0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.5.2291–2302.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Microarray-Based Detection of 90 Antibiotic Resistance Genes of Gram-Positive Bacteria
Vincent Perreten,
1* Lorianne Vorlet-Fawer,
1Peter Slickers,
2Ralf Ehricht,
2Peter Kuhnert,
1and Joachim Frey
1Institute of Veterinary Bacteriology, University of Berne, CH-3001 Bern, Switzerland,1and Clondiag Chip Technologies GmbH, D-07743 Jena, Germany2
Received 23 July 2004/Returned for modification 8 September 2004/Accepted 5 January 2005
A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, andLactococcus lactisand an avirulent strain ofBacillus anthracisharboring the broad- host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in aStaphylococcus haemolyticusstrain isolated from mastitis milk and 6 resistance genes in aClostridium perfringensstrain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance.
The intensive use of antibiotics in both public health and animal husbandry has selected for antibiotic-resistant bacteria (39). Under antibiotic selective pressure, bacteria have the abil- ity to develop and exchange resistance genes, making them non- susceptible to the antimicrobial substances deployed. While antibiotic resistance has emerged in some important animal and human gram-positive pathogens, such asStaphylococcus andStreptococcusspp. andClostridium perfringens, others, such asBacillus anthracis, are currently still sensitive to antibiotics (15, 24). Nevertheless,B. anthraciscan acquire resistance genes from other gram-positive bacteria in vitro, as previously de- scribed (30, 46) and as demonstrated in this study. It is there- fore important to follow the evolution of antibiotic resistance in the bacterial population in order to prevent and repress the emergence of multidrug-resistant strains of those bacteria that can still be treated with antibiotics.
Furthermore, commensal bacteria represent a reservoir of antibiotic resistance genes that have the potential to be trans- ferred to human and animal pathogens. An effort has therefore been made in Europe to reduce the emergence and spread of resistant bacteria. The use of antimicrobial substances for non- therapeutic purposes in animal husbandry has been banned, and surveillance programs for antibiotic-resistant bacteria among both human and animal isolates have been implemented (40).
Additionally, it has been proposed that bacteria used as pro- biotics in food or feed or as starter cultures for the food in- dustry must be free of antibiotic resistance genes (http://europa
.eu.int/comm/food/fs/sc/scf/out178_en.pdf). Bacteria used in food preparation are mainly gram positive and includeLacto- coccus, Lactobacillus, Pediococcus, Leuconostoc, Carnobacte- rium, Enterococcus,Micrococcus,Streptococcus, Staphylococcus, andPropionibacteriumspp. Animal probiotics consist mainly of strains ofBacillus,Enterococcus faecium,Pediococcus, Lacto- bacillus, andStreptococcus.
A simple method which allows the rapid detection of anti- biotic resistance genes would complement the standard MIC determination for pathogenic and commensal bacteria. In the clinic, this would have the advantage of detecting silent anti- biotic resistance genes which might be turned on in vivo or spread to other bacteria and would help in prescribing the appropriate antibiotic. Such a method could also be applied to slow-growing bacteria, for which the MIC determination may cause problems. In the food industry, it would help to deter- mine whether antibiotic-susceptible starter cultures harbor si- lent antibiotic resistance genes which could directly reach con- sumers through the food chain. This technology could be used as a tool to survey the antibiotic resistance gene situation in specific bacteria and would enable rapid tracking of newly emerging resistance genes. For these purposes, a convenient and affordable technology should be available.
Today, PCR and hybridization analysis are common meth- ods used to detect antibiotic resistance genes in bacteria. How- ever, the detection of specific resistance genes remains a tre- mendous amount of work if every possible resistance gene has to be assessed, and therefore microarray technology is most suitable for resistance gene analysis (28). The few microarrays that have been developed to date for identification of antibi- otic resistance genes are either restricted to a class of drug or
* Corresponding author. Mailing address: Institute of Veterinary Bacteriology, University of Berne, La¨ngass-Strasse 122, Postfach, CH- 3001 Bern, Switzerland. Phone: 41 31 631 2430. Fax: 41 31 631 2634.
E-mail: vincent.perreten@vbi.unibe.ch.
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limited to a certain number of genes. Call et al. developed a microarray for detecting 17 tetracycline resistance genes and one-lactamase gene (8). Recently, a microarray-based system has been optimized for the detection of genes specific toStaph- ylococcus aureus, including 12 resistance genes known to occur occasionally in this species (37).
In this report we describe the first hybridization system using microarray technology for routine microbial investigations that allows rapid and efficient screening of gram-positive bacteria for the presence of up to 90 of the most prevalent and trans- ferable antibiotic resistance genes.
MATERIALS AND METHODS
Bacterial strains and growth conditions.The bacterial strains and plasmids used in this study are listed in Table 1. Strains harboring well-characterized resistance genes as well as field strains were used to test the specificity and sensitivity of the microarray-based hybridization system. Hybridization results are shown only for some selected strains (see Fig. 2 and 3). The completely
sequenced broad-host-range enterococcal plasmid pRE25 (48), which contains five resistance genes [catpIP501,erm(B),sat4,aph(3⬘)-III, andant(6)-Ia], was used as a gene target to reveal the presence of resistance genes inEnterococcusand in an avirulent strain ofB. anthracis.Lactococcus lactisK214, harboring the mosaic resistance plasmid pK214 [tet(S),cat-LM,mdt(A), andstr] (43), was used as an example of a starter culture. The array was also tested with a vancomycin- resistantE. faeciumstrain harboring avan(A) gene and with strains showing a multidrug resistance phenotype but an unknown genotype. For this purpose, one Staphylococcus haemolyticusstrain isolated from mastitis milk and oneC. per- fringensisolate from cattle were investigated.
All the strains were grown on tryptone soya agar containing 5% defibrinated sheep blood (Oxoid Ltd., Basingstoke, England) at 37°C unless otherwise indi- cated.C. perfringenswas incubated under anaerobic conditions.L. lactiswas grown on M17 agar (Oxoid) at 30°C.Escherichia coliandB. anthracisstrains were grown on Luria-Bertani (LB) agar plates at 37°C. In liquid media,Enterococcus andStaphylococcuswere grown in brain heart infusion broth,Bacillusstrains in LB broth, andL. lactisin GM17 broth.C. perfringenswas grown in Scha¨dler broth (Oxoid) supplemented with 0.05% (vol/vol)L-cysteine at 37°C under anaerobic conditions. The assays involvingB. anthracisstrains were performed in a bio- safety level 3 laboratory using avirulent strains.
TABLE 1. Bacterial strains and plasmids
Strain Characteristic(s)a Reference or sourceb
Enterococcus faecalisRE25 pRE25 [erm(B),catpIP501,aph(3ⴕ)-III,sat4,ant(6)-Ia];tet(M) 48
Enterococcus faecalisJH2-2 RifrFusr 31
Enterococcus faecalisJHRE25-2 JH2-2 containing pRE25 [erm(B),catpIP501,aph(3⬘)-III,ant(6)-Ia, sat4]; RifrFusr 48
Lactococcus lactisK214 pK214 [tet(S),cat-LM,mdt(A),str] 43, 44
Clostridium perfringensMLP26c tetA(P)erm(B)sat4catPaph(3⬘)-III ant(6⬘)-Ia This study
Staphylococcus haemolyticusVPS617d tet(K)mph(C)erm(C)msr blaZ mecA dfr(A)aph(3ⴕ)-III aph(2ⴕ)-Ia aac(6ⴕ)-Ie ant(6ⴕ)-IaInorAsat4
This study
Bacillus anthracis4230 pXO2⫹[⌬cap::ant(9)-Ia,acpA];pX01ⴚ;bla1 bla2 23
Bacillus anthracisBR4253 4230 containing pRE25 [erm(B),catpIP501,aph(3⬘)-III,ant(6)-Ia,sat4]; pXO2⫹ [⌬cap::ant(9⬘)-Ia,acpA]; pXO1⫺;bla1 bla2
This study Enterococcus faeciumSF11770 aac(6⬘)-Im aph(2⬘)-Ib aac(6⬘)-Ii ant(4⬘)-Ia ant(6)-Ia aph(3⬘)-III erm(B)sat4
tet(L)-1tet(M)van(A)van(Z)
11
Enterococcus gallinarumSF9117 aph(2⬘)-Ic van(C-1)erm(B) 12
Enterococcus casseliflavusUC73 aph(2⬘)-Id van(C) 53
Bacillus subtilisBR151 pPL708 [cat-86,ant(4⬘)-Ia] 21
Bacillus subtilisDSM4393 pC194 (cat-TC);tet(L)-2aadK DSMZ
Escherichia coliJIR1905 pWD212 (catB) 29
Escherichia coliJIR1597 pJIR235 (catQ) 3
Staphylococcus aureusNCTC50582 pC221 (catpC221);norA NCTC
Listeria monocytogenesBM4293 dfr(D) 9; CIP
Bacillus subtilisEC101 pEC101 [erm(D),cat-TC];tet(L)-2aadK 35
Escherichia coliVA831 pVA831 [erm(F)] 35
Escherichia coli/pGERM pGERM [erm(G)] 50
Staphylococcus warneriVC5 pVC5 [Inu(A)];blaZ 41
Escherichia coliDB10 Inu(B) 7
Streptococcus salivariusSp6 mef(A)erm(B) 51
Streptococcus pyogenesA498 tet(T) 14; CIP
Escherichia coliSC1 pSC1 [tet(W)] 4
Escherichia coliAGHD1 pAGHD1 [tet(Z)] 52
Enterococcus faecium70/90 van(A)van(Z)aac(6ⴕ)-Iitet(M) erm(B) 33; this study
Enterococcus faecalisDSM12956 van(B)sat4 ant(6)-Ia aph(3⬘)-III erm(B) DSMZ
Enterococcus casseliflavusDSM20680 van(C) DSMZ
Enterococcus gallinarumBM4174 van(C-1)tet(L)-1tet(U)tet(M)ant(6)-Ia aph(3⬘)-III erm(B)sat4 20
Enterococcus faecium10/96A van(D4) 17
Enterococcus faeciumN0-0072 van(D5)sat4 erm(B)ant(6)-Ia 6
Enterococcus faecalisBM4405 van(E) 22
Enterococcus faecalisBM4518 van(G)aac(6⬘)-Ie aph(2⬘)-Ia erm(B) 18
Staphylococcus aureusBM3093 pIP680 [vat(A),vgb(A),vga(A)];norA 1; CIP
Staphylococcus aureusBM3318 vat(B)vga(B)erm(A)vga(A)v aac(6⬘)-Ie ant(4⬘)-Ia ant(6)-Ia ant(9)-Ia aph(2⬘)-Ia
aph(3⬘)-III blaZ mecA sat4 norA 27; CIP
Staphylococcus cohniiBM10711 pIP1714 [vat(C),vgb(B)];erm(C)mecA tet(K) 2
Lactobacillus fermentumROT1 pLME300 [vat(E),erm(LF)]e 26
aThe genes highlighted in bold are those used as references to validate the microarray. The other genes are those that were additionally detected in the reference strains with the microarray. Rifr, rifampin resistance; Fusr, fusidic acid resistance.
bNCTC, National Collection of Type Cultures, Centre for Infections, Colindale, London, England; DSMZ, Deutsche Sammlung von Mikroorganismen und Zell- kulturen GmbH, Braunschweig, Germany; CIP, Collection de l’Institut Pasteur, Paris, France.
cC. perfringensMLP26 was isolated from the intestines of a calf.
dS. haemolyticusVPS617 was isolated from the milk of a cow with mastitis.
eerm(LF) is anerm(T)-like gene which contains a 260-bp 3⬘fragment identical toerm(B).
Conjugal transfer.The transfer of plasmid pRE25 (48) fromE. faecalisRE25 toB. anthracis4230 was performed by filter mating as described previously (42).
The transconjugants were selected on LB agar plates containing 19.2g of the combination trimethoprim-sulfamethoxazole (1:5) (3.2g:16g) and 10g of erythromycin per milliliter. The transconjugants were identified by colony mor- phology and by the detection of both thecatpIP501anderm(B) resistance genes present on plasmid pRE25 by PCR.
Antimicrobial susceptibility tests.The MICs of erythromycin, clindamycin, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, the combina- tion quinupristin-dalfopristin, enrofloxacin, vancomycin, oxacillin, penicillin, the sulfonamide sulfisoxazole, trimethoprim, and the combination amoxicillin-clavu- lanic acid were determined in Mueller-Hinton broth using custom Sensititre sus- ceptibility plates (Trek Diagnostics Systems, East Grinstead, England; MCS Diag- nostics BV, Swalmen, The Netherlands) according to NCCLS guidelines (38).
PCR techniques.The antibiotic resistance genes were amplified by PCR using TaqDNA polymerase in accordance with the supplier’s directions (Roche Di- agnostics, Basel, Switzerland) and using an annealing temperature of 54°C. The oligonucleotides used for PCRs are listed in Table 2.
Genomic DNA isolation.Total DNA was obtained after half a loopful of bacterial cells was lysed in a lysis buffer (0.1 M Tris-HCl, pH 8.5, 0.05% Tween 20, 0.24 mg/ml proteinase K) for 1 h at 60°C, followed by a 15-min denaturation step at 95°C. The lysate was filtered through a 0.2-m HT Tuffryn membrane (Acrodisc Syringe Filter; Pall Gelman Laboratory, Ann Arbor, MI). Alterna- tively, DNA was isolated using the guanidium thiocyanate method (45) and was extracted with phenol-chloroform. After addition of ammonium acetate, the cell
lysates were purified with 1 volume of phenol:chloroform:isoamyl alcohol (49.5:
49.5:1 [vol/vol/vol]). After 5 min of centrifugation at 14,000 rpm (Centrifuge Ep- pendorf 5415; Eppendorf AG, Hamburg, Germany), the water phase was treated with 1 volume of chloroform:isoamyl alcohol (49.5:1 [vol/vol]). The DNA was pre- cipitated by the addition of 0.6 volume of isopropanol to the aqueous phase and then centrifuged. The DNA pellet was washed once with 80% ethanol and, after a 5-min centrifugation, was dried under a vacuum and resuspended in water.
DNA labeling.The quality of each DNA preparation was assessed by agarose gel electrophoresis using 5l of the DNA sample and subsequent ethidium bromide staining. The concentration of DNA was determined spectrophoto- metrically at 260 nm. Genomic DNA (10 to 100 ng) was labeled by a randomly primed polymerization reaction using Sequenase, version 2.0 (USB Corporation, Cleveland, Ohio) and consisted of three cycles of enzymatic reactions. The labeling reactions were based on the method of Bohlander et al. (5). The protocol, as modified by the DeRisi Laboratory (University of California, San Francisco; www.microarrays.org/pdfs/Round_A_B_C.pdf), was altered as fol- lows. Round A was used unmodified. During Round B, 25 instead of 35 PCR cycles were performed. In Round C, end concentrations of 0.1 mM (each) dATP, dCTP, and dGTP, 0.065 mM dTTP, and 0.035 mM biotin-16-dUTP (Roche Diagnostics) were used instead of the concentrations stated. Furthermore, 35 PCR cycles were run, and a fraction (10 to 20l) of the finished reaction product was used for hybridization analysis without further purification steps.
DNA array preparation.The gene sequences and the derived specific oligo- nucleotides used to prepare the microarray are listed in Table 3. The oligonu- cleotides were designed from published DNA sequences using the Array Design TABLE 2. Oligonucleotides used for the detection of resistance genes by PCR analysis
Gene Primer name Sequence (5⬘33⬘) Primer design reference or source
catpIP501 catF CCTGCGTGGGCTACTTTA This study
catR CAAAACCACAAGCAACCA
erm(B) erm(B)-F GAAAAGGTACTCAACCAAATA 13
erm(B)-R GTAAACAATTTAAGTACCATTACT
erm(C) erm(C)-F AATCGGCTCAGGAAAAGG This study
erm(C)-R ATCGTCAATTCCTGCATG
mecA mecA-1 AAAATCGATGGTAAAGGTTGGC 34
mecA-2 AGTTCTGCAGTACCGGATTTGC
tet(K) tet(K)-1 TTAGGTGAAGGGTTAGGTCC This study
tet(K)-2 GCAAACTCATTCCAGAAGCA
tetA(P) tetA(P)F CACAGATTGTATGGGGATTAGG 36
tetA(P)R CATTTATAGAAAGCACAGTAGC
tet(L) tetLF GTGAATACATCCTATTCA This study
tetLR TTAGAAATCCCTTTGAGA This study
tet(U) tetU-F ATGCAGCTAAGACGTGGC This study
tetU-R TTATTCGGTATCACTTCTCTGTC
sat4 sat4-F CGATAAACCCAGCGAACC This study
sat4-R ATAACATAGTATCGACGG
aph(3⬘)-IIIa aph3-III-F CCGCTGCGTAAAAGATAC This study
aph3-III-R GTCATACCACTTGTCCGC
ant(6)-Ia ant6-I-F AATTGTGACCCTTGAGGG This study
ant6-I-R GGCATATGTGCTATCCAG
aac(6⬘)-Ie-aph(2⬘)-Ia aac6-aph2-F CAGAGCCTTGGGAAGATGAAG 54
aac6-aph2-R CCTCGTGTAATTCATGTTCTGGC
aac(6⬘)-Ii aac(6)-Ii-F GAGATACTGATTGGTAGC This study
aac(6)-Ii-R TCTTCACTGACTTCTGCC
dfr(A) dfrA-F CCTTGGCACTTACCAAATG This study
dfrA-R CTGAAGATTCGACTTCCC
blaZ blaZ-F CAGTTCACATGCCAAAGAG This study
blaZ-R TACACTCTTGGCGGTTTC
mph(C) mphC-F CATTGAATGAATCGGGAC This study
mphC-R TTCATACGCCGATTCTCC
van(E) vanE-F AGAATGGTGCTATGCAGG This study
vanE-R TCATGATTTTCCACCGCC
msr(A) msrA-F GCTTAACATGGATGTGG This study
msr(SA) msrA-R GATTGTCCTGTTAATTCCC
msr(SA⬘)
catD catDPS-F CCTTGYACATACAGYATGAC This study
catP catDPS-R AACTTGRATKGCSARAGGAAG
catS
vgb(B) vgb(B)-F GTCTATTCCCGATTCAGG This study
vgb(B)-R TGCAAACCATACGGATCC
TABLE3.Oligonucleotidesequencesoftheprobesandcharacteristicsandsourcesoftheantibioticresistancegenesrepresentedonthemicroarray Spot no.IdentificationSequence(5⬘33⬘)GenotypeResistance phenotypeaMechanismGenBank accession no.
Gene positionbSource 1be_AAC6-Ie_144ACATTATACAGAGCCTTGGGAAGATGAAGTaac(6⬘)-IeTob,Dbk,Ntl,Amk,Ast, 2⬘Ntl,5-epi,SisoAcetyltransferaseM180861725–2412Staphylococcusaureus 2be_AAC6-Ie_475TTGCCAGAACATGAATTACACGAGGGCAAA 3be_AAC6-Ii_71CTTGGCCGGAAGAATATGGAGACAGCTCGGaac(6⬘)-IiTob,Dbk,Ntl,Amk,2⬘Ntl, 5-epi,SisoAcetyltransferaseL12710169–717Enterococcusfaecium 4be_AAC6-Ii_396AGTGGCTTCCATCCAGAACCTTCGTGAACA 5be_AAC6-Im_15GCGAGTTTCCTTTCGCCCGATGAATGAGGAaac(6⬘)-ImTob,Dbk,Ntl,Amk,2⬘Ntl, 5-epi,SisoAcetyltransferaseAF3379471215–1751Enterococcusfaecium,E.coli 6be_AAC6-Im_286GCGATGGACCAATTTATCGGTGAGCCGGAA 7be_ANT4-Ia_118CTTGGTCGTCAGACTGATGGGCCCTATTCGant(4⬘)-IaTob,Amk,Isp,DbkAdenylyltransferaseNC_0015651390–2151Staphylococcus,Bacillus 8be_ANT4-Ia_197ATGAATGGACAACCGGTGAGTGGAAGGTGG 9be_ANT6-Ia_433CCAAGCGCAAGGGAGTATGATGATTGCTGCant(6)-IaSmAdenylyltransferaseAF51633514900–15808Enterococcus,Staphylococcus 10be_ANT6-Ia_576ATCATGGAAGGTCGGCATCGAAACAGGCTT 11be_ANT9-Ia_278GGAGTGAAGTTGTCCCTTGGCAATATCCTCCAant(9)-IaSpcAdenylyltransferaseX02588331–1113Staphylococcusaureus 12be_ANT9-Ia_560ACCCTAGCTCGAATGTGGCAAACAGTGACT 13be_APH2-Ia_149AAGACAAATGCACGGTTTAGATTATACAGAaph(2⬘)-IaKm,Tob,Nm,Liv,GmCPhosphotransferaseM180862494–3164Staphylococcusaureus 14be_APH2-Ia_292TTATGGAAAGACTAAATGCAACAACAGTTT 15be_APH2-Ib_317AGGATGCCCTTGCATATGATGAAGCGACGTaph(2⬘)-IbKm,Tob,Nm,Liv,GmCPhosphotransferaseAF207840122–1021Enterococcusfaecium, Escherichiacoli16be_APH2-Ib_737ATCAGCATAAGGCGCCGGAAGTAGCAGAAA 17be_APH2-Ic_58AGCATACAATCCGTCGAGTCGCTTGGTGAGaph(2⬘)-IcKm,Tob,Nm,Liv,GmCPhosphotransferaseU51479196–1116Enterococcusgallinarum 18be_APH2-Ic_346CTGGCGCTGCAACTTGCTGAGTTCATGAAT 19be_APH2-Id_249GCCATCAGAAACGTACCAAATGTCTTTCGCAGGaph(2⬘)-IdKm,Tob,GmC,2⬘Ntl, 5-epi,Amk,DbkPhosphotransferaseAF016483131–1036Enterococcuscasseliflavus 20be_APH2-Id_354GGCAGCTAAGGACCTGGCCCGATTTCTAAG 21be_APH3-III_136ACGGACAGCCGGTATAAAGGGACCACCTATaph(3⬘)-IIIKm,Nm,Prm,Rsm,Liv, GmBPhosphotransferaseM36771293–1084Staphylococcusaureus, Enterococcusfaecalis22be_APH3-III_332TTATCGAGCTGTATGCGGAGTGCATCAGGC 23be_APH3-IVa_20ATTGGCCGGAGGAACTTCTTGAGCTTCTCGaph(3⬘)-IVaKm,Nm,Prm,Rsm,ButPhosphotransferaseX03364277–1065Bacilluscirculans 24be_APH3-IVa_474GGAGTACGATTGCACGCCGGAGGAATTGTA 25be_NorA_426AGGACCAGGGATTGGTGGATTTATGGCAGAAnorANor,dEno,dOfl,dCipdQuinolones—effluxD90119478–1644Staphylococcusaureus 26be_aadK_61ATCCGATTGGTCACTTTGGAAGGGTCACGTaadKSmAdenylyltransferaseM2687990–944Bacillussubtilis 27be_aadK_175GATCAGTGGCTCGAAATCTTTGGGAAGCGC 28be_bla1_201AGGTGTATATGCGATTGATACTGGTACAAAbla1Amp,cAmox/clav,cPipcBeta-lactamaseAF367983626–1555Bacillusanthracis 29be_bla1_366AGTGGATTATTCACCTGTTACAGAGAAACA 30be_bla2_192CGGAGAAGCAGTTCCTTCGAACGGTTTAbla2Amp,cAmox/clav,cCfx,c Cpd,Cft,Caz,CaxBeta-lactamaseAF367984791–1561Bacillusanthracis 31be_bla2_246ACTTGTCGATTCTTCTTGGGATGATAAGTT 32be_blaZ_718TTTGTTTATCCTAAGGGCCAATCTGAACCTblaZBeta-lactamsBeta-lactamaseM60253142–987Enterococcusfaecalis, Staphylococcusaureus33be_blaZ_811AGTGAAACCGCCAAGAGTGTAATGAAGGAA 34be_cat-86_367AGCAGCAACCTATTTCCGAAACCTCATATGCCAcat-86CmAcetyltransferaseK00544145–807Bacilluspumilus 35be_cat-86_605TGAGGTGGCTTATTGAACATTGTGACGAGTGGT 36be_cat-DPS_set_114ATTTGCAGAAAGGATATGATTATTTGATTCCTcatDCmAcetyltransferaseX1510091–729Clostridiumdifficile catPCmAcetyltransferaseU150272953–3576Clostridiumperfringens catSCmAcetyltransferaseX749481–492Streptococcuspyogenes 37be_cat-LM_set_135AGGATATGAACTGTATCCTGCTTTGAcat-LMCmAcetyltransferaseX684121328–1975Listeriamonocytogenes catpC223CmAcetyltransferaseAY3552851000–1647Staphylococcusaureus catpSCS5CmAcetyltransferaseM58515213–872Staphylococcushaemolyticus catpSCS7CmAcetyltransferaseM5851690–719Staphylococcusaureus 38be_cat-TC_set_170TGACAAGGGTGATAAACTCAAATACAGCTcat-TCand catpC194CmAcetyltransferaseU75299657–1373Lactobacillusreuteri 39be_cat-TC_set_232GGTTATTGGGATAAGTTAGAGCCACTTTATCmAcetyltransferaseNC_0020131260–1910Staphylococcusaureus CmAcetyltransferaseNC_0020131260–1910Staphylococcusaureus 40be_catB_27TCATTGGAGTAGAAAGCCATACTTTGAACAcatBCmAcetyltransferaseM93113145–804Clostridiumbutyricum 41be_catB_233TAGGATATTGGGATAGCATGAATCCAAGCT 42be_catDP_set_281TTTCCAGCCTTTGGACTGAGTGTAAGTCcatPandcatDCmAcetyltransferaseU150272953–3576Clostridiumperfringens 43be_catDP_set_416CTATGATACCGTGGTCAACCTTCGATGGCmAcetyltransferaseX1510091–729 CmAcetyltransferaseX1510091–729 44be_catQ_66TGCGGTTAGGTGCACTTACAGTATGACTGCAcatQCmAcetyltransferaseM55620459–1118Clostridiumperfringens 45be_catQ_186TAACCGTCACAAGGAGTTCCGCACCTGTTT 46be_catS_228CCTTTGGACACCATACATACCAGATTTcatSCmAcetyltransferaseX749481–492Streptococcuspyogenes 47be_catS_383GCTTTAATCTGAATTTGCAGAAAGGATATGA 48be_catpXX_set_196GTGTTTAGAACAGGAATTAATAGTGAGAATAAcatpSCS1CmAcetyltransferaseM64281208–855Staphylococcusintermedius
catpSCS6CmAcetyltransferaseX6082788–735Staphylococcusaureus catpIP501CmAcetyltransferaseX65462208–855Streptococcusagalactiae catpC221CmAcetyltransferaseX025292267–2914Staphylococcusaureus catpUB112CmAcetyltransferaseX02872208–855Staphylococcusaureus 49be_cfr_466GGAATGGGTGAAGCTCTAGCCAACCGTCAAcfrCm,FfcUnknownAJ249217570–1619Staphylococcussciuri 50be_cfr_908GAGAAGCAAACGAAGGGCAGGTAGAAGCCT 51be_dfrA_20TCGCTCACGATAAACAAAGAGTCATTGGGTdfr(A)TmpDihydrofolatereductaseAF0519162823–3308:rStaphylococcusaureus 52be_dfrA_172AGACGTAACGTCGTACTCACTAACCAAGCT 53be_dfrD_140ACCTTCAATCAATCGGAAGGGCTTTACCTGACAdfr(D)TmpDihydrofolatereductaseZ5014194–582Staphylococcushaemolyticus 54be_ermA_193TGTCAAGTGACTAAAGAAGCGGTAAACCerm(A)MLSBMethylaseX032164551–5282:rStaphylococcusaureus 55be_ermA_590AGTGGGTAAACCGTGAATATCGTGTTCT 56be_ermB_112ACAGGTAAAGGGCATTTAACGACGAAACTGGCerm(B)MLSBMethylaseY00116262–999Enterococcusfaecalis 57be_ermB_520AAACTTACCCGCCATACCACAGATGTTCCAGA 58be_ermC_149AGAGGTGTAATTTCGTAACTGCCATTGAerm(C)MLSBMethylaseJ017552004–2738:rStaphylococcusaureus 59be_ermC_372TTTAATCGTGGAATACGGGTTTGCTAAAerm(C)MLSBMethylase 60be_ermD_555AGTGGACTCGGCAATGGTCAGAATAACACGAerm(D)MLSBMethylaseM29832430–1293Bacilluslicheniformis 61be_ermF_231TGCCCGAAATGTTCAAGTTGTCGGTTGTGAerm(F)MLSBMethylaseM14730241–1041Bacteroidesfragilis, Streptococcus 62be_ermF_494GTCCTGAAAGTTTCTTGCCACCGCCAACTG 63be_ermG_98ACATCTTTGAAATAGGTGCAGGGAAAGGTCerm(G)MLSBMethylaseM15332672–1406Bacillussphaericus 64be_ermG_296TTGGCAGCATACCTTACAACATAAGCACAA 65be_ermQ_521ACTTCCATCCCATGCCTAGTGTAGATTGCGTerm(Q)MLSBMethylaseL22689262–1035Clostridiumperfringens 66be_ermT_104TTGAGATTGGTTCAGGGAAAGGTCATTTerm(T)MLSBMethylaseM64090168–902Lactobacillusreuteri 67be_ermT_149AAAGGTGTAATTATGTAACCGCCATTGAAA 68be_ermX_231GGCGGTCGAAGTGGTCCATGATGATTTCCTerm(X)MLSBMethylaseM36726296–1150Corynebacteriumdiphtheriae 69be_ermX_282TCCCTGCGTCATTGTGGGAAACATTCCCTT 70be_ermY_122AAGGGCATTTCACACTAGAACTGGTTCAerm(Y)MLSBMethylaseAB014481556–1290Staphylococcusaureus 71be_ermY_258ACAGTTTAAGTTCCCAAACAACAAAGCA 72be_lnuA_115AAACAACAAAGAGAACACAGAGATATAGATlnu(A)LmTransferaseJ03947645–1130Staphylococcusaureus 73be_lnuA_218ATTGGATGCCTTCACGTATGGAACTTAA 74be_lnuB_169TCATCCAACTGGTTGTTTGACGTAGCTCCGTlnu(B)LmTransferaseAJ238249127–930Enterococcusfaecium 75be_mdtA_355CAGACCGCTCAGATGCCAACAGTCCAATCTmdt(A)MLSB,Tet,MinEffluxX9294610534–11790Lactococcuslactis 76be_mdtA_571GTCAGGATACCAGAAGTCGCTTCACAGGGC 77be_mecA_871AGCTCCAACATGAAGATGGCTATCGTGTCACAmecAMet,OxaPenicillin-binding protein2⬘AB09621720340–22346Staphylococcusaureus 78be_mecA_1042GCTCAGGTACTGCTATCCACCCTCAAACAGG 79be_mef_set_39AATATGGGCAGGGCAAGCAGTATCATTAmef(A)and mef(B)MMajorfacilitatorU70055314–1531Streptococcuspyogenes 80be_mef_set_193GGTGTGCTAGTGGATCGTCATGATAGGMMajorfacilitatorU836671–1218Streptococcuspneumoniae MMajorfacilitatorU836671–1218Streptococcuspneumoniae 81be_mphC_281CAGGTAAACCCGCAGCCACAATAGATCCAGAmph(C)MPhosphorylaseAF1671615665–6564Staphylococcusaureus 82be_mphC_555CGAACTATGGCCTCGACATGCGACCATGAT 83be_msr_set_289ATGCATACAACCGACAGTATGAGTGGTGmsr(A)and msr(SA)M,SATP-bindingtransporterX52085343–1809Staphylococcusepidermidis 84be_msr_set_655GCTAAACGAAATCAAGCGCAACAAATGGmsr(SA)M,SATP-bindingtransporterAB0166132005–3471Staphylococcusaureus msr(SA⬘)M,SATP-bindingtransporterAB013298487–1953Staphylococcusaureus msr(B)M,SATP-bindingtransporterM8180294–624Staphylococcusxylosus 85be_sat4_161AGGATGAAGAGGATGAGGAGGCAGATTGCCsat4SthAcetyltransferaseAF51633515805–16347Enterococcusfaecium 86be_sat4_338GCAAGGCATAGGCAGCGCGCTTATCAAT 87be_tetK_259AGTTTGAGCTGTCTTGGTTCATTGATTGCtet(K)TetEffluxM16217305–1684Staphylococcusaureus 88be_tetK_351TGCTGCATTCCCTTCACTGATTATGGT 89be_tetL_1_151ACAAACTGGGTGAACACAGCCTTTATGTtet(L)TetEffluxM11036189–1565Bacillusstearothermophilus 90be_tetL_1_676TCTTATCGTTAGCGTGCTGTCATTCCTG 91be_tetL_2_269GCTTAGGGTCGATCATTGGATTTGTTGGtet(L)TetEffluxX08034188–1564Bacillussubtilis 92be_tetL_2_504GTCGTATTTGCTGCTTATTCCAACTGCA 93be_tetM_1033CTGCTGCAAACGACTGTTGAACCGAGCAAAtet(M)Tet,MinRibosomalprotectionX04388131–2050Enterococcusfaecalis 94be_tetM_1308TCCACCGAATCCTTTCTGGGCTTCCATTGG 95be_tetAP_1193TATCAGTGGCTCGCTTGAAGCTTGGATTGCtetA(P)Tet,MinRibosomalprotectionL20800207–2120Clostridiumperfringens 96be_tetAP_1266GGAGCACAAGCAGGGCAGATAGGAGCATTT 97be_tetS_18CGGTATCTTAGCACATGTTGATGCAGGAtet(S)Tet,MinRibosomalprotectionL09756447–2372Listeriamonocytogenes 98be_tetS_776CAGATGATGGTCAACGGCTTGTCTATGT Continuedonfollowingpage
TABLE1—Continued Spot no.IdentificationSequence(5⬘33⬘)GenotypeResistance phenotypeaMechanismGenBank accession no.
Gene positionbSource 99be_tetT_232CACATGGATTTCATAGCCGAAGTTGAGCtet(T)Tet,MinRibosomalprotectionL42544478–2433Streptococcuspyogenes 100be_tetT_1326GGTTCCACCAAATCCTTATTGGGCATCT 101be_tetU_133GCTGAGCCTTCTAATTGGTCGATAATTGCTtet(U)Tet,MinUnknownU01917413–730Enterococcusfaecium 102be_tetW_66CCTGCTATATGCCAGCGGAGCCATTTCAGAtet(W)Tet,MinRibosomalprotectionAJ222769192–2111Butyrivibriofibrisolvens 103be_tetW_455TTATCATCAAGCAGACGGTGTCGCTGTCCC 104be_tetZ_43GTGATGCCGATCTTGCCTACCCTTCTCGACtet(Z)TetEffluxAF12100011880–13034:rCornynebacteriumglutamicum 105be_tetZ_93CATGATCCCACTGCACGTCGGACTACTGAC 106be_vanA_192CTATTCAGCTGTACTCTCGCCGGATAAAvan(A)Van,TeiLigaseM972976979–8010Enterococcusfaecium 107be_vanA_884TACAAGATAACGGCCGCATTGTACTGAA 108be_vanB_set_65AATCCGCAATAGAAATTGCTGCGAACATvan(B)and van(B2)VanLigaseU0045662–1090Enterococcusfaecalis 109be_vanB_set_151CTATGCAAGAAGCCATGTACGGAATGGGVanLigaseAF3109531–1029Enterococcusfaecium VanLigaseAF3109531–1029Enterococcusfaecium 110be_vanC-1_77TCCAAGCTATTGACCCGCTGAAATATGAvan(C-1)VanLigaseAF1626941411–2442Enterococcusgallinarum 111be_vanC-1_497ACCATGGATTCCCGATCTTTATCAAGCC 112be_vanC_set_37CCGGAATACACCGTTTCTTTAGCTTCAGvan(C-2)and van(C-3)VanLigaseL2963833–1085Enterococcuscasseliflavus 113be_vanC_set_184CAAGACACGTGGTTGTTGGATACGAAACVanLigaseAY03376426–1078Enterococcusflavescens VanLigaseAY03376426–1078Enterococcusflavescens 114be_vanD4-5_183CTATGCGGGATACCCGGCTGTGATTTCTCCvan(D4)and van(D5)VanLigaseAF2775711262–2293Enterococcusfaecium 115be_vanD4-5_267GCCTGTAGACGTGGTGCTTCCGATGATTCAVanLigaseAY4890454010–5041Enterococcusfaecium VanLigaseAY4890454010–5041Enterococcusfaecium 116be_vanE_298GGAGGTTATGGTGAGAATGGTGCTATGCAGGGvan(E)VanLigaseAF4308072976–4034Enterococcusfaecalis 117be_vanE_357TGTAGGTTGTGGTATCGGAGCTGCAGCAAT 118be_vanG_362TGGCAGGAATACCTGTTGTTGGCTGCGATAvan(G)VanLigaseAF2535623715–4764Enterococcusfaecalis 119be_vanG_549ACCTGTTCGTGCAGGCTCTTCCTTTGGAAT 120be_vanZ_328ACAAATACTGTTGGAGGCTTTCTTGGACTGvan(Z)TeiUnknownM9729710116–10601Enterococcusfaecium 121be_vatA_288TCATCTATTCAGGATGGGTTGGGAGAAGTvat(A)SATransferaseL07778258–917Staphylococcusaureus 122be_vatA_429AATCATTGCTGCAGAAGCTGTTGTCAC 123be_vatB_9TGGCCCTGATCCAAATAGCATATATCCACAvat(B)SATransferaseU1945967–705Staphylococcusaureus 124be_vatB_109ACTTACTATTCCGATGTTAACGGAGCTGAA 125be_vatC_474TTCAGTTGTTGGCGGTAATCCTTCACGATTvat(C)SATransferaseAF0156281307–1945Staphylococcusaureus 126be_vatC_552AAGGTGGTGGGACCTAGAGATAGAGACGAT 127be_vatD_453GCCATACATGTTAGCTGGAGGAAATCCTvat(D)SATransferaseL12033162–791Enterococcusfaecium 128be_vatE_349TGTAGTCGGAAATGACGTGTGGTTTGGGCAvat(E)SATransferaseAF13972563–707Enterococcusfaecium 129be_vatE_409AGGTGACGGTGCCATTATCGGAGCAAATAGT 130be_vgaA_834CTCGGGTACAATTGAAGGACGGGTATTGTGGAvga(A)SBATP-bindingtransporterM90056909–2477Staphylococcusaureus 131be_vgaA_886CGCGGAGGAGACAAGATGGCAATTATCGGA 132be_vgaB_569TGCTTCTACGAAAGCAACAAGAAGAATACGvga(B)SBATP-bindingtransporterU82085629–2287Staphylococcusaureus 133be_vgaB_649GAGAATAAGGCGCAAGGAATGATTAAGCCC 134be_vgbA_142ACAGAGTACCCACTACCGACACCAGATGCAvgb(A)SBHydrolaseM20129641–1540Staphylococcusaureus 135be_vgbA_281TGCCTAACCCAGATTCAGCACCCTACGGTA 136be_vgbB_273ATATCCATTGCCACAGCCGGATTCTGGTCCvgb(B)SBLactonaseAF015628399–1286Staphylococcuscohnii 137be_vgbB_539CAAATGCAGCGGCTCCAGTGGGTATCACTA 1381⫻Spottingpuffer 139Marken-Mix aAminoglycosides:Tob,tobramycin;Dbk,dibekacin;Ntl,netilmicin;Amk,amikacin;2⬘Ntl,2⬘-N-ethylnetilmicin;5-epi,5-episisomicin;Siso,sisomicin;Isp,isepamicin;Sm,streptomycin;Spc,spectinomycin;Ast, Astromicin(fortimicin);Km,kanamycin;Nm,neomycin;Liv,lividomycin;GmB,gentamicinB;GmC,gentamicinC;Prm,paromomycin;Rsm,ribostamycin;But,butirosin;Thephenotypeswerefoundinreferences49 and56.Fluoroquinolones:Nor,norfloxacin;Eno,enoxacin;Ofl,ofloxacin;Cip,ciprofloxacin.Beta-LactamsandCephem:Amp,ampicillin;Amox/clav,amoxicillin-clavulanicacid;met,methicillin;Oxa,oxacillin;Ctx, cefoxitin;Cpd,cefpodoxime;Cft,cefotaxime;Caz,ceftazidime;Cax,ceftriaxone.Phenicols:Cm,chloramphenicol;Ffc,florfenicol.Folatepathwayinhibitors:Tmp,trimethoprim.MLS:M,macrolides,L,lincosamides; SB,streptograminsB;SA,streptograminA;Lm,lincomycin.Tetracyclines:Tet,tetracycline,Min,minocycline.Glycopeptides:Van,vancomycin;Tei,teicoplanin.Others:Sth,streptothricin. b:r,thegeneisfoundonthecomplementarystrand. cWhenexpressedinE.coli(10). dWhenoverexpressedinS.aureus(32).
Software Package (Clondiag Technologies, Jena, Germany). They consist of 26- to 33-mers with similar physicochemical parameters. The probes were spotted onto a 3- by 3-mm glass surface with a Microgrid II spotting machine (BioRobotics Inc./Apogent Discoveries Europe, Cambridge, England) as described previ- ously (37). The glass substrates were incorporated into standard microreac- tion tubes. The layout of the spotted probes in the microarray is shown in Fig. 1.
DNA hybridization and detection.The microarray tubes were positioned in a Thermomixer comfort (Eppendorf AG, Hamburg, Germany) and washed twice with QMT hybridization buffer (Quantifoil, Jena, Germany) for 5 min at 30°C and 550 rpm. The labeled genomic DNA (10 to 20l) was mixed with QMT hybridization buffer to obtain a final volume of 100l, denatured for 5 min at 94°C, kept on ice for 3 min, and hybridized for 1 h at 60°C and 550 rpm. The arrays were washed in 500l 2⫻SSC (1⫻SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0) containing 0.2% sodium dodecyl sulfate solution for 5 min at 30°C and 550 rpm, in 500l 2⫻SSC for 5 min at 20°C and 550 rpm, and in 500l 0.2⫻SSC for 5 min at 20°C and 550 rpm. The arrays were blocked with 100l 6⫻SSPE (60 mM sodium phosphate, 1.08 M NaCl, 6 mM EDTA, pH 7.4) solution containing 0.005% Triton X-100 and 2% (wt/vol) milk powder for 15 min at 30°C and 550 rpm; then 100l of conjugate buffer (6⫻SSPE, 0.005%
Triton X-100, 100 pg/l of streptavidin-peroxidase conjugate [Clondiag]) was added, and the array tubes were incubated for 15 additional minutes at 30°C and 550 rpm. The arrays were washed in 2⫻SSC–0.01% Triton X-100 at 30°C for 5 min and in 2⫻SSC and then 0.2⫻SSC for 5 min at 20°C. The arrays were kept at 20°C in the last washing solution until visualization. The hybridized probes were enhanced using 100l of tetramethylbenzidine peroxidase substrate (Clon- diag). The peroxidase staining procedure and the online detection were per- formed in anatr01array tube reader (Clondiag) for 15 min at 25°C according to the manufacturer’s specifications. The hybridization analyses were performed in duplicate.
The data were analyzed using Iconoclust software (Clondiag). Signal intensity and local background were measured for each spot on the array. Extinctions of local backgrounds were subtracted from extinctions of spots. A threshold was determined so that each value above zero was considered a signal. Resulting values below 0.1 were considered negative (⫺), and those above 0.3 were con- sidered positive (⫹). Values between 0.1 and 0.3 were regarded as ambiguous (⫹/⫺).
RESULTS
Construction of the gene array. A total of 90 resistance genes that had already been characterized in gram-positive bacteria were selected from the GenBank database to be rep- resented on the microarray (Table 3). Only extrinsic poten- tially transmissible resistance genes were included. Antibiotic resistance due to single-base mutations of the target genes could not be considered, since highly stringent annealing tem- peratures would be necessary to obtain a specific hybridization with these oligonucleotides. Each antibiotic resistance gene or group of genes was represented on the array by two different oligonucleotides situated apart from each other within the pro- tein coding sequence. The oligonucleotides were chosen ac- cording to their high specificity for the related resistance genes.
Consensus sequences were used to design the oligonucleotides specific for several subtypes of resistance genes sharing DNA identities higher than 89%. Hence, the chloramphenicol acet- yltransferase genescatDandcatP(99.5% DNA identitity) were represented by the catDP oligonucleotides be_catDP_set_281 and be_catDP_set_416, the genescat-LM, catpC223, catpSCS5, andcatpSCS7(DNA identity,ⱖ90.6%) by the oligonucleotide be_cat-LM_set_135, the genescat-TCandcatpC194(99.7%) by the cat-TC oligonucleotides cat-TC_set_170 and cat-TC_set_
232, the genes catpC221, catpUB112, catpSCS1, catpSCS6, and catpIP501 (ⱖ96.9%) by the oligonucleotide be_catpXX_set_196, the macrolide efflux genesmef(A) andmef(E) (89.9%) by the mef oligonucleotides be_mef_set_39 and be_mef_set_193, the vancomycin resistance genesvan(B) andvan(B2) (95.6%) by the vanB oligonucleotides be_vanB_set_65 and be_vanB_set_
151, the van(C-2) and van(C-3) genes (98.7%) by the vanC FIG. 1. Distribution layout of the oligonucleotides on the microarray. The detectable genes are italicized, and details are given in Table 3. The following gene abbreviations include a family of genes:catDPSdetectscatD,catP, andcatS;catDPdetectscatDandcatP;catpXXdetectscatpC221, catpUB112,catpSCS1,catpSCS6, andcatpIP501;cat-LMdetectscat-LM,catpSCS5, andcatpSCS7;cat-TCdetectscat-TCandcatpC194;mefdetectsmef(A) and mef(B);msrdetectsmsr(A),msr(SA),msr(SA⬘), andmsr(B);van(B) detectsvan(B) andvan(B2);van(C) detectsvan(C-2) andvan(C-3). The position controls (ctrl) consist of biotin-labeled oligonucleotides.
