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Development of a DNA Microarray to Detect Antimicrobial Resistance Genes Identified in the National Center for Biotechnology Information Database

Development of a DNA Microarray to Detect Antimicrobial Resistance Genes Identified in the National Center for Biotechnology Information Database
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  Development of a DNA Microarray to DetectAntimicrobial Resistance Genes Identifiedin the National Center for Biotechnology Information Database Jonathan G. Frye, 1 Rebecca L. Lindsey, 1 Gaelle Rondeau, 2 Steffen Porwollik, 2 Fred Long, 2 Michael McClelland, 2 Charlene R. Jackson, 1 Mark D. Englen, 1 Richard J. Meinersmann, 1 Mark E. Berrang, 1 Johnnie A. Davis, 1 John B. Barrett, 1 Jennifer B. Turpin, 1 Sutawee N. Thitaram, 1 and Paula J. Fedorka-Cray 1 To understand the mechanisms and epidemiology of antimicrobial resistance (AR), the genetic elements re-sponsible must be identified. Due to the myriad of possible genes, a high-density genotyping technique isneeded for initial screening. To achieve this, AR genes in the National Center for Biotechnology InformationGenBank database were identified by their annotations and compiled into a nonredundant list of 775 genes. ADNA microarray was constructed of 70mer oligonucelotide probes designed to detect these genes encodingresistances to aminoglycosides,  b -lactams, chloramphenicols, glycopeptides, heavy metals, lincosamides, mac-rolides, metronidazoles, polyketides, quaternary ammonium compounds, streptogramins, sulfonamides, tetra-cyclines, and trimethoprims as well as resistance transfer genes. The microarray was validated with two fullysequenced control strains of   Salmonella enterica : Typhimurium LT2 (sensitive) and Typhi CT18 (multidrug re-sistance [MDR]). All resistance genes encoded on the MDR plasmid, pHCM1, harbored by CT18 were detectedin that strain, whereas no resistance genes were detected in LT2. The microarray was also tested with a variety of  bacteria, including MDR  Salmonella enterica  serovars,  Escherichia coli, Campylobacter  spp.,  Enterococcus  spp.,methicillin-resistant  Staphylococcus aureus, Listeria  spp., and  Clostridium difficile . The results presented heredemonstrate that a microarray can be designed to detect virtually all AR genes found in the National Center forBiotechnology Information database, thus reducing the subsequent assays necessary to identify specific resis-tance gene alleles. Introduction A ntimicrobial resistance  (AR) in bacteria is anongoing problem in human and animal health. Vir-tually from the inception of antimicrobial chemotherapies totreat bacterial infections, resistance was found and began toexpand. 1 Despite regulations and controls of antimicrobialuse designed to reduce its development and spread, bacterialresistance to antimicrobials continues to increase. 1 To helpunderstandthedevelopmentofARanditsspread,thegeneticmechanisms must be identified. These studies often requireassaying for dozens or hundreds of possible resistance-encoding genes to investigate the underlying genetics behindthe phenotypic resistance observed in bacteria. 12 This prob-lem has been compounded by the growth of multidrugresistance (MDR) in important pathogenic bacteria, oppor-tunistic pathogens, commensal bacteria, and environmental bacteria. To address this issue, many researches have turnedto high-density gene detection techniques, primarily DNAmicroarrays. 2,5,7,9,10,13,16,24,26,37,46 Several studies have demonstrated that DNA microarrayscan be used to detect resistance genes as effectively as standardtechniques such as polymerase chain reaction, sequencing,conjugation, and Southern hybridization. 2,5,7,9,10,13,16,24,26,37,46 Most of these microarrays rely on short to medium-length(20mer–80mer) oligonucleotides as detection probes becausethey are easily synthesized without the requirement of template DNA and can be cheaply made and arrayed onto avariety of substrates such as glass slides. 13,34 Modified mi-croscope glass slides have become the most widespreadformat for custom microarrays, with most universitiesand research institutes having access to printing robots and 1 Bacterial Epidemiology and Antimicrobial Resistance Research Unit, Richard B. Russell Research Center, Agriculture Research Service,U.S. Department of Agriculture, Athens, Georgia. 2 Sidney Kimmel Cancer Center, San Diego, California. MICROBIAL DRUG RESISTANCEVolume 16, Number 1, 2010 ª  Mary Ann Liebert, Inc.DOI: 10.1089 = mdr.2009.0082 9  Table  1.  Summary of Gene Probes with Positive Hybridizations to the Control Strains and Test Isolates  Aminoglycoside Beta-lactam Chloramphenicol Efflux Glycopeptide Heavy metal Macrolide MetronidazolePoly-ketideQuaternaryammonium Sulfonamides Tetracycline Transfer associated Trimethoprim LT2  aadA1 acrR, emrR, marA,marB nitroimidazoleresistanceCT18  aac(3), aadA, strA,strB bla, bla PER2, bla TEM-1,  temcat, cat4, catIII acrR, corA, emrR,marA, marB, msrAble merACDRT sulII tet (A) ,tet (A)(B) ,tet (R) htdAFT, InsA,IntI, parB,  putativetransposase,  repC,repE, stbA, tnpA,tnpAIS26, tnpR,traI, trhBCFNUV dfrA, dfrV,dfrV, dfrB SE Tc JF201  aac(3), aadA, aadA, aadAb,aadA2*, aadA2, aadB, aadE,acc(6)-Ib*, aph(3)-I, aph2,aphA7, aphAI, ksgA, ntpII,strA*, strB, str3K ampC, ampR,blaL2, bla, PER2 , cmy-2*,sme-1*, temcat, cmlA5, flo* acrR, corA, EmrA,emrR, marA, marB,msrA,  ABCtransporter,  ybitble merACFT nonR qac,qacEdeltasul, sulII tet (A) ,tet (A)(B) ,tet (C) , tet (D) ,tet (R) InsA, IntI,  putativetransposase,  tnpA,tnpAIS26, tnpM,tnpRdfr, dfr6,dfrA SE Hd JF210  aac(3), aadA, aadA, aadAb,aadA2*, aadA2, aadA7,aadB, acc(6)-Ib*, aph(3)-I,aphA7, aphAI, ksgA, ntpII,orfE, SAT-2, strA*, strB, str3K ampC, bla,blaL2,bla TEM-1 ,cmy-2*, sme-1*,temcat, cmlA5, flo* acrR, corA, emrR,marA, marB, msrA, ABC transporter,  ybitble merACDFPRRT,orf5nonR qac,qacEdeltasul, sulII tet (A) ,tet (A)(B) ,tet (C) , tet (D) ,tet (R) InsA, IntI, mobA, putativetransposase, tnp A, tnpAIS26,tnpM, tnpR,trhCFdfr, dfr6,dfrA EC JF220  aac(3), aac(6), aadA, aadAb,aadA2*, aadE, aph(3)-I,aphA7, aphAI, ksgA, strA*,strB, str3K ampC, ampR,bla, blaL2,bla TEM-1 ,cfxA, cmy-2*, penA,sme-1*, tem flo* acrR, corA, EmrA,emrR,marA, marB,msrA,  potassium-transportingATPase, ABCtransporter,  ybitble, vanH merACDFPRRT,orf5nonR qac,qacEdeltasul, sulII tet (A) , tet (C) ,tet (D) , tet (R) InsA, IntI,  putativetransposase,  tnpA,tnpAIS26, tnpM,tnpRdfrA, dhf  EC JF227  aacC3, aadA, aadAb, aadA2*,aadE, aph(3)-I, aphA7, aphAI,ksgA, strA*, strB, str3K ampC, ampR,bla, blaL2,bla TEM-1 ,cfxA, cmy-2*,sme-1*, temcat, flo* acrR, corA, EmrA,emrR, marA, marB,msrA,  potassium-transportingATPase, ABCtransporter,  ybitble, vanH merADFRRT,orf5nonR qac,qacEdeltasul, sulII tet (A) ,tet (A)(B) ,tet (C) , tet (D) ,tet (R) InsA, IntI,  putativetransposase,  tnpA,tnpAIS26, tnpMdfrA, dhf  CC 14-22  aad9, aphA-3 cam, cfiA cmeBC, hsdR,marRtet (O)  btgA, trfA2, trhU  CC 141-27  aphA-3 blaL2, cam cmeBC, hsdR, potassium-transportingATPase tet (O)  htdT  ECF ATCC25788  vanC* merT tnpA, trans 1   0     Aminoglycoside Beta-lactam Chloramphenicol Efflux Glycopeptide Heavy metal Macrolide MetronidazolePoly-ketideQuaternaryammonium Sulfonamides Tetracycline Transfer associated Trimethoprim EGM ATCC49573  blaL2, MecR acrR, EmrA, potassium-transportingATPase, ABCtransporter,  ybitble, vanC* tet (M) , tet (O) ,tet (S)putativetransposase, tnpA, tnpR,trans LI03  msrC,  potassium-transportingATPase,  ybitble tet (M) , tet (O) ,tet (S)putativetransposase, recP LI04  bla TEM-1  msrC,  potassium-transportingATPase,  ybitble  nitroimidazoleresistance tet (M) , tet (O) putativetransposaseMRSA G10-C87-2  blaI, mecA,mecI, mecR, MecR, pre, penicillinaserepressor,  spcmarR,  potassium-transportingATPase ble ermA  putativetransposase, tnpA, tnpAIS26,tnpB, tnpC, transposase BMRSA DN  aadE, aphA-3, sat4 blaI, mecA,mecR, MecR, pre, penicillinaserepressor,  spccat marR,  potassium-transportingATPase ble ermA  putativetransposase, tnpA, tnpB, tnpC, transposase BMRSA 36-1  aadE, aphA-3, sat4 blaI, mecA, MecR, preermC  putativetransposase, repC MRSA 59  aadE, aphA-3, sat4 mecA, MecR  putativetransposase, repC CD 70CD 98  orfE CD 112  blaL2 tet (C)  parB, tnpB,tnpR,trhC CD 113Complete data in Supplemental Table S1 (available online at = mdr).*Indicates multiple identical probes for this gene. Genes with more than one different probe are listed for each positive hybridization detected.Isolates: SE, Salmonella enterica; Hd, Heidelberg; Tc, serovar Typhimurium variant Copenhagen; EC, Escherichia coli; CC, Campylobacter coli; ECF, Enterococcus casseliflavus; EGM, Enterococcus gallinarum; LI, Listeria Innocua; MRSA, MethicillinResistant Staphylococcus aureus; CD, Clostridium difficile. 1  1    scanners designed to manufacture and analyze these arrays.With the advent of this technology, it should be theoreticallypossible to design microarrays for the detection of all knownsequenced AR genes available in the public domain andcheaply construct them in many research facilities worldwide. 13,23 It is important to note that this microarray is notintended to replace phenotypic testing in diagnostic andclinical settings, although there has been considerable prog-ress in the development of diagnostic microarrays. 2,4,15,31 Microarray data are difficult to interpret in a clinical setting because the detection of a gene is a potentially nebulousresult. Indeed, genes detected may not be functional or ex-pressed, and negative hybridization results are even moredifficult to interpret, as previously uncharacterized resistancemechanisms could lead to failure of a selected treatment. Inthe clinical setting, the goal is to select a successful treatmentregimen, thus making phenotypic testing more informativethan gene detection. However, when the goal is to study themolecular epidemiology of AR, DNA microarrays are anexceptional tool for detecting multiple AR genes in a singleassay.In the present study, a DNA microarray was designed todetect as many resistance genes as possible for use as ascreening technique in studies of prevalence, epidemiology,and spread of resistance genes. The National Center forBiotechnology Information (NCBI) databases and simple bioinformatics processes were used to build a local databaseof target gene sequences. Unique oligonucleotide probeswere designed for the detection of each AR-associated genein this database. 13,43 Probes 70 bases in length were chosen because of their ability to tolerate possible mismatcheswithin the probe regions and to detect as many alleles of resistance genes as possible. These sequences were used toconstruct the microarray utilizing the most widely availablemicroarray format, glass slides, which was then tested oncontrol strains and a variety of MDR bacteria. The resultsdemonstrate that these simple and relatively inexpensivetechniques yielded a highly useful research tool to study theepidemiology of AR genes in a wide range of important bacteria. This report supplies researchers in the field withinformation they can use to build their own arrays and im-prove upon this technique. Materials and Methods Identification and selection of target genes  Previous work demonstrated that oligonucleotide micro-arrays can be used to detect resistance genes in a wide va-riety of bacteria. 13,25,26,37,41 The major short coming of thesemicroarrays as well as our previous array (described in Frye et al. 13 ) was the limited number of genes they were designedto detect and thus the limited number of bacteria they could be used to investigate. To design a microarray representingthe most comprehensive set of genes associated with AR,sequences available in the NCBI GenBank database wereidentified by employing several database search strategies. Aquery designed to yield the maximum number of nonre-dundant genes annotated as ‘‘bacteria & antibiotic & resist*’’yielded 3,391 genes from the nonredundant DNA sequencedatabase as well as 1,115 genes from the translated proteindatabase. All of these sequences were sorted by nucleotidecoding sequence enabling the identification and eliminationof identical duplicate genes, leaving 3,751 genes. These se-quences were downloaded into a local database, and 70meroligonucleotides were designed as described below. 35,43 Allprobe sequences likely to detect a homologous gene in theprobe set ( > 90% identity over the length of the 70mer) wereeliminated from the list for a final total of 1,224 gene probes.Probe sequences that would detect any of the 94 genes fromour previous work (Frye  et al. 13 ) were excluded, as thosepreviously designed probes were included on the new mi-croarray and used as working control probes. 13 Next, theannotation of each gene was examined, and genes notlikely to be related to AR or AR gene transfer were deletedleaving 681 genes. The sequences of these probes as well asthe probes from the previous array (total  n ¼ 775) are in-cluded in Supplemental Table S1 (available online at = mdr). Finally, the probe sequences wereBLAST-Like Alignment Tool (BLAT) searched against theentire NCBI database to identify all close homologs to whichthey would likely hybridize. 18 This yielded 24,489 genes inthe complete NCBI database that were likely to be detected by the microarray at the time of the query (data not shown). Oligonucleotide probe design and microarray construction  Sequences for the 681 genes were used to design an opti-mized unique 70mer oligonucleotide probe for each geneusing the program OligoWiz 2.0 following methods andsettings recommended by the authors (probe sequences arepresented in Supplemental Table S1). 35,43 Oligonucleotideprobes of this length were selected, as previous work haddemonstrated that 70mers have good specificity, can tolerateseveral nucleotide mismatches in the target sequence, requireno chemical modification to adhere to the slide surface, andgive good results without the need for problematic labelingor amplification of DNA samples. 11,13,19,23 These probes weresynthesized (Operon, Huntsville, AL), diluted in printing buffer (35 m M in 50% dimethyl sulfoxide [DMSO]), and ar-rayed in triplicate onto UltraGaps amino silane–coated slides(Corning, Life Sciences, Acton, MA) with an Omnigrid robot(Genemachines, San Carlos, CA), and postprocessed as pre-viously described. 13,38 As stated above, the 94 probes fromour test microarray were added for a total of 775 unique AR-associated genes. 13 Additional controls included 3 positivecontrol probes designed to detect bacterial 16s rDNA se-quences, 22 2 Cy3-labeled and 1 Cy5-labeled Lambda DNAcontrols, 12 buffer (50% DMSO) only spots, and 130 empty(background) spots. Twelve probes were also synthesized induplicate or triplicate as controls (indicated in Table 1 andSupplemental Table S1 with an ‘‘*’’). Strains, growth conditions,and antimicrobial susceptibility  The fully sequenced control strains were  Salmonella enterica serovar Typhimurium LT2 28 and  Salmonella enterica  serovarTyphi CT18 ( S.  Typhi CT18). 36 Enterococcus  control strainsobtained from the American Type Culture Collection (ATCC,Manassas, VA) are indicated by their ATCC numbers inTables 1 and 2. Test isolates of   Salmonella  serovars,  Escherichiacoli, Enterococcus  spp.,  Campylobacter coli, Listeria innocua ,methicillin-resistant  Staphylococcus aureus  (MRSA), and Clostridium difficile  were obtained from the National Anti- 12 FRYE ET AL.  Table  2.  Antimicrobial Susceptibility Phenotypes of Test Isolates Used in This Study  Antimicrobial class Antimicrobial  S    E    T   c    J    F    2    0    1     b     S    E    H    d    J    F    2    1    0    E    C    J    F    2    2    0    E    C    J    F    2    2    7    C    C    1    4       -     2    2    C    C    1    4    1       -     2    7    E    C    F    A    T    C    C    2    5    7    8    8    E    G    M    A    T    C    C    4    9    5    7    3    L    I    0    3    L    I    0    4    M    R    S    A    G    1    0       -     C    8    7       -     2    M    R    S    A    D    N    M    R    S    A    3    6       -     1    M    R    S    A    5    9    C    D    7    0    C    D    9    8    C    D    1    1    2    C    D    1    1    3 Aminoglycosides AMK S S S SAPR R SGEN R R R R S S S ID S S S S S SKAN R R R R S IDSTR R R R R S ID S S S S S SBacitracin BAC R RBeta-lactams AMC R R R R S S S S S SAMP R R R R S S R R R R S R S SCEF R R R RCRO S I S I R S R R S RFEP S SFOX R R RIPM S SOXA R R R R R RPEN S R R R R R R RTIO R R S RChloramphenicol CHL R R R R S S S SPhosphoglycolipid FLA R IDGlycopeptides VAN S I S S S S S S S S S SLincosamides CLI S R R S R R R R R R S RLIN R R S S S SLipopeptide DAP S S S S S SMacrolides AZM S RERY S R I S S S R R R R S R S STYL S SMetronidazole MET R R R RNitrofurantoin NIT S SOxazolidinone LZD S ID S S S S S SQuinolones CIP S S R R S S I S S S R S R SGAT S S R S S SLEV S S R S S S S S S SNAL S S R R S SRifamycin RIF S S S S S S R S R SStreptogramin Q-D I ID S S S S S SSulfanilamide NIL R R R RTetrcycline TET R R R R S R S R R S S S R STrimeth/sulfa SXT R R S S S S S S S S S SClinical and Laboratory Standards Institute (formerly NCCLS) breakpoints were used to determine resistance phenotype.Antimicrobials: AMK, amikacin; APR, apramycin; GEN, gentamicin; KAN, kanamycin; STR, streptomycin; BAC, bacitracin; AMC, amoxicillin-clavulanic acid; AMP, ampicillin; CEF, cephalothin; CRO,ceftriaxone; FEP, cefepime; FOX, cefoxitin; IPM, imipenem; OXA, oxacillin; PEN, penicillin; TIO, ceftiofur; CHL, chloramphenicol; FLA, flavomycin; VAN, vancomycin; CLI, clindamycin; LIN, lincomycin; DAP,daptomycin; AZM, azithromycin; ERY, erythromycin; TYL, tylosin; MET, metronidazole; NIT, nitrofurantoin; LZD, linezolid; CIP, ciprofloxacin; GAT, gatifloxicin; LEV, levofloxicin; NAL, nalidixic acid; RIF,rifamampin; Q-D, quinupristin-dalfopristin (Synercid); NIL, sulfanilamide; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole.Isolates: SE,  Salmonella enterica ; Hd, Heidelberg; Tc, serovar Typhimurium variant Copenhagen; EC,  Escherichia coli ; CC,  Campylobacter coli ; ECF,  Enterococcus casseliflavus ; EGM , Enterococcus gallinarum ; LI,  ListeriaInnocua ; MRSA, Methicillin Resistant  Staphylococcus aureus ; CD,  Clostridium difficile. R, resistant; S, susceptible; I, intermediate; ID, indeterminate (due to lack of CLSI standard); empty block, not assayed. 1   3  
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