin; TOB, tobramycin; AZM, azithromycin; MEM, meropenem; CAZ, ceftazidime; SXT, co-trimoxazole; CIP, ciprofloxacin.Jassem et al. Annals of Clinical Microbiology and Antimicrobials 2014, 13:2 Page 2 of 5http://www.ann-clinmicrob.com/content/13/1/2NC_009256.1) contains (determined as previously [17]),following sequence alignment only Bcep1808_1575showed high identity, 71%, 85%, and 92%, with the char-acterized transporters MexY (accession NC_008463.1)and AmrB (accession NC_007434.1), and their homologueBCAL1675 in B. cenocepacia (accession NC_011000.1),respectively. These transporters are part of an operon alsoencoding a repressor, membrane fusion protein, and outermembrane channel [7]. PCR product analysis revealedthat B. vietnamiensis clinical isolates C8395, D0774,D0072, and D2910 contained these efflux system genes inthe same order (data not shown).To evaluate the expression of RND pump genes in B.vietnamiensis, triplicate overnight cultures were diluted1:100 into CAMHB, LB medium, or synthetic cysticfibrosis sputum medium (SCFM) [18] with or withouttobramycin and ceftazidime at half MIC or azithromycin,meropenem, and co-trimoxazole at a quarter MIC, andgrown to an optical density at 600 nm (OD600) of 0.5 or0.8. RNA was extracted using an RNeasy Plus Mini kit(Qiagen, Toronto, Canada), and treated with RNase-freeDNase (Promega, San Luis Obispo, USA). Reverse tran-scription was performed using SuperScript II ReverseTranscriptase (Invitrogen, Carlsbad, USA) according tothe manufacturer’s protocol. Amplification of the resultantDNA was quantified in a 7300 Real-Time PCR System(Applied Biosystems, Carlsbad, USA) in the presence ofSybrGreen (Invitrogen), with primers for Bcep1808_1575(5’-CCGAACGACATCTACTTCAAGGTCGG-3’, 5’-ATCCTTCGCGACTTCGACGATCAG-3’), Bcep1808_1573 (pu-tative repressor gene) (5’-TGCAGATCCTGCGGATCAC-GAAA-3’, 5’-TTCGAGCAACGACACCAGATAGACG-3’),and 16S (for normalization) (5’-CACGCTTTACGCCCAGTAATTCCG-3’, 5’-CCGGAAGAATAAGCACCGGCTAAC-3’). Denaturation occurred at 95°C for 10 minutes,followed by 40–50 cycles of 15 seconds at 95°C and 1minute at 60°C.The late, aminoglycoside-resistant isolate D0774expressed 11.4-, 9.6-, and 8.0-fold more Bcep1808_1575,herein “amrB”, than the early, aminoglycoside-susceptibleisolate C8395, at OD600 of 0.8 in CAMHB, LB medium,and SCFM, respectively (P < 0.01) (Figure 1A). amrBexpression between D0774 and C8395 was, however, 2.7times less at OD600 of 0.5 vs 0.8 (P < 0.001) (Figure 1A),owing to D0774 expressing less amrB earlier (P < 0.01)(data not shown). Compared with C8395, D0774 alsoexpressed 3.6- and 3.2-fold more Bcep1808_1573,herein “amrR”, at OD600 of 0.8 in CAMHB and SCFM,respectively (P < 0.01) (Figure 1A). In another set of se-quential isolates, the late, aminoglycoside-resistant isolateD2910 also overexpressed amrB (by 5.3-fold) and amrR(by 2.4-fold) compared with the early, aminoglycoside-susceptible D0072 at OD600 of 0.8 in CAMHB (data notshown).After a single exposure of C8395 to subinhibitoryconcentrations of test antimicrobials in CAMHB, amrBexpression did not change (data not shown). After expos-ure of C8395 to serially doubling concentrations of tobra-mycin or azithromycin but not to other antibiotics orhydrogen peroxide (i.e. in C8395TE and C8395AE),expression of amrB increased 9.9- and 8.6-fold, respect-ively (P < 0.01) (Figure 1B, data not shown). Comparedwith C8395, C8395TE also expressed 14.2-fold lessamrR (P < 0.01) (Figure 1B).Active efflux via a RND efflux system is, therefore,probably involved in the decreased drug accumulationobserved in B. vietnamiensis strains that acquired ami-noglycoside resistance during infection and after expos-ure to tobramycin and azithromycin in vitro, owing tothe correlation between resistance and amrB expression.Other aminoglycoside resistance determinants exist,since exposure to hydrogen peroxide did not induceamrB expression, and future tests with minimal mediumwould demonstrate that absoluteness of these observa-tions. As proposed previously [19], amrB upregulationonly in response to ribosome-targeting agents suggests itis a response to this interaction, not to antibiotics per se.Moreover, amrB overexpression was not sufficient tocause resistance to non-aminoglycoside antibiotics,supporting the notion that they are not substrates forthe putative AmrAB-OprA efflux system [13]. Lastly,there was no association between amrR expressionand aminoglycoside resistance or amrB expression, asis also true for mexZ [20].To determine if mutations in amrR were responsible forthe observed overexpression of amrB in B. vietnamiensis,sequences of the putative repressor were examined. DNAisolation, PCR using Phusion High-Fidelity DNA Poly-merase (New England Biolabs, Ipswich, USA) withspecific primers (5’-TTCAAAGAGGTGTGGGCAGGA-3’,5’-CCGAAACCCGTGTTGTTCATC-3’), and product ana-lysis by agarose gel electrophoresis were done using stand-ard protocols [21]. PCR products were purified with aWizard SV Gel and PCR Clean-Up System (Promega) andcloned into One Shot TOP10 E. coli cells with a Zero BluntTOPO PCR Cloning Kit (Invitrogen). Plasmid DNA wasisolated using a QIAprep Miniprep Kit (Qiagen), andM13 primers amplified amrR. Resultant products weresequenced at the UBC Centre for Molecular Medicineand Therapeutics.C8395 and D0774 amrR differed from that of G4by two silent mutations (data not shown). The late,aminoglycoside-resistant isolate D0774 also contained asubstitution at position 425 (T→ C), that at residue 142of the protein, in the suggested ligand binding alphahelix region [22], replaces a leucine with a proline. D0072and D2910 amrR sequences also differed: at position 156,or amino acid residue 52 amid the predicted DNA andJassem et al. Annals of Clinical Microbiology and Antimicrobials 2014, 13:2 Page 3 of 5http://www.ann-clinmicrob.com/content/13/1/2C-terminal ligand binding domains [22], there was a~2000 bp insertion in the late, aminoglycoside-resistantD2910. Only silent mutations were observed in amrRamong C8395, C8395TE, C8395AE, C8395PE, andC8395PC (data not shown).The amrR mutations identified likely influenced theexpression of the putative B. vietnamiensis amrB trans-porter gene. The change in D0774 AmrR may indirectlyaffect DNA binding to the transcription factor [22], whilethe large insertion within D2910 amrR likely inactivatesthe repressor altogether. As per the in vitro derived isolatefindings, aminoglycoside-resistant P. aeruginosa isolatesoverexpressing mexXY without mutations in mexZ alsoexist [23,24].In conclusion, in B. vietnamiensis, oxidative stresscan induce aminoglycoside resistance, while active effluxvia the putative AmrAB-OprM efflux system is likelyinvolved in clinical and in vitro antimicrobial-inducedFigure 1 Expression of the putative RND efflux system genes amrB and amrR in clinical CF and in vitro antibiotic or hydrogen peroxideexposed B. vietnamiensis isolates. Expression was determined by real-time reverse transcription PCR and compared (A) between the early,aminoglycoside-susceptible isolate C8395, and the late, aminoglycoside-resistant D0774, in various types of media and stages of growth, and (B)between C8395 before and after its exposure to various antimicrobials, peroxide, or passage alone in CAMHB. The averages of three technical repeatswere taken for each biological replicate. Fold change means were calculated by comparing the mean expression in C8395 to each biological replicateof (A) D0774 or (B) condition. Data points represent the averages of three biological replicates ± standard errors. (A) **, P< 0.01; *** P< 0.001 by unpairedStudent’s t-test. (B) *, P< 0.05; **, P< 0.01 by Dunnett’s Multiple Comparison Test after one-way ANOVA. Abbreviations: amrB, Bcep1808_1575; amrR,Bcep1808_1573; RND, resistance-nodulation-division; CAMHB, cation-adjusted Mueller-Hinton broth; LB, Luria-Bertani medium; SCFM, synthetic cystic fibrosissputum medium; OD600, optical density at 600 nm.Jassem et al. Annals of Clinical Microbiology and Antimicrobials 2014, 13:2 Page 4 of 5http://www.ann-clinmicrob.com/content/13/1/2aminoglycoside resistance. Such elucidation of resistanceinducing conditions and resistance factors may improvetherapeutic regimens against infection with this species.Additional mechanisms of aminoglycoside resistanceshould be investigated next. The contribution of resistancedeterminants to aminoglycoside inefficacy may explain theobserved varied degrees of resistance.Availability of supporting dataThe data supporting the results of this study is includedwithin the article.AbbreviationsBCC: Burkholderia cepacia complex; CF: Cystic fibrosis; RND: Resistance-nodulation-division; CAMHB: Cation-adjusted Mueller-Hinton broth;MIC: Minimum inhibitory concentration; LB: Luria-Bertani; SCFM: Syntheticcystic fibrosis sputum medium; OD600: Optical density at 600 nm.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsANJ designed the study, performed and analyzed experiments not done byCMF, and wrote the manuscript. CMF serially exposed C8395 to hydrogenperoxide and performed most of the susceptibility and expression tests. DPSparticipated in the design and coordination of the study and criticallyreviewed the manuscript. All authors read and approved the finalmanuscript.Authors’ informationPart of this work was presented at the American Association for theAdvancement of Science Annual Meeting, Vancouver, BC, Canada, 16 to 20February 2012.AcknowledgementsSupport was provided by Cystic Fibrosis Canada (D.P.S. grant; A.N.J., C.M.Fstudentships) and the Child & Family Research Institute (C.M.F. studentship).We thank Trevor Hird for technical assistance.Author details1Department of Pathology and Laboratory Medicine, University of BritishColumbia, 950 W 28th Avenue, Vancouver, British Columbia V5Z 4H4,Canada. 2Department of Pediatrics, University of British Columbia, 950 W28th Avenue, Vancouver, British Columbia V5Z 4H4, Canada. 3Centre forUnderstanding and Preventing Infection in Children, Child & Family ResearchInstitute, University of British Columbia, 950 W 28th Avenue, Vancouver,British Columbia V5Z 4H4, Canada.Received: 30 October 2013 Accepted: 1 January 2014Published: 6 January 2014References1. 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Antimicrob Agents Chemother 2003, 47(10):3202–3207.doi:10.1186/1476-0711-13-2Cite this article as: Jassem et al.: Investigation of aminoglycosideresistance inducing conditions and a putative AmrAB-OprM efflux system inBurkholderia vietnamiensis. Annals of Clinical Microbiology and Antimicrobials2014 13:2.Jassem et al. Annals of Clinical Microbiology and Antimicrobials 2014, 13:2 Page 5 of 5http://www.ann-clinmicrob.com/content/13/1/2

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