Vol. 35, No. 2
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1991, p. 387-389
0066-4804/91/020387-03$02.00/0 Copyright © 1991, American Society for Microbiology
4-Quinolone Resistance Mutations in the DNA Gyrase of Escherichia coli Clinical Isolates Identified by Using the Polymerase Chain Reaction MARK ORAM AND L. MARK FISHER* Molecular Genetics Group, Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London SW17 ORE, United Kingdom Received 19 June 1990/Accepted 30 November 1990
Seven nalidixic acid-resistant clinical isolates of Escherichia coli were shown to carry resistance mutations in their gyrase A proteins. Six had serine-83 to leucine or tryptophan changes; the seventh had an aspartate-87 to valine substitution. The frequent occurrence of a mutation at serine-83 implies a key role for this residue in quinolone action.
High-molecular-weight chromosomal DNA was prepared from each strain (17) and subjected to PCR by using two 20-mer oligonucleotide primers, 5'-TACACCGGTCAACAT TGAGG and 5'-TTAATGATTGCCGCCGTCGG, which were identical in sequence to nucleotide positions 24 to 43 or complementary in sequence to positions 652 to 671 of the E. coli K-12 gyrA gene, respectively (21) (Fig. 1). Reactions were performed in a DNA Thermal Cycler (Perkin ElmerCetus, Beaconsfield, Bucks, United Kingdom) in 10 mM Tris hydrochloride (pH 8.3)-50 mM KCl-1.5 mM MgCl2-0.01% gelatin containing 200 ,uM (each) deoxynucleoside triphosphate (Perkin Elmer-Cetus), 1 to 1.5 U of Taq DNA polymerase, 10 ng of each oligonucleotide primer, and 10 ng of bacterial DNA (final volume, 50 ,ul). Thirty cycles were used for each reaction, with the following temperature profiles: 92°C, 25 s; 64°C, 1 min; 74°C, 2.5 min. DNA analysis by agarose gel electrophoresis revealed in each case amplification of the expected 648-bp fragment. PCR products were precipitated with ethanol and digested with Sacl and SmaI, generating 582-bp gyrA fragments (encoding gyrase A amino acid residues 19 to 213) which were ligated into Sacl-SmaI-cut M13mpl8 and M13mpl9 replicative form DNA (Pharmacia, Milton Keynes, Bucks, United Kingdom). Transformation of DNA into E. coli XL1 recA cells, selection of colorless recombinant plaques, and purification of single-stranded M13 DNA were done as recommended by Amersham International, Little Chalfont, Bucks, United Kingdom. DNA sequencing of complementary strands in two independent clones was by the chain
Antimicrobial 4-quinolones exert their potent antagonistic effects on bacterial DNA replication by inhibiting DNA gyrase, an A2B2 tetrameric enzyme that catalyzes ATPdependent DNA supercoiling (4, 6-8, 10, 13, 23). Gyrase promotes the negative supercoiling of DNA and the formation and resolution of interlocked DNA circles and knotted DNA by passing a DNA duplex through a transient enzymebridged double-strand break in DNA (22). Studies with purified Escherichia coli DNA gyrase have shown that quinolones interfere with DNA breakage and reunion mediated by Tyr-122 of the 875-residue gyrase A subunits (6, 12). Genetic studies of 4-quinolone resistance in laboratory strains of E. coli K-12 have implicated a number of chromosomal loci in low-level resistance, whereas high-level resistance invariably maps at gyrA, which encodes the gyrase A protein (1, 6, 9, 15, 24-26). The following quinolone resistance mutations in the gyrase A protein have been identified from sequence analysis of quinolone-resistant gyrA genes in E. coli KL16: Ser-83--Leu, Ser-83--*Trp, Asp-87--Asn, Gly-81--Cys, Ala-84--*Pro, Ala-67-*Ser, and Gln-106-->His (25). In the only molecular study thus far of a resistant clinical isolate, we showed that mutation of Ser-83 to Trp in the gyrase A protein was responsible for quinolone resistance in a uropathogenic E. coli strain, 227, which was obtained from a patient treated with enoxacin (1). To provide much needed information on the molecular basis of resistance in clinical isolates, we describe here the analysis of an additional seven E. coli strains. Uropathogenic clinical isolates of E. coli resistant to quinolones were obtained by Laura Piddock from several independent sources in Birmingham, England, between June 1984 and April 1985. Strains 58, 158, 202, 218, 227, 231, 233, and 235 were selected for study on the basis of their highlevel resistance to nalidixic acid (MICs, 31 to >1,000 ,g/ml) (2) (see Fig. 3). Several of the strains, 58, 202, and 227, were shown to contain a DNA gyrase A subunit that reconstituted a quinolone-resistant gyrase activity (2). Using a restriction fragment length polymorphism analysis, we showed that all strains except strain 202 carried an amino acid substitution of unknown identity at Asp-82 or Ser-83 of the gyrase A protein (5). We applied the polymerase chain reaction (PCR) in concert with rapid cloning-DNA sequence analysis to identify these gyrA mutations at the nucleotide level. *
Sac I
*v
Sma I 67 83
106
Tyr122
a
4.
gyrA 100 bp
FIG. 1. PCR amplification of a 5' gyrA gene region from clinical isolates of E. coli. The diagram shows the 5' end of the E. coli gyrA gene (heavy line). Arrows denote two synthetic 20mer oligonucleotides positioned next to their homologous sequences on complementary strands of the gyrA gene. Triangles indicate unique sites for Sacd and SmaI located at nucleotide positions 54 and 636, respectively. Tyr-122 denotes the coding sequence for the catalytic tyrosine; locations of the coding sequences for residues whose mutations confer quinolone resistance are also shown.
Corresponding author. 387
388
ANTIMICROB. AGENTS CHEMOTHER.
NOTES
FIG. 2. Mutations in the gyrA gene from clinical isolates identified on DNA sequencing gels. PCR products were digested with Sacl and SmaI, cloned into M13mpl8, and sequenced by the dideoxy chain-termination method. Gels show a region encompassing coding sequence for residues 82 and 83. The bracket defines a Hinfl site (GACTC) at this position in the strain 202 gyrA sequence. Asterisks indicate nucleotide changes from the wild-type sequence.
termination technique (19) with [a-35S]dATP and T7 DNA polymerase by the rapid Amersham Multiwell sequencing method (Fig. 2). Although DNA sequences of cloned 582-bp gyrA fragments exhibited several nucleotide differences compared with that of the quinolone-susceptible E. coli K-12 gyrA gene, only a single coding change was present in each case (Fig. 3). The gyrA genes from strains 58, 158, 218, 231, and 235 each exhibited a C-+T transition at nucleotide STRAIN
MIC (Ug/mi) NAL
KL-16
position 248 leading to a Ser-83--Leu substitution at the protein level; strain 233, like strain 227, had a C-+G transversion at this nucleotide position, generating a Ser-83-+Trp substitution. In contrast, isolate 202 retained a wild-type C base at position 248 (i.e., a Ser-83 in the gyrase A protein) but, instead, had an A--T transversion at position 260, producing an Asp-87--->Val substitution (Fig. 2 and 3). The nature of these DNA sequence changes is consistent with our previous restriction fragment length polymorphism results (Fig. 3) (5). Mutation of Ser-83 to either Leu or Trp has been shown to confer quinolone resistance in nonclinical isolates of E. coli (25, 26), e.g., N-51 and P-18 (Fig. 3). We found the same mutations in seven nalidixic acid-resistant clinical isolates (MICs, 125 to >1,000 ,ug/ml), including the previously described strain 227 (1). We cannot exclude the possibility that other mutations contribute to resistance in these clinical isolates. Indeed, among the Ser-83--Leu gyrA mutants, strain 218 exhibited higher resistance to nalidixic acid and other quinolones, including ciprofloxacin, (Fig. 3) and grew less well than the other strains did, suggesting the presence of additional mutations. However, we note that E. coli KL16 strains N-51 and P-18 had levels of quinolone resistance broadly similar to those of our clinical isolates (Fig. 3) (1, 25, 26). Thus, the Ser-83 to Leu or Trp gyrase A mutations that we have identified may, in large part, be responsible for the resistance of the clinical strains. Strain 202 carried an Asp-87--+Val substitution in its gyrase A protein which maps in the same region of the polypeptide chain as the other known resistance mutations, i.e., adjacent to catalytic Tyr-122. For this strain the quinolone MICs were the lowest of those for the clinical isolates examined here (Fig. 3), and the gyrase A protein partially purified from the strain was less resistant to quinolone inhibition than was the Ser-83--Trp protein (2). It is significant that mutation of Asp-87 to Asn in the gyrase A protein
3
CIP 0.0125
58 158 218 231 235
500 125 >1000 250 500
0.5 0.25 1 0.25 0.25
227 233
500 250
0.25 0.25
202
31
0.06
GYR A GENE 87 83 .Gly Asp SER Ala Val Tyr Asp Thr Ile .GGT GAC TCG GCG GTC TAT GAC ACG ATC
1*
T
T*
T*
T* T* (Leu)
G*
G*
T T T T T
T T
(Trp)
N-51 P-18 N-113
400 400 200
0.39 0.39 0.2
C Ser
11
T
T* (Val)
A*
(Asn) FIG. 3. Nucleotide changes in E. coli gyrA genes and inferred amino acid substitutions in the DNA gyrase A proteins of clinical isolates. Asterisks indicate nucleotide differences with the E. coli KL16 gyrA sequence (top) (26) that result in amino acid changes (parentheses). Silent base changes are also indicated (the KL16 gyrA sequence [26] itself exhibits a silent change at nucleotide position 267 compared with that of another K-12 strain [21]). Underlining denotes a Hinfl site, and numbers identify the positions of the deduced amino acid residues. The mutation in strain 227 conferring quinolone resistance has been determined previously (1). Strains N-51, N-113, and P-18 are nalidixic acidand pipemidic acid-resistant KL16 strains whose quinolone resistance mutation has been mapped as shown previously (25). The MICs of nalidixic acid (NAL) and ciprofloxacin (CIP) have been described previously for clinical isolates (2) and KL16 strains (26). The MICs of nalidixic acid determined here (12a) confirmed the results of previous work (2), except for the somewhat higher values for strains 58, 231, and 233. Both nalidixic acid and ciprofloxacin MICs are in broad agreement with those determined independently by L. Piddock (16).
VOL. 35,
NOTES
1991
TABLE 1.
Patterns of silent nucleotide changes seen in gyrA genes from clinical isolates Nucleotide change at the following nucleotide
positions in gyrAa:
Strain (amino acid)
255 273 300 304 333 408 453 468 594
158, 218, 231, and 235 (Leu-83) 58 (Leu-83) 227 and 233 (Trp-83) 202 (Val-87)
+
+
+
-
+
+
+
-
+
+ + +
+ + +
+ + +
+ -
+ + +
-
+ -
+
+ + +
a The presence or absence of the nucleotide change is indicated by + and -, respectively. Compared with the E. coli KL16 gyrA gene (26) there were C- T substitutions at positions 255, 273, 304, 408, and 453; T-+C substitutions at positions 300, 333, and 594; and a G-*C change at position 468.
is responsible for quinolone resistance in KL16 strain N-113 (Fig. 3) (25). Although genetic proof is awaited, these considerations identify the Asp-87-Val substitution as a strong candidate as a resistance mutation. Interestingly, examination of silent nucleotide changes in the gyrA genes revealed four different patterns of DNA polymorphism (Table 1). Four strains bearing the gyrase A Ser-83-*Leu mutation shared the same polymorphisms. Those gyrA genes encoding the Ser-83--Trp mutation shared another pattern of changes. It is conceivable that the mutations to Leu and Trp codons arose in the gyrA genes of distinct precursor strains. The main conclusion of this study is that mutation of Ser-83 in the gyrase A protein frequently confers clinical resistance to quinolones, suggesting that it has a key role in quinolone action. This residue and others involved in resistance are located in a compact domain of the gyrase A protein responsible for enzymatic DNA breakage and reunion (Fig. 1) (18). Serine-83 appears to fulfill an important function because it is conserved in the gyrase A proteins of E. coli, Bacillus subtilis, and Staphylococcus aureus, while the Klebsiella pneumoniae protein has an analogous threonine residue which, likewise, carries a small aliphatic side chain bearing a hydroxyl group (3, 11, 14, 21). It is not clear how substitution with Leu or Trp relieves quinolone inhibition of the gyrase-DNA complex (20). These changes introduce a bulkier hydrophobic side chain which could interfere with quinolone binding. Alternatively, the serine (threonine) OH group could participate in a key hydrogen-bonding or metal-liganding interaction essential for quinolone action. Resolution of these questions requires structural information on the gyrase complex and its mutants. We are especially grateful to Laura Piddock for generously providing clinical isolates and drug susceptibility data, Mike Cheetham for synthesizing oligonucleotides, and Maree Bagmara and Tim Rutherford for advice on PCR and access to equipment. This work was supported in part by the SERC. REFERENCES 1. Cullen, M. E., A. W. Wyke, R. Kuroda, and L. M. Fisher. 1989. Cloning and characterization of a DNA gyrase A gene from
Escherichia coli that confers clinical resistance to 4-quinolones.
Antimicrob. Agents Chemother. 33:886-894. 2. Cullen, M. E., A. W. Wyke, F. McEachern, C. A. Austin, and L. M. Fisher. 1989. Inhibition of DNA gyrase: bacterial sensitivity and clinical resistance to 4-quinolones. Curr. Top. Infect. Dis. Clin. Microbiol. 2:41-47. 3. Dimri, G. P., and H. K. Das. 1990. Cloning and sequence analysis of the gyrA gene of Klebsiella pneumoniae. Nucleic Acids Res. 18:151-156.
389
4. Drlica, K., and R. J. Franco. 1988. Inhibitors of DNA topoisomerases. Biochemistry 27:2253-2259. 5. Fisher, L. M., J. M. Lawrence, I. C. Josty, R. Hopewell, E. E. C. Margerrison, and M. E. Cullen. 1989. Ciprofloxacin and the fluoroquinolones. Am. J. Med. 87(Suppl. 5A):2S-8S. 6. Gellert, M., K. Mizuuchi, M. H. O'Dea, T. Itoh, and J.-I. Tomizawa. 1977. Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc. Natl. Acad. Sci. USA 74:4772-4776. 7. Gellert, M., K. Mizuuchi, M. H. O'Dea, and H. A. Nash. 1976. DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc. Natl. Acad. Sci. USA 73:3872-3876. 8. Goss, W. A., W. H. Deitz, and T. M. Cook. 1965. Mechanism of action of nalidixic acid on Escherichia coli. II. Inhibition of DNA synthesis. J. Bacteriol. 89:1068-1074. 9. Hane, M. W., and T. H. Wood. 1969. Escherichia coli K-12 mutants resistant to nalidixic acid: genetic mapping and dominance studies. J. Bacteriol. 99:238-241. 10. Higgins, N. P., C. L. Peebles, A. Sugino, and N. R. Cozzarelli. 1978. Purification of subunits of Escherichia coli DNA gyrase and reconstitution of enzyme activity. Proc. Natl. Acad. Sci. USA 75:1773-1777. 11. Hopewell, R., M. Oram, R. Briesewitz, and L. M. Fisher. 1990. DNA cloning and organization of the Staphylococcus aureus gyrA and gyrB genes: close homology between gyrase proteins and implications for 4-quinolone action and resistance. J. Bacteriol. 172:3481-3484. 12. Horowitz, D. S., and J. C. Wang. 1987. Mapping the active site tyrosine of Escherichia coli DNA gyrase. J. Biol. Chem. 262: 5339-5344. 12a.Lawrence, J. M., and L. M. Fisher. Personal communication. 13. Mizuuchi, K., M. H. O'Dea, and M. Gellert. 1978. DNA gyrase: subunit structure and ATPase activity of the purified enzyme. Proc. Natl. Acad. Sci. USA 75:5960-5963. 14. Moriya, S., N. Ogasawara, and H. Yoshikawa. 1985. Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. Nucleotide sequence of some 10,000 base pairs in the origin region. Nucleic Acids Res. 13:2251-2265. 15. Nakamura, S., M. Nakamura, T. Kojima, and H. Yoshida. 1989. gyrA and gyrB mutations in quinolone-resistant strains of Escherichia coli. Antimicrob. Agents Chemother. 33:254-255. 16. Piddock, L. 1990. Personal communication. 17. Pritchard, R. H., and I. B. Holland. 1985. Basic cloning techniques. Blackwell Scientific Publications, Ltd., Oxford. 18. Reece, R. J., and A. Maxwell. 1989. Tryptic fragments of the Escherichia coli DNA gyrase A protein. J. Biol. Chem. 264: 19648-19653. 19. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5436-5467. 20. Shen, L. L., L. A. Mitscher, P. N. Sharma, T. J. O'Donnell, D. W. T. Chu, C. S. Cooper, T. Rosen, and A. G. Pernet. 1989. Mechanisms of inhibition of DNA gyrase by quinolone antibacterials: a cooperative drug-DNA binding model. Biochemistry
28:3886-3894.
21. Swanberg, S. L., and J. C. Wang. 1987. Cloning and sequencing of the Escherichia coli gyrA gene coding for the A subunit of DNA gyrase. J. Mol. Biol. 197:729-736. 22. Wang, J. C. 1985. DNA topoisomerases. Annu. Rev. Biochem. 54:665-697. 23. Wolfson, J. S., and D. C. Hooper. 1985. The fluoroquinolones: structures, mechanisms of action and resistance, and spectra of activity in vitro. Antimicrob. Agents Chemother. 28:581-586. 24. Yamagishi, J.-I., H. Yoshida, M. Yamayoshi, and S. Nakamura. 1986. Nalidixic acid mutations of the gyrB gene of Escherichia coli. Mol. Gen. Genet. 204:367-373. 25. Yoshida, H., M. Bogaki, M. Nakamura, and S. Nakamura. 1990. Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli. Antimicrob. Agents Chemother. 34:1271-1272. 26. Yoshida, H., T. Kojima, J.-I. Yamagishi, and S. Nakamura. 1988. Quinolone-resistant mutations of the gyrA gene of Escherichia coli. Mol. Gen. Genet. 211:1-7.
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1991, p. 387-389
0066-4804/91/020387-03$02.00/0 Copyright © 1991, American Society for Microbiology
4-Quinolone Resistance Mutations in the DNA Gyrase of Escherichia coli Clinical Isolates Identified by Using the Polymerase Chain Reaction MARK ORAM AND L. MARK FISHER* Molecular Genetics Group, Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, London SW17 ORE, United Kingdom Received 19 June 1990/Accepted 30 November 1990
Seven nalidixic acid-resistant clinical isolates of Escherichia coli were shown to carry resistance mutations in their gyrase A proteins. Six had serine-83 to leucine or tryptophan changes; the seventh had an aspartate-87 to valine substitution. The frequent occurrence of a mutation at serine-83 implies a key role for this residue in quinolone action.
High-molecular-weight chromosomal DNA was prepared from each strain (17) and subjected to PCR by using two 20-mer oligonucleotide primers, 5'-TACACCGGTCAACAT TGAGG and 5'-TTAATGATTGCCGCCGTCGG, which were identical in sequence to nucleotide positions 24 to 43 or complementary in sequence to positions 652 to 671 of the E. coli K-12 gyrA gene, respectively (21) (Fig. 1). Reactions were performed in a DNA Thermal Cycler (Perkin ElmerCetus, Beaconsfield, Bucks, United Kingdom) in 10 mM Tris hydrochloride (pH 8.3)-50 mM KCl-1.5 mM MgCl2-0.01% gelatin containing 200 ,uM (each) deoxynucleoside triphosphate (Perkin Elmer-Cetus), 1 to 1.5 U of Taq DNA polymerase, 10 ng of each oligonucleotide primer, and 10 ng of bacterial DNA (final volume, 50 ,ul). Thirty cycles were used for each reaction, with the following temperature profiles: 92°C, 25 s; 64°C, 1 min; 74°C, 2.5 min. DNA analysis by agarose gel electrophoresis revealed in each case amplification of the expected 648-bp fragment. PCR products were precipitated with ethanol and digested with Sacl and SmaI, generating 582-bp gyrA fragments (encoding gyrase A amino acid residues 19 to 213) which were ligated into Sacl-SmaI-cut M13mpl8 and M13mpl9 replicative form DNA (Pharmacia, Milton Keynes, Bucks, United Kingdom). Transformation of DNA into E. coli XL1 recA cells, selection of colorless recombinant plaques, and purification of single-stranded M13 DNA were done as recommended by Amersham International, Little Chalfont, Bucks, United Kingdom. DNA sequencing of complementary strands in two independent clones was by the chain
Antimicrobial 4-quinolones exert their potent antagonistic effects on bacterial DNA replication by inhibiting DNA gyrase, an A2B2 tetrameric enzyme that catalyzes ATPdependent DNA supercoiling (4, 6-8, 10, 13, 23). Gyrase promotes the negative supercoiling of DNA and the formation and resolution of interlocked DNA circles and knotted DNA by passing a DNA duplex through a transient enzymebridged double-strand break in DNA (22). Studies with purified Escherichia coli DNA gyrase have shown that quinolones interfere with DNA breakage and reunion mediated by Tyr-122 of the 875-residue gyrase A subunits (6, 12). Genetic studies of 4-quinolone resistance in laboratory strains of E. coli K-12 have implicated a number of chromosomal loci in low-level resistance, whereas high-level resistance invariably maps at gyrA, which encodes the gyrase A protein (1, 6, 9, 15, 24-26). The following quinolone resistance mutations in the gyrase A protein have been identified from sequence analysis of quinolone-resistant gyrA genes in E. coli KL16: Ser-83--Leu, Ser-83--*Trp, Asp-87--Asn, Gly-81--Cys, Ala-84--*Pro, Ala-67-*Ser, and Gln-106-->His (25). In the only molecular study thus far of a resistant clinical isolate, we showed that mutation of Ser-83 to Trp in the gyrase A protein was responsible for quinolone resistance in a uropathogenic E. coli strain, 227, which was obtained from a patient treated with enoxacin (1). To provide much needed information on the molecular basis of resistance in clinical isolates, we describe here the analysis of an additional seven E. coli strains. Uropathogenic clinical isolates of E. coli resistant to quinolones were obtained by Laura Piddock from several independent sources in Birmingham, England, between June 1984 and April 1985. Strains 58, 158, 202, 218, 227, 231, 233, and 235 were selected for study on the basis of their highlevel resistance to nalidixic acid (MICs, 31 to >1,000 ,g/ml) (2) (see Fig. 3). Several of the strains, 58, 202, and 227, were shown to contain a DNA gyrase A subunit that reconstituted a quinolone-resistant gyrase activity (2). Using a restriction fragment length polymorphism analysis, we showed that all strains except strain 202 carried an amino acid substitution of unknown identity at Asp-82 or Ser-83 of the gyrase A protein (5). We applied the polymerase chain reaction (PCR) in concert with rapid cloning-DNA sequence analysis to identify these gyrA mutations at the nucleotide level. *
Sac I
*v
Sma I 67 83
106
Tyr122
a
4.
gyrA 100 bp
FIG. 1. PCR amplification of a 5' gyrA gene region from clinical isolates of E. coli. The diagram shows the 5' end of the E. coli gyrA gene (heavy line). Arrows denote two synthetic 20mer oligonucleotides positioned next to their homologous sequences on complementary strands of the gyrA gene. Triangles indicate unique sites for Sacd and SmaI located at nucleotide positions 54 and 636, respectively. Tyr-122 denotes the coding sequence for the catalytic tyrosine; locations of the coding sequences for residues whose mutations confer quinolone resistance are also shown.
Corresponding author. 387
388
ANTIMICROB. AGENTS CHEMOTHER.
NOTES
FIG. 2. Mutations in the gyrA gene from clinical isolates identified on DNA sequencing gels. PCR products were digested with Sacl and SmaI, cloned into M13mpl8, and sequenced by the dideoxy chain-termination method. Gels show a region encompassing coding sequence for residues 82 and 83. The bracket defines a Hinfl site (GACTC) at this position in the strain 202 gyrA sequence. Asterisks indicate nucleotide changes from the wild-type sequence.
termination technique (19) with [a-35S]dATP and T7 DNA polymerase by the rapid Amersham Multiwell sequencing method (Fig. 2). Although DNA sequences of cloned 582-bp gyrA fragments exhibited several nucleotide differences compared with that of the quinolone-susceptible E. coli K-12 gyrA gene, only a single coding change was present in each case (Fig. 3). The gyrA genes from strains 58, 158, 218, 231, and 235 each exhibited a C-+T transition at nucleotide STRAIN
MIC (Ug/mi) NAL
KL-16
position 248 leading to a Ser-83--Leu substitution at the protein level; strain 233, like strain 227, had a C-+G transversion at this nucleotide position, generating a Ser-83-+Trp substitution. In contrast, isolate 202 retained a wild-type C base at position 248 (i.e., a Ser-83 in the gyrase A protein) but, instead, had an A--T transversion at position 260, producing an Asp-87--->Val substitution (Fig. 2 and 3). The nature of these DNA sequence changes is consistent with our previous restriction fragment length polymorphism results (Fig. 3) (5). Mutation of Ser-83 to either Leu or Trp has been shown to confer quinolone resistance in nonclinical isolates of E. coli (25, 26), e.g., N-51 and P-18 (Fig. 3). We found the same mutations in seven nalidixic acid-resistant clinical isolates (MICs, 125 to >1,000 ,ug/ml), including the previously described strain 227 (1). We cannot exclude the possibility that other mutations contribute to resistance in these clinical isolates. Indeed, among the Ser-83--Leu gyrA mutants, strain 218 exhibited higher resistance to nalidixic acid and other quinolones, including ciprofloxacin, (Fig. 3) and grew less well than the other strains did, suggesting the presence of additional mutations. However, we note that E. coli KL16 strains N-51 and P-18 had levels of quinolone resistance broadly similar to those of our clinical isolates (Fig. 3) (1, 25, 26). Thus, the Ser-83 to Leu or Trp gyrase A mutations that we have identified may, in large part, be responsible for the resistance of the clinical strains. Strain 202 carried an Asp-87--+Val substitution in its gyrase A protein which maps in the same region of the polypeptide chain as the other known resistance mutations, i.e., adjacent to catalytic Tyr-122. For this strain the quinolone MICs were the lowest of those for the clinical isolates examined here (Fig. 3), and the gyrase A protein partially purified from the strain was less resistant to quinolone inhibition than was the Ser-83--Trp protein (2). It is significant that mutation of Asp-87 to Asn in the gyrase A protein
3
CIP 0.0125
58 158 218 231 235
500 125 >1000 250 500
0.5 0.25 1 0.25 0.25
227 233
500 250
0.25 0.25
202
31
0.06
GYR A GENE 87 83 .Gly Asp SER Ala Val Tyr Asp Thr Ile .GGT GAC TCG GCG GTC TAT GAC ACG ATC
1*
T
T*
T*
T* T* (Leu)
G*
G*
T T T T T
T T
(Trp)
N-51 P-18 N-113
400 400 200
0.39 0.39 0.2
C Ser
11
T
T* (Val)
A*
(Asn) FIG. 3. Nucleotide changes in E. coli gyrA genes and inferred amino acid substitutions in the DNA gyrase A proteins of clinical isolates. Asterisks indicate nucleotide differences with the E. coli KL16 gyrA sequence (top) (26) that result in amino acid changes (parentheses). Silent base changes are also indicated (the KL16 gyrA sequence [26] itself exhibits a silent change at nucleotide position 267 compared with that of another K-12 strain [21]). Underlining denotes a Hinfl site, and numbers identify the positions of the deduced amino acid residues. The mutation in strain 227 conferring quinolone resistance has been determined previously (1). Strains N-51, N-113, and P-18 are nalidixic acidand pipemidic acid-resistant KL16 strains whose quinolone resistance mutation has been mapped as shown previously (25). The MICs of nalidixic acid (NAL) and ciprofloxacin (CIP) have been described previously for clinical isolates (2) and KL16 strains (26). The MICs of nalidixic acid determined here (12a) confirmed the results of previous work (2), except for the somewhat higher values for strains 58, 231, and 233. Both nalidixic acid and ciprofloxacin MICs are in broad agreement with those determined independently by L. Piddock (16).
VOL. 35,
NOTES
1991
TABLE 1.
Patterns of silent nucleotide changes seen in gyrA genes from clinical isolates Nucleotide change at the following nucleotide
positions in gyrAa:
Strain (amino acid)
255 273 300 304 333 408 453 468 594
158, 218, 231, and 235 (Leu-83) 58 (Leu-83) 227 and 233 (Trp-83) 202 (Val-87)
+
+
+
-
+
+
+
-
+
+ + +
+ + +
+ + +
+ -
+ + +
-
+ -
+
+ + +
a The presence or absence of the nucleotide change is indicated by + and -, respectively. Compared with the E. coli KL16 gyrA gene (26) there were C- T substitutions at positions 255, 273, 304, 408, and 453; T-+C substitutions at positions 300, 333, and 594; and a G-*C change at position 468.
is responsible for quinolone resistance in KL16 strain N-113 (Fig. 3) (25). Although genetic proof is awaited, these considerations identify the Asp-87-Val substitution as a strong candidate as a resistance mutation. Interestingly, examination of silent nucleotide changes in the gyrA genes revealed four different patterns of DNA polymorphism (Table 1). Four strains bearing the gyrase A Ser-83-*Leu mutation shared the same polymorphisms. Those gyrA genes encoding the Ser-83--Trp mutation shared another pattern of changes. It is conceivable that the mutations to Leu and Trp codons arose in the gyrA genes of distinct precursor strains. The main conclusion of this study is that mutation of Ser-83 in the gyrase A protein frequently confers clinical resistance to quinolones, suggesting that it has a key role in quinolone action. This residue and others involved in resistance are located in a compact domain of the gyrase A protein responsible for enzymatic DNA breakage and reunion (Fig. 1) (18). Serine-83 appears to fulfill an important function because it is conserved in the gyrase A proteins of E. coli, Bacillus subtilis, and Staphylococcus aureus, while the Klebsiella pneumoniae protein has an analogous threonine residue which, likewise, carries a small aliphatic side chain bearing a hydroxyl group (3, 11, 14, 21). It is not clear how substitution with Leu or Trp relieves quinolone inhibition of the gyrase-DNA complex (20). These changes introduce a bulkier hydrophobic side chain which could interfere with quinolone binding. Alternatively, the serine (threonine) OH group could participate in a key hydrogen-bonding or metal-liganding interaction essential for quinolone action. Resolution of these questions requires structural information on the gyrase complex and its mutants. We are especially grateful to Laura Piddock for generously providing clinical isolates and drug susceptibility data, Mike Cheetham for synthesizing oligonucleotides, and Maree Bagmara and Tim Rutherford for advice on PCR and access to equipment. This work was supported in part by the SERC. REFERENCES 1. Cullen, M. E., A. W. Wyke, R. Kuroda, and L. M. Fisher. 1989. Cloning and characterization of a DNA gyrase A gene from
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