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. 1988 Apr;32(4):477–484. doi: 10.1128/aac.32.4.477

Molecular cloning and mechanism of trimethoprim resistance in Haemophilus influenzae.

R de Groot 1, J Campos 1, S L Moseley 1, A L Smith 1
PMCID: PMC172205  PMID: 2837138

Abstract

We studied 10 trimethoprim-resistant (Tmpr) Haemophilus influenzae isolates for which agar dilution MICs were 10 to greater than 200 micrograms/ml. Trimethoprim resistance was transferred from two Tmpr H. influenzae isolates to a Tmps strain by conjugation or transformation. Wild-type Tmpr strains and Tmpr transcipients did not contain detectable plasmid DNA. The trimethoprim resistance gene was cloned into a cosmid vector, and recombinant plasmids were transduced into Escherichia coli. A 0.50-kilobase intragenic probe derived from a 12.9-kilobase fragment which encoded trimethoprim resistance hybridized with whole-cell DNA from Tmps and Tmpr strains. Southern blot analysis of restricted DNA from isogenic Tmps and Tmpr H. influenzae indicated that acquisition of trimethoprim resistance involved a rearrangement or change in nucleotide sequence. Hybridization was not seen with DNA derived from Tmpr E. coli containing dihydrofolate reductase I, II, and III genes or with Tmpr Neisseria meningitidis, Neisseria gonorrhoeae, and Pseudomonas cepacia. Southern hybridization with 12 multiply resistant encapsulated H. influenzae strains confirmed that the trimethoprim resistance gene was chromosomally mediated. Dihydrofolate reductase activity was significantly greater in cell sonicate supernatants of Tmpr strains in comparison with isogenic Tmps recipients. Differences were not found in the trimethoprim inhibition profile of dihydrofolate reductase activity in Tmps and Tmpr strains. We conclude that the mechanism of trimethoprim resistance in H. influenzae is overproduction of chromosomally located dihydrofolate reductase.

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Selected References

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  1. Albritton W. L., Setlow J. K., Slaney L. Transfer of Haemophilus influenzae chromosomal genes by cell-to-cell contact. J Bacteriol. 1982 Dec;152(3):1066–1070. doi: 10.1128/jb.152.3.1066-1070.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amyes S. G., Smith J. T. R-factor trimethoprim resistance mechanism: an insusceptible target site. Biochem Biophys Res Commun. 1974 May 20;58(2):412–418. doi: 10.1016/0006-291x(74)90380-5. [DOI] [PubMed] [Google Scholar]
  3. Archer G. L., Coughter J. P., Johnston J. L. Plasmid-encoded trimethoprim resistance in staphylococci. Antimicrob Agents Chemother. 1986 May;29(5):733–740. doi: 10.1128/aac.29.5.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Birnboim H. C. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. doi: 10.1016/0076-6879(83)00059-2. [DOI] [PubMed] [Google Scholar]
  5. Bolivar F., Rodriguez R. L., Greene P. J., Betlach M. C., Heyneker H. L., Boyer H. W., Crosa J. H., Falkow S. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene. 1977;2(2):95–113. [PubMed] [Google Scholar]
  6. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  7. Braveny I., Machka K. Multiple resistant Haemophilus influenzae and parainfluenzae in West Germany. Lancet. 1980 Oct 4;2(8197):752–753. doi: 10.1016/s0140-6736(80)91980-7. [DOI] [PubMed] [Google Scholar]
  8. Burchall J. J. Comparative biochemistry of dihydrofolate reductase. Ann N Y Acad Sci. 1971 Nov 30;186:143–152. doi: 10.1111/j.1749-6632.1971.tb46965.x. [DOI] [PubMed] [Google Scholar]
  9. Campos J., Garcia-Tornel S., Sanfeliu I. Susceptibility studies of multiply resistant Haemophilus influenzae isolated from pediatric patients and contacts. Antimicrob Agents Chemother. 1984 Jun;25(6):706–709. doi: 10.1128/aac.25.6.706. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Campos J., García-Tornel S., Gairí J. M., Fábregues I. Multiply resistant Haemophilus influenzae type b causing meningitis: comparative clinical and laboratory study. J Pediatr. 1986 Jun;108(6):897–902. doi: 10.1016/s0022-3476(86)80923-4. [DOI] [PubMed] [Google Scholar]
  11. Catlin B. W., Bendler J. W., 3rd, Goodgal S. H. The type b capsulation locus of Haemophilus influenzae: map location and size. J Gen Microbiol. 1972 May;70(3):411–422. doi: 10.1099/00221287-70-3-411. [DOI] [PubMed] [Google Scholar]
  12. Catlin B. W. Nutritional profiles of Neisseria gonorrhoeae, Neisseria meningitidis, and Neisseria lactamica in chemically defined media and the use of growth requirements for gonococcal typing. J Infect Dis. 1973 Aug;128(2):178–194. doi: 10.1093/infdis/128.2.178. [DOI] [PubMed] [Google Scholar]
  13. De Graaff J., Elwell L. P., Falkow S. Molecular nature of two beta-lactamase-specifying plasmids isolated from Haemophilus influenzae type b. J Bacteriol. 1976 Apr;126(1):439–446. doi: 10.1128/jb.126.1.439-446.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Doern G. V., Chapin K. C. Susceptibility of Haemophilus influenzae to amoxicillin/clavulanic acid, erythromycin, cefaclor, and trimethoprim/sulfamethoxazole. Diagn Microbiol Infect Dis. 1986 Jan;4(1):37–41. doi: 10.1016/0732-8893(86)90054-4. [DOI] [PubMed] [Google Scholar]
  15. Flensburg J., Sköld O. Massive overproduction of dihydrofolate reductase in bacteria as a response to the use of trimethoprim. Eur J Biochem. 1987 Feb 2;162(3):473–476. doi: 10.1111/j.1432-1033.1987.tb10664.x. [DOI] [PubMed] [Google Scholar]
  16. Fling M. E., Elwell L. P. Protein expression in Escherichia coli minicells containing recombinant plasmids specifying trimethoprim-resistant dihydrofolate reductases. J Bacteriol. 1980 Feb;141(2):779–785. doi: 10.1128/jb.141.2.779-785.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Fling M. E., Richards C. The nucleotide sequence of the trimethoprim-resistant dihydrofolate reductase gene harbored by Tn7. Nucleic Acids Res. 1983 Aug 11;11(15):5147–5158. doi: 10.1093/nar/11.15.5147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fling M. E., Walton L., Elwell L. P. Monitoring of plasmid-encoded, trimethoprim-resistant dihydrofolate reductase genes: detection of a new resistant enzyme. Antimicrob Agents Chemother. 1982 Nov;22(5):882–888. doi: 10.1128/aac.22.5.882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Grey D., Hamilton-Miller J. M., Brumfitt W. Incidence and mechanisms of resistance to trimethoprim in clinically isolated gram-negative bacteria. Chemotherapy. 1979;25(3):147–156. doi: 10.1159/000237834. [DOI] [PubMed] [Google Scholar]
  20. Gutmann L., Williamson R., Moreau N., Kitzis M. D., Collatz E., Acar J. F., Goldstein F. W. Cross-resistance to nalidixic acid, trimethoprim, and chloramphenicol associated with alterations in outer membrane proteins of Klebsiella, Enterobacter, and Serratia. J Infect Dis. 1985 Mar;151(3):501–507. doi: 10.1093/infdis/151.3.501. [DOI] [PubMed] [Google Scholar]
  21. Hansen J. B., Olsen R. H. Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5. J Bacteriol. 1978 Jul;135(1):227–238. doi: 10.1128/jb.135.1.227-238.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Herriott R. M., Meyer E. M., Vogt M. Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J Bacteriol. 1970 Feb;101(2):517–524. doi: 10.1128/jb.101.2.517-524.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  24. Hull R. A., Gill R. E., Hsu P., Minshew B. H., Falkow S. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect Immun. 1981 Sep;33(3):933–938. doi: 10.1128/iai.33.3.933-938.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kao J. C., Perry K. L., Kado C. I. Indoleacetic acid complementation and its relation to host range specifying genes on the Ti plasmid of Agrobacterium tumefaciens. Mol Gen Genet. 1982;188(3):425–432. doi: 10.1007/BF00330044. [DOI] [PubMed] [Google Scholar]
  27. Koch A. E., Burchall J. J. Reversal of the antimicrobial activity of trimethoprim by thymidine in commercially prepared media. Appl Microbiol. 1971 Nov;22(5):812–817. doi: 10.1128/am.22.5.812-817.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  29. Mariani B. D., Schimke R. T. Gene amplification in a single cell cycle in Chinese hamster ovary cells. J Biol Chem. 1984 Feb 10;259(3):1901–1910. [PubMed] [Google Scholar]
  30. Maskell R., Okubadejo O. A., Payne R. H., Pead L. Human infections with thymine-requiring bacteria. J Med Microbiol. 1978 Feb;11(1):33–45. doi: 10.1099/00222615-11-1-33. [DOI] [PubMed] [Google Scholar]
  31. McDade R. L., Jr, Johnston K. H. Characterization of serologically dominant outer membrane proteins of Neisseria gonorrhoeae. J Bacteriol. 1980 Mar;141(3):1183–1191. doi: 10.1128/jb.141.3.1183-1191.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mendelman P. M., Chaffin D. O., Stull T. L., Rubens C. E., Mack K. D., Smith A. L. Characterization of non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae. Antimicrob Agents Chemother. 1984 Aug;26(2):235–244. doi: 10.1128/aac.26.2.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meyers J. A., Sanchez D., Elwell L. P., Falkow S. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J Bacteriol. 1976 Sep;127(3):1529–1537. doi: 10.1128/jb.127.3.1529-1537.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Michalka J., Goodgal S. H. Genetic and physical map of the chromosome of Hemophilus influenzae. J Mol Biol. 1969 Oct 28;45(2):407–421. doi: 10.1016/0022-2836(69)90115-6. [DOI] [PubMed] [Google Scholar]
  36. OSBORN M. J., HUENNEKENS F. M. Enzymatic reduction of dihydrofolic acid. J Biol Chem. 1958 Oct;233(4):969–974. [PubMed] [Google Scholar]
  37. Pattishall K. H., Acar J., Burchall J. J., Goldstein F. W., Harvey R. J. Two distinct types of trimethoprim-resistant dihydrofolate reductase specified by R-plasmids of different compatibility groups. J Biol Chem. 1977 Apr 10;252(7):2319–2323. [PubMed] [Google Scholar]
  38. Portnoy D. A., Moseley S. L., Falkow S. Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis. Infect Immun. 1981 Feb;31(2):775–782. doi: 10.1128/iai.31.2.775-782.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rigby P. W., Burleigh B. D., Jr, Hartley B. S. Gene duplication in experimental enzyme evolution. Nature. 1974 Sep 20;251(5472):200–204. doi: 10.1038/251200a0. [DOI] [PubMed] [Google Scholar]
  40. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  41. Roberts M. C., Swenson C. D., Owens L. M., Smith A. L. Characterization of chloramphenicol-resistant Haemophilus influenzae. Antimicrob Agents Chemother. 1980 Oct;18(4):610–615. doi: 10.1128/aac.18.4.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Roberts M., Stull T. L., Smith A. L. Comparative virulence of Haemophilus influenzae with a type b or type d capsule. Infect Immun. 1981 May;32(2):518–524. doi: 10.1128/iai.32.2.518-524.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Setlow J. K., Boling M. E., Beattie K. L., Kimball R. F. A complex of recombination and repair genes in Haemophilus influenzae. J Mol Biol. 1972 Jul 21;68(2):361–378. doi: 10.1016/0022-2836(72)90218-5. [DOI] [PubMed] [Google Scholar]
  44. Shurin P. A., Pelton S. I., Donner A., Finkelstein J., Klein J. O. Trimethoprim-sulfamethoxazole compared with ampicillin in the treatment of acute otitis media. J Pediatr. 1980 Jun;96(6):1081–1087. doi: 10.1016/s0022-3476(80)80649-4. [DOI] [PubMed] [Google Scholar]
  45. Smith A. L., Smith D. H., Averill D. R., Jr, Marino J., Moxon E. R. Production of Haemophilus influenzae b meningitis in infant rats by intraperitoneal inoculation. Infect Immun. 1973 Aug;8(2):278–290. doi: 10.1128/iai.8.2.278-290.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Smith D. R., Rood J. I., Bird P. I., Sneddon M. K., Calvo J. M., Morrison J. F. Amplification and modification of dihydrofolate reductase in Escherichia coli. Nucleotide sequence of fol genes from mutationally altered plasmids. J Biol Chem. 1982 Aug 10;257(15):9043–9048. [PubMed] [Google Scholar]
  47. So M., Dallas W. S., Falkow S. Characterization of an Escherichia coli plasmid encoding for synthesis of heat-labile toxin: molecular cloning of the toxin determinant. Infect Immun. 1978 Aug;21(2):405–411. doi: 10.1128/iai.21.2.405-411.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  49. Stone D., Smith S. L. The amino acid sequence of the trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67. J Biol Chem. 1979 Nov 10;254(21):10857–10861. [PubMed] [Google Scholar]
  50. Sundström L., Vinayagamoorthy T., Sköld O. Novel type of plasmid-borne resistance to trimethoprim. Antimicrob Agents Chemother. 1987 Jan;31(1):60–66. doi: 10.1128/aac.31.1.60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Then R. L., Angehrn P. Low trimethoprim susceptibility of anaerobic bacteria due to insensitive dihydrofolate reductases. Antimicrob Agents Chemother. 1979 Jan;15(1):1–6. doi: 10.1128/aac.15.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Then R. L. Mechanisms of resistance to trimethoprim, the sulfonamides, and trimethoprim-sulfamethoxazole. Rev Infect Dis. 1982 Mar-Apr;4(2):261–269. doi: 10.1093/clinids/4.2.261. [DOI] [PubMed] [Google Scholar]
  53. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  55. Zackrisson G., Brorson J. E. Antibiotic sensitivity of Haemophilus influenzae strains including three recent chloramphenicol-resistant isolates. Acta Pathol Microbiol Scand B. 1980 Aug;88(4):193–198. doi: 10.1111/j.1699-0463.1980.tb02628.x. [DOI] [PubMed] [Google Scholar]
  56. Zolg J. W., Hänggi U. J. Characterization of a R plasmid-associated, trimethoprim-resistant dihydrofolate reductase and determination of the nucleotide sequence of the reductase gene. Nucleic Acids Res. 1981 Feb 11;9(3):697–710. doi: 10.1093/nar/9.3.697. [DOI] [PMC free article] [PubMed] [Google Scholar]

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