Quinolone Resistance Mutations DNA Gyrase gyrA …aac.asm.org/content/38/9/2014.full.pdfQuinolone Resistance Mutations in the ... byinserting the cloned genes into theEscherichia coli-S

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1994, p. 2014-2023 Vol. 38, No. 90066-4804/94/$04.00+0Copyright X 1994, American Society for Microbiology

Quinolone Resistance Mutations in the DNA Gyrase gyrAand gyrB Genes of Staphylococcus aureus

HIDEAKI ITO, HIROAKI YOSHIDA,* MAYUMI BOGAKI-SHONAI, TOSHIYUKI NIGA,HIROAKI HAYTORI, AND SHINICHI NAKAMURA

Bioscience Research Laboratories, Dainippon Pharmaceutical Co., Ltd., Enoki 33-94, Suita, Osaka 564, Japan

Received 8 March 1994/Returned for modification 1 June 1994/Accepted 4 July 1994

A 6.4-kb DNA fragment containing the DNA gyrase gyrA and gyrB genes was cloned and sequenced from thequinolone-susceptible Staphylococcus aureus type strain ATCC 12600. An expression plasmid was constructedby inserting the cloned genes into the Escherichia coli-S. aureus shuttle vector pAT19, and deletion plasmidscarrying only functional gyrA and gyrB genes were derived from this plasmid. An efficient transformationsystem for S. aureus RN4220 was established by using these plasmids. Quinolone-resistant mutants of S. aureusRN4220 were isolated by three-step selection with quinolones. The first- and second-step mutants wereconsidered to be transport mutants, and the third-step mutants were divided into five groups with respect totheir resistance patterns and transformation results with gyrA and gyrB genes. Sequencing analysis of theresulting mutant gyrase genes showed that they had the following point mutations: group 1, Ser-84 (TCA) toLeu (TTA) in GyrA; group 2, Ser-84 (TCA) to Ala (GCA), Ser-85 (TCT) to Pro (CCT), or Glu-88 (GAA) to Lys(AAA) in GyrA; group 3, Asp-437 (GAC) to Asn (AAC) in GyrB; group 4, Arg-458 (CGA) to Gln (CAA) in GyrB;and group 5, Ser-85 (TCT) to Pro (CCT) in GyrA and Asp-437 (GAC) to Asn (AAC) in GyrB. When the gyrAand/or gyrB mutants were transformed with the wild-type gyrA and/or gyrB plasmids, they became quinolonesusceptible, but transformants with the plasmids having the same mutations on the gyrA and/orgyrB genes didnot confer susceptibility. These results indicate that mutations in both gyrA and gyrB can be responsible forquinolone resistance in S. aureus.

Fluoroquinolones are synthetic chemotherapeutic agentswith broad and potent antibacterial activity against gram-negative and -positive organisms and have been used againstvarious bacterial infections. Staphylococcus aureus is an impor-tant pathogenic organism causing a variety of local and sys-temic infections and was originally susceptible to quinolones(5). Therefore, it was considered that quinolones might beuseful for treating S. aureus infections, including those withmethicillin-resistant S. aureus (MRSA), which are resistant tomost available antibacterial agents. However, the usefulness ofquinolones for MRSA infections has been limited, since theemergence of quinolone resistance has been found to berelatively rapid (27, 28).

Unusual findings as to quinolone resistance in S. aureus arethat some MRSA resistant to fluoroquinolones remain suscep-tible to sparfloxacin (17) and other newer quinolones (26) andthat quinolone-resistant mutants can easily be obtained bysingle-step selection with ciprofloxacin and norfloxacin, but notwith sparfloxacin (17).

Three genes related to quinolone resistance in S. aureushave been reported so far: gyrA (6, 7, 30, 31), norA (40), andflq(33). The gyrase genes of S. aureus have been cloned andsequenced (2, 21), and analysis of PCR-generated fragmentsfrom quinolone-resistant S. aureus revealed that the gyrAmutations were similar to those responsible for quinoloneresistance in Escherichia coli (7, 30, 31). Since DNA gyrasereconstituted from the A subunit from quinolone-resistant S.aureus and the wild-type B subunit is quinolone resistant (24,25), it is suggested that gyrA mutations might cause quinoloneresistance in S. aureus. However, transformation with the

* Corresponding author. Mailing address: Bioscience Research Lab-oratories, Dainippon Pharmaceutical Co., Ltd., Enoki 33-94, Suita,Osaka 564, Japan. Phone: 81.6.3375909. Fax: 81.6.3387656.

gyrase gene has not been successfully performed for S. aureus,and it is still unclear whether the gyrA mutations reported arereally responsible for quinolone resistance in S. aureus. ThenorA gene has been cloned and sequenced, and it is consideredto encode a quinolone efflux pump (40). Interestingly, organ-isms transformed with a plasmid carrying the norA gene areresistant to relatively hydrophilic quinolones, such as norfloxa-cin, enoxacin, and ciprofloxacin, but are susceptible or lessresistant to relatively hydrophobic ones, such as nalidixic acid,oxolinic acid, and sparfloxacin (40). These results are ac-counted for by efficient excretion of hydrophilic quinolonesand by inefficient excretion of hydrophobic quinolones throughthe pump (40). The flq gene (33) located near the thr and trpgenes on the S. aureus chromosome has not been cloned yetand its function is not known.

In order to clarify the relationship between gyrase mutationsand quinolone resistance, we cloned the wild-type gyrA andgyrB genes of S. aureus type strain ATCC 12600, developed anefficient transformation system for quinolone-resistant sponta-neous mutants of S. aureus RN4220, and analyzed the muta-tions in their gyrA and gyrB genes by both transformation andDNA sequencing.

MATERUILS AND METHODS

Bacterial strains and vectors. S. aureus type strain ATCC12600 was purchased from Japanese Society for Bacteriology.S. aureus RN4220 (18) was kindly provided by R. P. Novick. E.coli HB101 and JM109, plasmids pBR322, pUC18, andpUC119, and phages M13mpl8 and M13mpl9 were purchasedfrom Takara Shuzo Co., Ltd. (Kyoto, Japan). The VCS-M13interference-resistant helper phage and a pT7Blue(R) T-vector kit were purchased from Stratagene cloning system (LaJolla, Calif.) and Novagen (Madison, Wis.), respectively. Plas-mid pAT19 (32) was kindly provided by P. Courvalin. E. coli

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QUINOLONE RESISTANCE MUTATIONS IN S. AUREUS DNA GYRASE 2015

p500301

p5A:ASOl

pSAMOUm

IVX a X V CZ PV C 3 XV C C NCR V * N

* . . .,

* *. .1

975 gyrA

o 2 3 4 5 6 7 S 9Length of MSA (kb)

FIG. 1. Restriction map of the gyrA and gyrB genes and adjoiningregions of S. aureus ATCC 12600. DNA inserts carried by the plasmidsare also shown. C, E, H, M, P, V, and X stand for cleavage sites forClaI, EcoRI, HindIII, MamI, PaM, EcoRV, and XbaI, respectively.

strains were cultivated in LB broth (22), and S. aureus strainswere cultivated in brain heart infusion broth (Difco Laborato-ries, Detroit, Mich.), unless otherwise indicated.

Materials. Oxolinic acid (15), ciprofloxacin (8), norfloxacin(16), and sparfloxacin (23) were synthesized at ExploratoryResearch Laboratories, Dainippon Pharmaceutical Co., Ltd.Oligonucleotides for PCR and DNA sequencing were synthe-sized by Takara Shuzo Co., Ltd. Restriction endonucleasesMaeII and MamI were purchased from Boehringer MannheimGmbH (Mannheim, Germany), and Pacl and PflMI werepurchased from New England Biolabs (Beverly, Mass.). Otherrestriction endonucleases, T4 DNA ligase, T4 polynucleotidekinase, the sequencing kits (7-DEAZA sequencing kit andBcaBEST dideoxy sequencing kit), a GeneAmp PCR reagentkit with AmpliTaq DNA polymerase, and a DNA blunting kitwere purchased from Takara Shuzo Co., Ltd. [a-32P]dCTP(>400 Ci/mmol and >3,000 Ci/mmol for DNA sequencing)and [y-32P]ATP (>5,000 Ci/mmol for labeling of oligonucleo-tide probes) were from Amersham International (Bucking-hamshire, United Kingdom). Sodium ampicillin was purchasedfrom Meiji Seika Kaisha, Ltd. (Tokyo, Japan). Erythromycin,lysozyme (grade I), and RNase A (type I-A) were purchasedfrom Sigma Chemical Co. (St. Louis, Mo.). Achromopeptidasewas purchased from Wako Pure Chemical Industries, Ltd.(Osaka, Japan), and the other reagents (guaranteed grade)were purchased from Nacalai Tesque, Inc. (Kyoto, Japan).

Isolation of spontaneous quinolone-resistant mutants of S.aureus RN4220. Spontaneous quinolone-resistant mutantswere selected on Mueller-Hinton agar (Difco Laboratories)containing quinolones. Drug susceptibility was tested by thetwofold serial agar dilution method, using Mueller-Hintonagar with an inoculum size of 105 CFU of bacteria. The drugsusceptibility tests of S. aureus strains containing pAT19 and itsderivatives were carried out in the presence of 8 jig oferythromycin per ml. Growth of the cells was evaluated afterincubation for 18 h at 37°C.

Preparation of chromosomal and plasmid DNA. Chromo-somal DNA was prepared by the method of Cosloy and Oishi(4). Small-scale plasmid DNA isolation was carried out by therapid boiling method described by Holmes and Quigley (11) or

by the method of Birnboim and Doly (1), except that lysozymewas replaced by achromopeptidase (500 U/ml) for S. aureus.

Large-scale plasmid DNA isolation was done by the method ofWilkie et al. (36).

Transformation of E. coli and S. aureus. Transformation ofE. coli was performed by the CaCl2 method (20), and trans-formants were selected on LB agar containing erythromycin at

Pat/Pac pwRB, K/lpnpZ88G01 7 _-gr YA= -

IITpI~AorPEa P!a So1 Pfl

pZSSGO1 7Ap p3SSG0) 73BP

FIG. 2. DNA fragment carried by pESSGO17. The gyrA and gyrBgenes are denoted by arrows. pESSGO17AP and pESSGO17ABP werederived from pESSGO17 by deletion of the 198-bp PmaCI-PmaCIfragment and the 345-bp BalI-PflMI fragment, respectively. P, pro-moter; RB, ribosome binding site; Aor,Aor5lHI; Bal, BalI; Kpn, KpnI;Mam, MamI; Nsp, NspV; Pac, PaM; Pfl, PflMI; Pma, PmaCI; Pst, PstI.

400 ,ug/ml for pAT19 and its derivatives and ampicillin at 25,ug/ml for the other plasmids. Transformation of S. aureus wascarried out as follows. Staphylococcal cells were prepared bythe method of K. Hiramatsu (Juntendo University, Tokyo)(10). One hundred microliters of the culture of S. aureusRN4220 and its quinolone-resistant mutants that had beengrown overnight were inoculated into 10 ml of brain heartinfusion broth and incubated at 37°C until an optical density at600 nm of 0.3 to 0.5 was attained. The cells were harvested bycentrifugation, suspended in 1 ml of 1.1 M sucrose, andtransferred into an Eppendorf centrifuge tube. The cells werecollected by centrifugation, washed twice in 0.5 ml of 1.1 Msucrose, and then resuspended in 80 RI of 1.1 M sucrose.Plasmid DNA (ca. 1 ,ug in a volume of 1 RI) was added to 20-plaliquots of the cell suspension, which were then subjected to ahigh-voltage electric pulse with the Cell-Porator apparatus(Bethesda Research Laboratories, Gaithersburg, Md.); theconditions used were as follows: capacitance, 330 ,uF; voltage,300 V; resistance, low fl, and booster, 2 kQi. The cells weremixed with 0.5 ml of brain heart infusion broth containing 1.1M sucrose, incubated at 37°C for 2 h, spread onto brain heartinfusion agar plates containing erythromycin at a concentra-tion of 8 ,ug/ml, and then cultured at 37°C for 24 h.DNA-DNA hybridization. Southern hybridization and colony

hybridization for cloning of S. aureus gyrase genes from strainATCC 12600 were carried out as described by Maniatis et al.(20). Oligonucleotide probes complementary to the sequencesof nucleotides 2155 to 2175 and 2314 to 2334 (see Fig. 3) weredesigned on the basis of information on the nucleotide se-quence of the portion of the gyrB-gyrA genes of a clinicalisolate of S. aureus (12) and were 2P labeled by using T4polynucleotide kinase.

Construction of the plasmids for transformation. PlasmidpESSGO17 used in transformation with gyrase genes wasconstructed by inserting the 6.4-kb KpnI-PstI fragment contain-ing the wild-type gyrB and gyrA genes ofpSAGBA64HPM (Fig.1) between the KpnI and PstI sites of an E. coli-S. aureusshuttle vector, pAT19 (Fig. 2). Plasmid pESSGO17AP carryingonly the functional gyrA gene was constructed by deleting the198-bp PmaCI-PmaCI segment (nucleotides 1160 to 1357) ofthe gyrB gene of pESSGO17 (Fig. 2). Plasmid pESSGO17ABPhaving only the functional gyrB gene was obtained by deletingthe 345-bp BalI and PflMI segment (nucleotides 2469 to 2814)of the gyrA gene of pESSGO17. gyrA and/or gyrB plasmids withthe mutations were obtained through replacement of a smallportion of the wild-type gyrA and/or gyrB genes by the corre-sponding fragments of the mutants. To replace the fragment inthe gyrA gene, the 351-bp Aor5lHI-BalI fragments (nucleo-tides 2118 to 2468) from the pT7Blue(R) derivatives contain-ing the PCR products of the gyrA mutants were ligated with the12.6-kb Aor5lHI-BalI fragments of pESSGO17 and pESSG017AP. Fragment replacement in the gyrB gene was carried

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ANTIMICROB. AGENTS CHEMOTHER.

1 TTAGACGATGTACTCAGTGAATTAGATGATTCGCGTCAAACGCATTTATTAAGTACGATTCAGCATAAAGTACAAACATTTGTCACTACGACATCTGTAG& gyrBM V T A L S D

101 ATGGTATTGATCATGAAATCATGAATAACGCTAAATTGTATCGTATTAATCAAGGTGAAATTATAAAGTAACAGAAAGCGATGGTGACTGCATTGTCAGA-35 -10 SD

V N N T D N Y G A G Q I Q V L E G L E A V R K R P G M Y I G S T S201 TGTAAACAACACGGATAATTATGGTGCTGGGCAAATACAAGTATTAGAAGGTTTAGAAGCAGTACGTAAAAGACCAGGTATGTATATAGGATCGACTTCA

E R G L H H L V W E I V D N S I D E A L A G Y A N Q I E V V I E K D301 GAGAGAGGTTTGCACCATTTAGTGTGGGAAATTGTCGATAATAGTATCGATGAAGCATTAGCTGGTTATGCAAATCAAATTGAAGTTGTTATTGAAAAAG

N W I K V T D N G R G I P V D I Q E K M G R P A V E V I L T V L H401 ATAACTGGATTAAAGTAACGGATAACGGACGTGGTATCCCAGTTGATATTCAAGAAAAAATGGGACGTCCAGCTGTCGAAGTTATTTTAACTGTTTTACA

A G G K F G G G G Y K V S G G L H G V G S S V V N A L S Q D L E V501 TGCTGGTGGTAAATTTGGCGGTGGCGGATACAAAGTATCTGGTGGTTTACATGGTGTTGGTTCATCAGTTGTAAACGCATTGTCACAAGACTTAGAAGTA

Y V H R N E T I Y H Q A Y K K G V P Q F D L K E V G T T D K T G T V601 TATGTACACAGAAATGAGACTATATATCATCAAGCATATAAAAAAGGTGTACCTCAATTTGACTTAAAAGAAGTTGGCACAACTGATAAGACAGGTACTG

I R F K A D G E I F T E T T V Y N Y E T L Q Q R I R E L A F L N K701 TCATTCGTTTTAAAGCAGATGGAGAAATCTTCACAGAGACAACTGTATACAACTATGAAACATTACAGCAGCGTATTAGAGAGCTTGCTTTCTTAAACAA

G I Q I T L R D E R D E E N V R E D S Y H Y E G G I K S Y V E L L801 AGGAATTCAAATCACATTAAGAGATGAACGTGATGAAGAAAACGTTAGAGAAGACTCCTATCACTATGAGGGCGGTATTAAATCGTACGTTGAGTTATTG

N E N K E P I H D E P I Y I H Q S K D D I E V E I A I Q Y N S G Y A901 AACGAAAATAAAGAACCTATTCATGATGAGCCAATTTATATTCATCAATCTAAAGATGATATTGAAGTAGAAATTGCGATTCAATATAACTCAGGATATG

T N L L T Y A N N I H T Y E G G T H E D G F K R A L T R V L N S Y1001 CCACAAATCTTTTAACTTACGCAAATAACATTCATACGTATGAAGGTGGTACGCATGAAGACGGATTCAAACGTGCATTAACGCGTGTCTTAAATAGTTA

G L S S K I M K E E K D R L S G E D T R E G M T A I I S I K H G D1101 TGGTTTAAGTAGCAAGATTATGAAAGAAGAAAAAGATAGACTTTCTGGTGAAGATACACGTGAAGGTATGACAGCAATTATATCTATCAAACATGGTGAT

P Q F E G Q T K T K L G N S E V R Q V V D K L F S E H F E R F L Y E1201 CCTCAATTCGAAGGTCAAACGAAGACAAAATTAGGTAATTCTGAAGTGCGTCAAGTTGTAGATAAATTATTCTCAGAGCACTTTGAACGATTTTTATATG

N P Q V A R T V V E K G I M A A R A R V A A K K A R E V T R R K S1301 AAAATCCACAAGTCGCACGTACAGTGGTTGAAAAAGGTATTATGGCGGCACGTGCACGTGTTGCTGCGAAAAAAGCGCGTGAAGTAACACGTCGTAAATC

A L D V A S L P G K L A D C S S K S P E E C E I F L V E G D S A G1401 AGCGTTAGATGTAGCAAGCCTTCCAGGTAAATTAGCCGATTGCTCTAGTAAAAGTCCTGAAGAATGTGAGATTTTCTTAGTCGAAGGGGACTCTGCCGGG

G S T K S G R D S R T Q A I L P L R G K I L N V E K A R L D R I L N1501 GGGTCTACAAAATCTGGTCGTGACTCTAGAACGCAGGCGATTTTACCATTACGAGGTAAGATATTAAATGTTGAAAAAGCACGATTAGATAGAATTTTGA

N N E I R Q M I T A F G T G I G G D F D L A K A R Y H K I V I M T1601 ATAACAATGAAATTCGTCAAATGATCACAGCATTTGGTACAGGAATCGGTGGCGACTTTGATCTAGCGAAAGCAAGATATCACAAAATCGTCATTATGAC

D A D V D G A H I R T L L L T F F Y R F M R P L I E A G Y V Y I A1701 TGATGCCGATGTGGATGGAGCGCATATTAGAACATTGTTATTAACATTCTTCTATCGATTTATGAGACCGTTAATTGAAGCAGGCTATGTGTATATTGCA

Q P P L Y K L T Q G K Q K Y Y V Y N D R E L D K L K S E L N P T P K1801 CAGCCACCGTTGTATAAACTGACACAAGGTAAACAAAAGTATTATGTATACAATGATAGGGAACTTGATAAACTTAAATCTGAATTGAATCCAACACCAA

W S I A R Y K G L G E M N A D Q L W E T T M N P E H R A L L Q V K1901 AATGGTCTATTGCACGATACAAAGGTCTTGGAGAAATGAATGCAGATCAATTATGGGAAACAACAATGAACCCTGAGCACCGCGCTCTTTTACAAGTAAA

L E D A I E A D Q T F E M L M G D V V E N R R Q F I E D N A V Y A2001 ACTTGAAGATGCGATTGAAGCGGACCAAACATTTGAAATGTTAATGGGTGACGTTGTAGAAAACCGTAGACAATTTATAGAAGATAATGCAGTTTATGCA

r+ gyrAN L D F * M A E L P Q S R I N E R N I T S E

2101 AACTTAGACTTCTAAGCGCTGTGAACTGAACTTTTGAAGGAGGAACTCTTGATGGCTGAATTACCTCAATCAAGAATAAATGAACGAAATATTACCAGTGSD

M R E S F L D Y A M S V I V A R A L P D V R D G L K P V H R R I L2201 AAATGCGTGAATCATTTTTAGATTATGCGATGAGTGTTATCGTTGCTCGTGCATTGCCAGATGTTCGTGACGGTTTAAAACCAGTACATCGTCGTATACT

Y G L N E Q G M T P D K S Y *K K S A R I V G D V M G K Y H P H G D2301 ATATGGATTAAATGAACAAGGTATGACACCGGATAAATCATATAAAAAATCAGCACGTATCGTTGGTGACGTAATGGGTAAATATCACCCTCATGGTGAC

S S I Y E A M V R M A Q D F S Y R Y P L V D G Q G N F G S M D G D G2401 TCATCTATTTATGAAGCAATGGTACGTATGGCTCAAGATTTCAGTTATCGTTATCCGCTTGTTGATGGCCAAGGTAACTTTGGTTCAATGGATGGAGATG

A A A M R Y T E A R M T K I T L E L L R D I N K D T I D F I D N Y2501 GCGCAGCAGCAATGCGTTATACTGAAGCGCGTATGACTAAAATCACACTTGAACTGTTACGTGATATTAATAAAGATACAATAGATTTTATCGATAACTA

FIG. 3. Nucleotide sequences of the gyrB and gyrA genes of S. aureus ATCC 12600 (from 180 bp upstream of the initiation codon of the gyrBgene to 135 bp downstream of the termination codon of the gyrA gene). The nucleotide sequences of the sense strands of the genes are shown fromthe 5' end (left) to the 3' (right) end. The deduced amino acid sequences are given below the DNA sequences (GyrB, nucleotides 181 to 2112;GyrA, nucleotides 2152 to 4812). The -35 and -10 sequences of the putative promoter, and the presumptive Shine-Dalgamo (SD)-like sequencesare underlined, and an inverted repeat which can act as a transcription terminator is underlined with arrows.

out in the same manner with pESSGO17 and pESSGO17A&BP, Amplification of the segments of the S. aureus gyrA and gyrBthe 12.1-kb NspV-Aor5lHI fragments of which were ligated genes corresponding to the quinolone resistance-determiningwith the 909-bp NspV-Aor5lHI fragments (nucleotides 1209 to regions of E. coli genes by PCR. One-hundred-microliter2117) of the PCR products. aliquots of cultures of S. aureus RN4220 and its quinolone-

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VOL. 38, 1994 QUINOLONE RESISTANCE MUTATIONS IN S. AUREUS DNA GYRASE 2017

D G N E R E P S V L P AR F P N L LA N G A S G I A VG M A T N I2601 TGATGGTAATGAAAGAGAGCCGTCAGTCTTACCTGCTCGATTCCCTAACTTATTAGCCAATGGTGCATCAGGTATCGCGGTAGGTATGGCAACGAATATT

P P H N L T E L I N G V L S L S K N P D I S I A E L M E D I E G P D2701 CCACCACATAACTTAACAGAATTAATCAATGGTGTACTTAGCTTAAGTAAGAACCCTGATATTTCAATTGCTGAGTTAATGGAGGATATTGAAGGTCCTG

F P T AG L I L G K S G I R R A Y E T G R G S I Q M R S R A V I E2801 ATTTCCCAACTGCTGGACTTATTTTAGGTAAGAGTGGTATTAGACGTGCATATGAAACAGGTCGTGGTTCAATTCAAATGCGTTCTCGTGCAGTTATTGA

E RG G G R Q R I V V T E I P F Q VN K A R M I E K I A E L V R D2901 AGAACGTGGAGGCGGACGTCAACGTATTGTTGTCACTGAAATTCCTTTCCAAGTGAATAAGGCTCGTATGATTGAAAAAATTGCAGAGCTCGTTCGTGAC

K K I D G I T D L R D E T S L R T G V R V V I D V R K D A NA S V I3001 AAGAAAATTGACGGTATCACTGATTTACGTGATGAAACAAGTTTACGTACTGGTGTGCGTGTCGTTATTGATGTGCGTAAGGATGCAAATGCTAGTGTCA

L N N L Y K Q T P L Q T S F G V N M I A L V N G R P K L I N L K E3101 TTTTAAATAACTTATACAAACAAACACCTCTTCAAACATCATTTGGTGTGAATATGATTGCACTTGTAAATGGTAGACCGAAGCTTATTAATTTAAAAGA

A L V H Y L E H Q K T V VR R R T Q Y N L R K A K D R A H I L E G3201 AGCGTTGGTACATTATTTAGAGCATCAAAAGACAGTTGTTAGAAGACGTACGCAATACAACTTACGTAAAGCTAAAGATCGTGCCCACATTTTAGAAGGA

L R I A L D H I DE I I S T I R ES D T D K V A M E S L Q Q R F K L3301 TTACGTATCGCACTTGACCATATCGATGAAATTATTTCAACGATTCGTGAGTCAGATACAGATAAAGTTGCAATGGAAAGCTTGCAACAACGCTTCAAAC

S E K Q A Q A I L D M R L R R L T G L E R D K I E A E Y N E L L N3401 TTTCTGAAAAACAAGCTCAAGCTATTTTAGACATGCGTTTAAGACGTCTAACAGGTTTAGAGAGAGACAAAATTGAAGCTGAATATAATGAGTTATTAAA

Y I S E L E A I L A D E E V L L Q L V R D E L T E I R D R F G D D3501 TTATATTAGTGAATTAGAAGCAATCTTAGCTGATGAAGAAGTGTTATTACAGTTAGTTAGAGATGAATTGACTGAAATTAGAGATCGTTTCGGTGATGAT

R R T E I Q L G G F E D L E D E D L I P E E Q I V I T L S H N N Y I3601 CGTCGTACAGAAATTCAATTAGGTGGATTTGAAGACTTAGAGGACGAAGACTTAATTCCAGAAGAACAAATAGTAATTACACTAAGCCATAATAACTACA

K R L P V S T Y R A Q N R G G R G V Q G M N T L E E D F V S Q L V3701 TTAAACGTTTGCCGGTATCTACATATCGTGCTCAAAACCGTGGTGGTCGTGGTGTTCAAGGTATGAATACATTGGAAGAAGATTTTGTCAGTCAATTGGT

T L S T H D H V L F F T N K G R V Y K L K G Y E V P E L S R Q S K3801 AACTTTAAGTACACATGACCATGTATTGTTCTTTACTAACAAAGGTCGTGTATACAAACTTAAAGGTTACGAAGTGCCTGAGTTATCAAGACAGTCTAAA

G I P V V N A I E L E N D E V I S T M I A V K D L E S E D N F L V F3901 GGTATTCCTGTAGTGAAT5CTATTGAACTTGAAAATGATGAAGTCATTAGTACAATGATTGCTGTTAAAGACCTTGAAAGTGAAGACAACTTCTTAGTGT

A T K R G V V K R S A L S N F S R I N R N G K I A I S F R E D D E4001 TTGCAACTAAACGTGGTGTCGTTAAACGTTCAGCATTAAGTAACTTCTCAAGAATAAATAGAAATGGTAAGATTGCGATTTCGTTCAGAGAAGATGATGA

L I A V R L T S G Q E D I L I G T S H A S L I R F P E S T L R P L4101 GTTAATTGCAGTTCGCTTAACAAGTGGTCAAGAAGATATCTTGATTGGTACATCACATGCATCATTAATTCGATTCCCTGAATCAACATTACGTCCTT.TA

G R T A T G V K G I T L R E G D E V V G L D V A H A N S V D E V L V4201 GGCCGTACAGCAACGGGTGTGAAAGGTATTACACTTCGTGAAGGTGACGAAGTTGTAGGGCTTGATGTAGCTCATGCAAACAGTGTTGATGAAGTATTAG

V T E N G Y G K R T P V N D Y R L S N R G G K G I K T A T I T E R4301 TAGTTACTGAAAATGGTTATGGTAAACGTACGCCAGTTAATGACTATCGCTTATCAAATCGTGGTGGTAAAGGTATTAAAACAGCTACGATTACTGAGCG

N G N V V C I T T V T G E E D L M I V T N A G V I I R L D V A D I4401 TAATGGTAATGTTGTATGTATCACTACAGTAACTGGTGAAGAAGATTTAATGATTGTTACTAATGCAGGTGTCATTATTCGACTAGATGTTGCAGATATT

S Q N G R A A Q G V R L I R L G D D Q F V S T V A K V K E D A E D E4501 TCTCAAAATGGTCGTGCAGCACAAGGTGTTCGCTTAATTCGCTTAGGTGATGATCAATTTGTTTCAACGGTTGCTAAAGTAAAAGAAGATGCAGAAGATG

T N E D E Q S T S T V S E D G T E Q Q R E A V V N D E T P G N A I4601 AAACGAATGAAGATGAGCAATCTACTTCAACTGTATCTGAAGATGGTACTGAACAACAACGTGAAGCGGTTGTAAATGATGAAACACCAGGAAATGCAAT

H T E V I D S E E N D E D G R I E V R Q D F M D R V E E D I Q Q S4701 TCATACTGAAGTGATTGATTCAGAAGAAAATGATGAAGATGGACGTATTGAAGTAAGACAAGATTTCATGGATCGTGTTGAAGAAGATATACAACAATCA

S D E E *4801 TCAGATGAAGAATAATAAAAAATAAGACTTCCCT ATAGTAGGGGAGTCTTATTTTTATGCTAGAAAGTAATGCTTTACTATATTCAATGATTAGTAATG

transcription terminator

4901 ACTAACTTTCTAATTGTTTCATTGCGTAAGGTATTTCATTGATAAGTCTT

FIG. 3-Continued.

resistant mutants grown overnight were centrifuged at 10,000 a BalI site) for amplification of 374-bp gyrA fragments; 5'-x g for 1 min. The cell pellets were washed once with 0.2-ml TGGTGATCCTCAATTCGAAG-3' (nucleotide positionsportions of distilled water, suspended in 100-,u portions of 1194 to 1213 with a NspV site) and 5'-CAGTTCACAGCGCTdistilled water, and then boiled at 95°C for 5 min. The boiled TAGAAG-3' (complementary to positions 2109 to 2128 withsuspensions were centrifuged at 10,000 x g for 10 min. Three an Aor51HI site) for amplification of 935-bp gyrB fragments.microliters of each supernatant was used as a template for Amplification was carried out in a GeneAmp PCR system 9600PCR. Four oligonucleotide primers were synthesized: 5'- (Perkin-Elmer Cetus) using a GeneAmp PCR reagent kit withGACl lCTAAGCGCTGTGAAC-3' (nucleotide positions 2107 AmpliTaq DNA polymerase and the primers shown above.to 2126 with an Aor5lHI site) and 5'-AAGTTACCTITGGC The PCR conditions were as follows: 2 cycles of 15 s at 97°C;CATCAAC-3' (complementary to positions 2461 to 2480 with 30 s at 63°C, 30 s at 74°C; 26 cycles of 15 s at 94°C; 30 s at 63°C;


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IAELPOSRINEN I TSERSFLDYAUSVIVAPDVM KPV1ILY "JGNTP

-SD SARE-ITPV-EE-LU SS 6 V -WV

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121 EYTEATKITLELLRDINKDTIDFIDNYDGNERPSVLPAWPILLAGAIAVA121121120 - I-A--LE-E-V-V-T-KI-D-U-TKI-V-S--

TN I PPINLTEL INGVLSLSKNPD IS I AELUEDI EGVPTAL I LGKSG IRAYET S

V -C-AYID-EG---HP AINl-RR-EE-R

IQMRSRAVIE*ERGGWt0RIVVTEIPFfO II ElEKIAELVRDKKIDGITD6JDETSLR

VYI-A--EV-VDAKT--ET-I-H---Y----L--- KE-RVE--SA-SDOK

TGVKVRKDANASVILWLTPLTFlTL IAILVNGRPLI 1.KMLYILEH

KTVVTIL KLRIAI EIIR....................

RE-T-IFE--R A-AV-P-ELH PAEAKTVANPWLG

* .... ESTDKYVA*ESLOW .* KLSEKOA ILCLLTGLERDK I EAEY

446446446479

506506506538

566566566598

626626626657

686686686717

746746746775

806806806835

NELLNYISELEAILADEEVLLOLVRDELTE IR GDOMRTEIOLGGEDLEDEOLIPEE

K-00-A--LR--GSA0R-UVEI-E-ELV-E--K-TANS A--INL-TO-

0IVITLSHNNYIKRLPVSTYRA0NRGGRGV0GkNTLEEDFVSQLVTLSTHO1VLFFTNKG

OV-V---QG-V-YO-L-E-E-R-K-KSAARIK--IDR-LVAN---I-C-SSR-

RVYKLKGYEVPELSROSKG IPl IELENDE ISTM IAKDLESEDLVFATKRGVVK

-S-V-4L-AT-GAR-R- I-LLP---R-TA ILP-TEF-EGVKFU-ANJ-T-

RSALSWFSR IM IAISFREDDEL IAV TSOED IL I GTSHASL I FSTLRPLGI

KTY-TE-N-LRTA-V-KLVDG----G D-EDEVYEFSAEGM--K-SV-A-C

TATGVKGI TLREGDEWGLDiMNSVDEVLWTENGTPVNDYRLSNRGKIKTAE

NT-R--R-G-K--S- -PR GAI-TA-0--- A-AE-PIKS-AT--VISI

TITERNGNYCITTVTGEEMUIVTNAGVIIRGRLDVD0S

KV--L--AVO-OL-UI-0--TLV-TR-SE-IV-NT-1-TAE-EN-V

TVAKVKEDAEDETMEDEOSTSTVSEDGTEOWREAVVNDETPGNAIHTEVIDSEENDEDGRE-T-D-

GLOR-A-PVE-LDT I GSAAE E IAPEVDVDDEP-EE...............

866 IE EDIO0SSOEE--866 L-DEE863 DEE

419 NVAAML-RAG-A--EPE-GRDGLYY-T-0------L - H-ELD- .................

FIG. 4. Comparison of the amino acid sequences of the S. aureus and E. coli GyrA polypeptides. The sequences of S. aureus 81231, S. aureus

601055, and E. coli KL16 are from references 21, 2, and 41, respectively. The dashes indicate that the amino acid residues are identical to thoseof S. aureus ATCC 12600. The dots indicate that the amino acid residues are absent. The sites of S. aureus corresponding to quinoloneresistance-determining sites (38) and the DNA-binding site (14) of E. coli are indicated by * and V above the amino acid residues, respectively.The putative leucine zipper structure (19) is overlined with arrows.

30 s at 74°C; 2 cycles of 15 s at 94°C; 30 s at 63°C; 3 min at 74°C.The PCR products were directly cloned into pT7Blue(R)T-vector.DNA sequencing. The DNA sequences of the gyrase genes

were determined by the dideoxy chain termination methodwith the phage M13mpl8 and M13mpl9 vectors and E. coliJM109. DNA sequencing of the PCR products was carried outas follows. The PCR products were cloned into plasmidpT7Blue(R) T-vector and transformed into E. coli JM109, andthen single-stranded DNA was prepared by the method ofVieira and Messing (34) using the VCS-M13 interference-resistant helper phage. The DNA sequences were determinedby the dideoxy chain termination method with a BcaBESTdideoxy sequencing kit and synthetic oligonucleotide primers.Other DNA techniques were done essentially as described byManiatis et al. (20).

Nucleotide sequence accession number. Nucleotide se-

quence accession number D10489 has been assigned to thesequence of strain ATCC 12600 containing the gyrB and gyrAgenes in the DDBJ, EMBL, and GenBank data bases.

RESULTS

Cloning of the DNA gyrase genes from quinolone-suscepti-ble S. aureus ATCC 12600. Southern hybridization analysisusing the oligonucleotide probes described in Materials andMethods showed that the 6.5-kb HindIII and 5-kb EcoRI DNAfragments derived from S. aureus ATCC 12600 might containthe gyrase genes, and these two fragments were inserted intothe HindIII and EcoRI sites, respectively, of pBR322. E. coliHB101 was transformed with the resultant plasmids, and about20 hybridization-positive clones were selected in each case.Restriction analysis and nucleotide sequencing suggested thatpSAGB301 and pSAGA501 had the gyrase genes (Fig. 1). A

S. arus ATCC12600S. aures 81231S.urets 601055co/i KL16

61616160

181181181180

241241241240

300300300299

360360360359

400400400

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1 MVTALSDVNNTDNYGAG0I0VLEGLEAVRKRPGMYIGSTSE RGLHHLVWE IVDNSIDEAI~~~~~~~~..cbe. __

.........BSNS-DSSS-K--KD0- --D-DDGT---U-F-V---A----

LAGYANOI EWVI EKDNMIKVTDNGRGIPVDIOEKMGRPAVEVILTVLHAGGKFGGGGYKV-K - - _ ____

a RSGGLHGVGSSVWNALSODLEVYVHRNET IYHOAYKKGVPOFDLKEVGTTDKTGTV IRFKA

DGE IFTETTVYNYETLOOR IRELAFLNKGIOI TLRDERDEENVREDSYHYEGGIKSYVEL

SL-T-V-EFE-- I -AK-L--S--S-VS-R--K-- ** GK--HF-------AF--Y

LNENKEPIHDEPIY HOSKDDI EVE IAIOYNSGYATNLLTYANN IIHTYEGGTHEDGFKRA

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TL-L-C--RDEYNPO-L--S-1I-------S------------O-PE IV-R-H--

IAOPPLYKLTOGKOKYYVYNDRELDKLKSELNPTPKWSIARYKGLGEWNADOLWETTMNP

-------VKK---EO-IKD-EAM-OYOI SIALDGATLHTNASAPALAGEALEKLVSEYN

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599 ._.____ _ ...............

589 ATOMIINRMERRYPKAMLKEL IYOPTLTEADLSDEOTVTRWVNALVSELNDKEOHIGSOWK

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649 FDVHTNAEONLFEPIVRVRTHGVDTDYPLDHEFI TGGEYRR ICTLGEKLRGLLEEDAF E

............................................................

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709 RGERROPVASFEOALDIILVKESRRGLS IORYKGLGEMNPEOLWIETTMIDPESRRMbLRVTVK

LTRVLNSYGLSSK IMKEEKDRLSGEDTREGMTAI SIKHGDPOFEGOTKTKLGNSEVROV

l-T-A-MDKEGYS-KA-VSAT-D-A-L I -VV-V-VP-K-SS--D--VS--KSA ....................................769 DAIAADOLFTTLIIGDAVEPRRAF IEENALKAMNID

VDKLFSEHFERFLYENPOVARTVVEKGIMAARARVAAKKAREVTRRKSALDVASLPGKLA

420 DCSSKSPEECE IFLVEGDSAGGSTKSGRDSRTOAILPLRGKILNVEKARLDRILNNNE IR416 0 _ _ _420

409 -OERD-ALS-LY A-0-ANRKN---K F-K SSO-VA

FIG. 5. Comparison of the amino acid sequences of the S. aureus and E. coli GyrB polypeptides. The sequences of S. aureus 81231, S. aureus

601055, and E. coli KL16 are from references 21, 2, and 37, respectively. The dashes indicate that the amino acid residues are identical to thoseof S. aureus ATCC 12600. The dots indicate that the amino acid residues are absent. The sites of S. aureus corresponding to quinoloneresistance-determining sites (39), the sites for ATP binding (35), and the sites causing coumarin antibiotic resistance (3) of E. coli are indicatedby *, *, and * above the amino acid residues, respectively.

4.2-kb XbaI-EcoRI fragment from pSAGA501 was subclonedbetween the XbaI and EcoRI sites of pUC18, and then a 4-kbXbaI fragment from pSAGB301 was inserted into the XbaI siteof the resultant plasmid in the right direction to obtainpSAGBA085 containing the entire gyrB and gyrA genes. Atruncated plasmid, pSAGBA64HPM, was constructed by in-serting a 6.4-kb filled-in PacI-MamI fragment from pSAGBA085 into the HincIl site of pUC119 (Fig. 1).

Nucleotide sequences of the gyrase genes of S. aureus ATCC12600. The nucleotide sequence of the cloned fragment con-

taining the gyrase genes in pSAGBA085 is shown with thededuced amino acid sequence in Fig. 3. This sequence hasputative promoter sequences (nucleotides 132 to 137 and 107to 114), two putative ribosomal binding sequences (nucleotides173 to 178 and 2137 to 2143), the gyrB gene (nucleotides 181 to2112), the gyrA gene (nucleotides 2152 to 4812), and an

inverted repeat (nucleotides 4818 to 4857), which might act as

a transcription terminator. The gyrA and gyrB genes encodepolypeptides of 887 and 644 amino acids with molecularweights of 99,350 and 72,539, respectively. The amino acidsequences of GyrA and GyrB are compared with those of S.aureus (2, 21) and E. coli reported previously (37, 41) in Fig. 4and 5. For the GyrA polypeptide (Fig. 4), our sequence was

different from that of S. aureus 81231 (clinical isolate) (21) atamino acids 709, 856, 860, 862, 884, and 887 and from that ofS. aureus 601055 (clinical isolate) (2) at amino acids 439, 483,594, 815, 818, 824, 825, and 887. These two strains had twoadditional glutamic acid residues at the C terminus of GyrA ofS. aureus ATCC 12600. For the GyrB polypeptide (Fig. 5), our

sequence was different from that of S. aureus 81231 (21) atamino acids 40, 41, 43, 46, 47, 49, 65, 312, 313, 314, 315, 318,319, 320, 321, 424, 522, and 579 and was identical to that of S.aureus 601055 (2). Our amino acid sequences exhibited 99.3and 99.1% amino acid identities for GyrA and 97.2 and 100%

S aureus ATCC12600S. aureus 81231S. ureus 601055E coli KL16

60586052

120118120112

180178180172

240238240229

300298300289

360356360349

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TABLE 1. Isolation of quinolone-resistant mutants from S.aureus RN4220

Parental Selective Parental Mutant Mutant MICMutants strain compounda strain MIC frequency, (pg/ml)

1st step RN4220 SPFX 0.1 <1.0 x i0-9CPFX 0.2 3.0 x 10-8 1.56NFLX 0.78 1.3 x 10-7 6.25-12.5cOA 1.56 <1.0 x 10-8

2nd step RCM101d CPFX 1.56 2.5 X 10-8 6.25RNM1Ole NFLX 6.25 1.1 x 10-6 50100c

3rd step RCM11Df SPFX 0.39 <1.1 x 10-9CPFX 6.25 >1.0x 10-4OA 3.13 2.8 x 10-8 25_100c

a CPFX, ciprofloxacin; NFLX, norfloxacin; SPFX, sparfloxacin; OA, oxolinicacid.

b Mutant frequencies were examined with a drug concentration of four timesthe MIC for the preceding step strains..

c Several strains with different MICs.d A first-step mutant selected by CPFX.e A first-step mutant selected by NFLX.fA second-step mutant selected by CPFX.

amino acid identities for GyrB with those of S. aureus 81231and S. aureus 601055, respectively. The functionally importantsites or regions in E. coli which had been disclosed werecompletely conserved between these three S. aureus gyrasesand highly conserved between the S. aureus and E. coli gyrasesand are as follows: the putative sites for ATP binding (35)(amino acids 14, 49, 74, 111, 117, 346, and 348 in GyrB of S.aureus ATCC 12600), the sites causing coumarin antibioticresistance (3) (amino acids 144 and 166 in GyrB), the sitescausing quinolone resistance (38, 39) (amino acids 68, 82, 84,85, 88, and 107 in GyrA and amino acids 437 and 458 in GyrB),the DNA-binding site (14) (amino acid 123 in GyrA), and theleucine zipper structure (19) (amino acids 417 to 455) of GyrA.

Selection of quinolone-resistant mutants of S. aureusRN4220. Spontaneous quinolone-resistant mutants were iso-lated from S. aureus RN4220, since an efficient transformationsystem was found with the combination of S. aureus RN4220and plasmid pAT19. The transformation frequency of S. aureusATCC 12600 from which the gyrA and gyrB genes were clonedwas too low.An attempt was first made to select spontaneous quinolone-

resistant mutants of S. aureus RN4220 with sparfloxacin,ciprofloxacin, norfloxacin, and oxolinic acid at four times theirMICs (Table 1). Mutants were detected at frequencies of 3 x

10-8 and 1.3 x 10-7, respectively, when ciprofloxacin andnorfloxacin were used as the selective agent, but no mutantswere detected when sparfloxacin or oxolinic acid was used as

the selective agent. The mutants selected by ciprofloxacin andnorfloxacin were 8 to 16 times more resistant to the quinolonesused for selection than the parental strain was (Table 1) butonly 2 times more resistant to sparfloxacin and oxolinic acidthan RN4220 was (Table 2 and data not shown). Second-stepmutants were obtained from representative mutants, RCM101and RNM101, by ciprofloxacin and norfloxacin selection (fourtimes the MICs for the first-step mutants) at frequencies of 2.5x 10-8 and 1.1 x 10-6, respectively. Ciprofloxacin-selectedsecond-step mutants were 32 times more resistant to cipro-floxacin than RN4220 was, and norfloxacin-selected second-step mutants were 64 to 128 times more resistant to norfloxacinthan RN4220 was (Table 1). These second-step mutants were

only two to four times more resistant to oxolinic acid andsparfloxacin than RN4220 was (Table 2 and data not shown).Third-step mutants were selected from a ciprofloxacin-selectedsecond-step mutant, RCM1O1D, by using sparfloxacin, cipro-floxacin, and oxolinic acid at four times their MICs. Mutantswere isolated by selection with ciprofloxacin and oxolinic acidat frequencies of >1.0 x 10-4 and 2.8 x 10-8, respectively.Oxolinic acid-selected mutants were 16 to 64 times moreresistant to oxolinic acid than RN4220 was and showed variouspatterns of resistance to sparfloxacin, ciprofloxacin, and nor-floxacin (Table 2). No mutants were detected on selection withsparfloxacin.

Quinolone resistance of oxolinic acid-selected third-stepmutants of S. aureus RN4220. The 35 third-step mutantsstudied were classified into five groups according to theirresistance patterns and the results of transformation with thegyrase genes described in the next paragraph. As shown inTable 2, compared with the parent second-step mutant,RCM1O1D, group 1 mutants were 16 to 64 times moreresistant to relatively hydrophobic quinolones, such as spar-floxacin and oxolinic acid, and 4 to 8 times more resistant torelatively hydrophilic quinolones, such as ciprofloxacin andnorfloxacin. Compared with RCM1O1D, group 2 and 3 mu-

tants were 16 to 32 times more resistant to oxolinic acid butonly 2 to 4 times more resistant to the other quinolones.Compared to RCM1O1D, group 4 mutants were 8 times moreresistant to an acidic quinolone like oxolinic acid but 4 to 8times more susceptible to amphoteric quinolones such assparfloxacin, ciprofloxacin, and norfloxacin. Compared withRCM1O1D, the group 5 mutant was 32 and 8 times moreresistant to oxolinic acid and sparfloxacin, respectively, but

TABLE 2. Quinolone susceptibility of various quinolone-resistant mutants of S. aureus RN4220

MIC (Lg/mJ)aStrain or group No. of strains

SPFX CPFX NFLX OA

StrainsRN4220 1 0.1 0.2 0.78 1.56RCM101 (1st-step mutant) 1 0.2 1.56 6.25 3.13RCM1O1D (2nd-step mutant) 1 0.39 6.25 25 3.13

Groups1 20 12.516, 254 2513, 507 1009, 2001, 506, 100142 5 0.781, 1.564 12.54, 251 501, 1004 501, 10043 7 0.781, 1.566 12.5 50 504 2 0.05 0.78 6.25 255 1 3.13 12.5 100 100

' CPFX, ciprofloxacin; NFLX, norfloxacin; SPFX, sparfloxacin; OA, oxolinic acid. The subscript numbers are the numbers of mutants with the indicated MICs.

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TABLE 3. Quinolone susceptibility changes followingtransformation of the mutants with the gyrase genes

Sparfloxacin MIC (p.g/ml)a for transformant with:Strain or No. ofgroup strains No pESSGO17 pESSGO17AP pESSGO17ABP

plasmid (gyrBA) (gyrA) (gyrB)

RCM 1 0.39 0.39 0.39 0.39lOlD

Groups1 20 12.516, 254 0.3915, 0.785 0.3915, 0.785 12.518, 2522 5 0.781, 0.22, 0.393 0.22, 0.393 0.782, 1.563

1.5643 7 0.781, 0.24, 0.393 1.56 0.23, 0.394

1.5664 2 0.05 0.39 0.05 0.395 1 3.13 0.39 0.78 0.78

a The subscript numbers are the numbers of transformants with the indicatedMICs.

only 2 to 4 times more resistant to ciprofloxacin and norfloxa-cin.Complementation test of the oxolinic acid-selected third-

step mutants of S. aureus RN4220 with the wild-type gyrasegenes. Table 3 shows the sparfloxacin susceptibilities of theoxolinic acid-selected third-step mutants of S. aureus RN4220before and after transformation with the plasmids having thewild-type gyrase genes of ATCC 12600. Transformation of thegroup 1 and 2 mutants with plasmid pESSGO17 carrying boththe gyrA and gyrB genes and plasmid pESSGO17AP carryingonly the functional gyrA gene made them as sparfloxacinsusceptible as the parent second-step mutant, RCM1O1D, butthe transformants with pESSGO17ABP carrying only the func-tional gyrB gene were still quinolone resistant, suggesting thatthe group 1 and 2 mutants might have a gyrA mutation.Transformation of the group 3 and 4 mutants with pESSGO17(gyrBA) and pESSGO17ABP (gyrB) made them sparfloxacinsusceptible or resistant, but transformation with pESSGO17AP(gyrA) did not, suggesting that they might have agyrB mutation.Transformation of the group 5 mutant with pESSGO17 (gyrBA)made it completely sparfloxacin susceptible, but transforma-tion with pESSGO17AP(gyrA) and pESSGO17ABP(yrB) madeit incompletely sparfloxacin susceptible, suggesting that itmight have mutations on both the gyrA and gyrB genes.

Identification of mutations in the gyrase genes. The DNA

sequences of S. aureus containing those corresponding to thequinolone resistance-determining regions of the E. coli gyrasegenes were amplified by PCR and sequenced (Table 4). Foreach mutant, at least three complete sets of PCR and sequenc-ing were carried out. The sequences of RN4220 andRCM1O1D were identical to those of ATCC 12600 (data notshown). Three of the group 1 mutants checked all had a Ser-84(TCA)-to-Leu (TTA) mutation in GyrA. The five group 2mutants were divided into three types; Ser-84 (TCA) to Ala(GCA) in GyrA in two strains, Ser-85 (TCT) to Pro (CCT) inGyrA in two strains, and Glu-88 (GAA) to Lys (AAA) in GyrAin one strain. Three of the group 3 mutants all possessed anAsp-437 (GAC)-to-Asn (AAC) mutation in GyrB, and one ofthe group 4 mutants possessed an Arg-458 (CGA)-to-Gln(CCA) mutation in GyrB. The one group 5 mutant concur-rently had a Ser-85 (TCT)-to-Pro (CCT) mutation in GyrA andan Asp-437 (GAC)-to-Asn (AAC) mutation in GyrB. Thesepoint mutations were identical or similar to those reportedpreviously for S. aureus (7, 30, 31) and E. coli (38, 39).Complementation test of the mutants with the same mutant

gyrase genes. In order to determine whether the gyrasemutations found were responsible for quinolone resistance,representatives of the oxolinic acid-selected third-step mutantswere transformed with plasmid derivatives having the gyrasegenes with the same mutations as in the hosts. All thetransformants tested remained sparfloxacin resistant (data notshown). These results indicate that the gyrase mutations foundare actually responsible for quinolone resistance.

DISCUSSION

Quinolone resistance in S. aureus is an important problemclinically, but the mechanism of the quinolone resistance hasnot been clearly elucidated. It has been reported that gyrAmutations in the PCR-generated fragments of S. aureus aresimilar to those of E. coli (7, 30, 31), but there have been fewfunctional analyses on the mutations which are responsible forquinolone resistance. In order to rectify this situation, weestablished an efficient transformation system in S. aureus withplasmids carrying the gyrase genes of S. aureus to carry outcomplementation tests. This system enabled us to analyzespontaneous quinolone-resistant mutants of S. aureus RN4220.

Quinolone-resistant mutants were obtained by ciprofloxacinand norfloxacin selection, but not by sparfloxacin or oxolinicacid selection. Kojima et al. (17) reported that quinolone-

TABLE 4. Quinolone resistance mutations in the gyrase genes

No. of Mutation in:Group strinGroupstrans S. aureus E. coli

1 3 Ser-84 (TCA)--Leu (TTA) in GyrA Ser-83 (TCG)-*Leu (TTG)a in GyrA

2 2 Ser-84 (TCA)-*Ala (GCA) in GyrA Ser-83 (TCG)-iAla (GCG)b in GyrA2 Ser-85 (TCT)-4Pro (CCT) in GyrA Ala-84 (GCG)-'Pro (CCG)' in GyrA1 Glu-88 (GAA)--Lys (AAA) in GyrA Asp-87 (GAC)-->Asn (AAC)a in GyrA

3 3 Asp-437 (GAC)->Asn (AAC) in GyrB Asp-426 (GAC)--Asn (AAC)C in GyrB

4 1 Arg-458 (CGA)-->Gln (CAA) in GyrB Lys-447 (AAG)-->Glu (GAG)C in GyrB

5 1 Ser-85 (TCT)--Pro (CCF) in GyrA and Asp-437(GAC)-iAsn (AAC) in GyrB

a The data are from reference 41.b The datum is from reference 9.c The data are from reference 39.

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resistant mutants of S. aureus can be isolated by ciprofloxacinand norfloxacin selection, but not by sparfloxacin selection,and Hori et al. (13) reported that quinolone-resistant mutantsof S. aureus can be selected by norfloxacin and at low frequencyby ofloxacin. Thus, in S. aureus, the frequency of isolation ofmutants differs significantly, depending on the quinolone usedfor selection.One of the second-step mutants, RCM1O1D, accumulated

ciprofloxacin to a lesser degree than RN4220 did (data notshown), and its level of quinolone resistance did not change ontransformation with pESSGO17 having the wild-type gyrB andgyrA genes, suggesting that the strain is a transport mutant.

Because the oxolinic acid-selected mutants (third-step mu-tants) showed a variety of resistance patterns compared withthose of the ciprofloxacin-selected mutants, they were used forfurther studies. They were considered to be gyrase mutantsbecause their transformants with plasmids carrying the wild-type gyrase gene were quinolone susceptible. It is not clear whygyrase mutants could be obtained on third-step selection butnot on first- and second-step selection. Hori et al. (13)suggested that gyrase mutations alone may be lethal andcannot exist without a suppressor mutation. However, this isunlikely because gyrase mutations alone are not lethal in E.coli (29, 41). Further studies are required to clarify this point.DNA sequence analyses of the 35 third-step mutants re-

vealed that these mutants had the mutations on the gyrA and/orgyrB genes (Table 4). These mutants transformed with theplasmids having the same mutations in the gyrA and/or gyrBgenes remained quinolone resistant, indicating that thesemutations were actually responsible for quinolone resistance.These complementation test results also indicate that thewild-type gyrA and gyrB genes are dominant over the corre-sponding mutant genes in S. aureus and thus can be used todetermine whether a mutant has a mutation(s) in the gyrAand/or gyrB genes.

All the mutations in the gyrA gene detected in this studywere reported previously for clinical isolates of S. aureus (7, 30,31), suggesting that the mechanism of quinolone resistancemutation in the gyrA gene of S. aureus is similar betweenlaboratory strains and clinical isolates. It is intriguing that themutants having Ser-84-to-Ala, Ser-85-to-Pro, and Glu-88-to-Lys mutations in GyrA had low-level resistance to sparfloxacin,while only the mutation of Ser-84 to Leu conferred high-levelresistance to sparfloxacin. Sparfloxacin resistance is considerednot to be affected largely by mutations other than Ser-84 toLeu. The bulkiness of the amino acid residue at this siteappears to be important for resistance to sparfloxacin. Muta-tions in the gyrB gene were found for the first time. Consider-ing that the levels of resistance to quinolones of the group 3mutants (Asp-437 to Asn in GyrB) are lower than those ofgyrAmutants, gyrA mutants seem to have a selective advantage andgyrB mutants may not be observed as often in clinical isolates.As a possible mechanism of action of quinolones, the

quinolone pocket model has been proposed for E. coli, inwhich quinolones exert their action through their binding to agyrase-DNA complex, and quinolone binding affinities for thecomplex are determined by both the GyrA and GyrB subunitsin concert (42). The existence of double mutations in the gyrAand gyrB genes may mean that the region around Ser-84 toGlu-88 in GyrA is in close proximity to Asp-437 and Arg-458 inGyrB. The quinolone resistance-determining regions of thegyrase subunits are presumed to constitute this putative quin-olone pocket (39). Mutations of these regions seem to preventquinolones from approaching the pocket or from interactingwith the DNA gyrase-DNA complex within the pocket toovercome the action of quinolones. In principle, the quin-

olone-gyrase interaction would be similar among bacterialorganisms.

REFERENCES1. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction

procedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

2. Brockbank, S. M. V., and P. T. Barth. 1993. Cloning, sequencing,and expression of the DNA gyrase genes from Staphylococcusaureus. J. Bacteriol. 175:3269-3277.

3. Contreras, A., and A. Maxwell. 1992. gyrB mutations which confercoumarin resistance also affect DNA supercoiling and ATP hydro-lysis byEscherichia coli DNA gyrase. Mol. Microbiol. 6:1617-1624.

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