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Volume 6, Issue 11

Exploration into the origins and mobilization of di-hydrofolate reductase genes and the emergence of clinical resistance to trimethoprim Open Access

Abstract

Trimethoprim is a synthetic antibacterial agent that targets folate biosynthesis by competitively binding to the di-hydrofolate reductase enzyme (DHFR). Trimethoprim is often administered synergistically with sulfonamide, another chemotherapeutic agent targeting the di-hydropteroate synthase (DHPS) enzyme in the same pathway. Clinical resistance to both drugs is widespread and mediated by enzyme variants capable of performing their biological function without binding to these drugs. These mutant enzymes were assumed to have arisen after the discovery of these synthetic drugs, but recent work has shown that genes conferring resistance to sulfonamide were present in the bacterial pangenome millions of years ago. Here, we apply phylogenetics and comparative genomics methods to study the largest family of mobile trimethoprim-resistance genes (). We show that most of the genes identified to date map to two large clades that likely arose from independent mobilization events. In contrast to sulfonamide resistance () genes, we find evidence of recurrent mobilization in genes. Phylogenetic evidence allows us to identify novel genes in the emerging pathogen , and we confirm their resistance phenotype . We also identify a cluster of homologues in cryptic plasmid and phage genomes, but we show that these enzymes do not confer resistance to trimethoprim. Our methods also allow us to pinpoint the chromosomal origin of previously reported genes, and we show that many of these ancient chromosomal genes also confer resistance to trimethoprim. Our work reveals that trimethoprim resistance predated the clinical use of this chemotherapeutic agent, but that novel mutations have likely also arisen and become mobilized following its widespread use within and outside the clinic. Hence, this work confirms that resistance to novel drugs may already be present in the bacterial pangenome, and stresses the importance of rapid mobilization as a fundamental element in the emergence and global spread of resistance determinants.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antibiotics , chemotherapeutic agent , evolution , phylogenetics , resistance , sulfonamides and trimethoprim
Funding
This study was supported by the:
  • Ministerio de Economía y Competitividad (Award BIO2016-77011-R)
    • Principle Award Recipient: Jordi Barbé
© 2020 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2020-09-24
2024-04-19
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References

  1. Letunic I, Bork P. Interactive tree of life (iTOL) v4: recent updates and new developments. Nucleic Acids Res 2019; 47:W256–W259 [View Article]
     [Google Scholar]
  2. Carlet J, Rambaud C, Pulcini C. Save antibiotics: a call for action of the World Alliance Against Antibiotic Resistance (WAAAR). BMC Infect Dis 2014; 14:436 [View Article]
     [Google Scholar]
  3. Rossolini GM, Arena F, Pecile P, Pollini S. Update on the antibiotic resistance crisis. Curr Opin Pharmacol 2014; 18:56–60 [View Article]
     [Google Scholar]
  4. Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010; 74:417–433 [View Article][PubMed]
     [Google Scholar]
  5. Baym M, Lieberman TD, Kelsic ED, Chait R, Gross R et al. Spatiotemporal microbial evolution on antibiotic landscapes. Science 2016; 353:1147–1151 [View Article]
     [Google Scholar]
  6. Hegreness M, Shoresh N, Damian D, Hartl D, Kishony R. Accelerated evolution of resistance in multidrug environments. Proc Natl Acad Sci USA 2008; 105:13977–13981 [View Article]
     [Google Scholar]
  7. Aminov RI, Mackie RI. Evolution and ecology of antibiotic resistance genes. FEMS Microbiol Lett 2007; 271:147–161 [View Article]
     [Google Scholar]
  8. Sengupta S, Chattopadhyay MK, Grossart H-P. The multifaceted roles of antibiotics and antibiotic resistance in nature. Front Microbiol 2013; 4:47 [View Article][PubMed]
     [Google Scholar]
  9. Kmeid JG, Youssef MM, Kanafani ZA, Kanj SS. Combination therapy for Gram-negative bacteria: what is the evidence?. Expert Rev Anti Infect Ther 2013; 11:1355–1362 [View Article]
     [Google Scholar]
  10. Williams KJ. The introduction of ‘chemotherapy’ using arsphenamine – the first magic bullet. J R Soc Med 2009; 102:343–348 [View Article]
     [Google Scholar]
  11. Aminov RI. A brief history of the antibiotic era: lessons learned and challenges for the future. Front Microbiol 2010; 1:134 [View Article][PubMed]
     [Google Scholar]
  12. Masters PA, O'Bryan TA, Zurlo J, Miller DQ, Joshi N. Trimethoprim-sulfamethoxazole revisited. Arch Intern Med 2003; 163:402–410 [View Article]
     [Google Scholar]
  13. Sköld O. Resistance to trimethoprim and sulfonamides. Vet Res 2001; 32:261–273 [View Article][PubMed]
     [Google Scholar]
  14. Quinlivan EP, McPartlin J, Weir DG, Scott J. Mechanism of the antimicrobial drug trimethoprim revisited. FASEB J 2000; 14:2519–2524 [View Article]
     [Google Scholar]
  15. Hitchings GH. Mechanism of action of trimethoprim-sulfamethoxazole-I. J Infect Dis 1973; 128:S433–S436 [View Article]
     [Google Scholar]
  16. Landy M, Larkum NW, Oswald EJ, Streightoff F. Increased synthesis of p-aminobenzoic acid associated with the development of sulfonamide resistance in Staphylococcus aureus . Science 1943; 97:265–267 [View Article]
     [Google Scholar]
  17. Huovinen P, Sundström L, Swedberg G, Sköld O. Trimethoprim and sulfonamide resistance. Antimicrob Agents Chemother 1995; 39:279–289 [View Article]
     [Google Scholar]
  18. Flensburg J, Sköld O. Massive overproduction of dihydrofolate reductase in bacteria as a response to the use of trimethoprim. Eur J Biochem 1987; 162:473–476 [View Article]
     [Google Scholar]
  19. Shin HW, Lim J, Kim S, Kim J, Kwon GC et al. Characterization of trimethoprim-sulfamethoxazole resistance genes and their relatedness to class 1 integron and insertion sequence common region in Gram-negative bacilli. J Microbiol Biotechnol 2015; 25:137–142 [View Article]
     [Google Scholar]
  20. Rådström P, Swedberg G, Sköld O. Genetic analyses of sulfonamide resistance and its dissemination in gram-negative bacteria illustrate new aspects of R plasmid evolution. Antimicrob Agents Chemother 1991; 35:1840–1848 [View Article]
     [Google Scholar]
  21. Perreten V, Boerlin P. A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland. Antimicrob Agents Chemother 2003; 47:1169–1172 [View Article][PubMed]
     [Google Scholar]
  22. Tagg KA, Watkins LF, Moore MD, Bennett C, Joung YJ et al. Novel trimethoprim resistance gene dfrA34 identified in Salmonella Heidelberg in the USA. J Antimicrob Chemother 2019; 74:38–41
     [Google Scholar]
  23. White PA, Rawlinson WD. Current status of the aadA and dfr gene cassette families. J Antimicrob Chemother 2001; 47:495–496 [View Article]
     [Google Scholar]
  24. Toulouse JL, Edens TJ, Alejaldre L, Manges AR, Pelletier JN. Integron-associated DfrB4, a previously uncharacterized member of the trimethoprim-resistant dihydrofolate reductase B family, is a clinically identified emergent source of antibiotic resistance. Antimicrob Agents Chemother 2017; 61:e02665-16 [View Article][PubMed]
     [Google Scholar]
  25. Sánchez-Osuna M, Cortés P, Barbé J, Erill I. Origin of the mobile di-hydro-pteroate synthase gene determining sulfonamide resistance in clinical isolates. Front Microbiol 2018; 9:3332 [View Article][PubMed]
     [Google Scholar]
  26. Eddy SR. Accelerated profile HMM searches. PLoS Comput Biol 2011; 7:e1002195 [View Article]
     [Google Scholar]
  27. Taly JF, Magis C, Bussotti G, Chang JM, Di Tommaso P et al. Using the T-Coffee package to build multiple sequence alignments of protein, RNA, DNA sequences and 3D structures. Nat Protoc 2011; 6:1669–1682 [View Article]
     [Google Scholar]
  28. O'Leary NA, Wright MW, Brister JR, Ciufo S, Haddad D et al. Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation. Nucleic Acids Res 2016; 44:D733–D745 [View Article]
     [Google Scholar]
  29. Benson DA, Cavanaugh M, Clark K, Karsch-Mizrachi I, Lipman DJ et al. GenBank. Nucleic Acids Res 2017; 45:D37–D42 [View Article]
     [Google Scholar]
  30. Holm L, Sander C. Removing near-neighbour redundancy from large protein sequence collections. Bioinformatics 1998; 14:423–429 [View Article]
     [Google Scholar]
  31. Altschul S et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997; 25:3389–3402 [View Article]
     [Google Scholar]
  32. Kaushik S, Mutt E, Chellappan A, Sankaran S, Srinivasan N et al. Improved detection of remote homologues using cascade PSI-BLAST: influence of neighbouring protein families on sequence coverage. PLoS One 8:e56449 [View Article]
     [Google Scholar]
  33. Brum JR, Ignacio-Espinoza JC, Roux S, Doulcier G, Acinas SG et al. Patterns and ecological drivers of ocean viral communities. Science 2015; 348:1261498 [View Article]
     [Google Scholar]
  34. Chen Q, Zobel J, Verspoor K. Benchmarks for measurement of duplicate detection methods in nucleotide databases. Database 2017; 2017:baw164 [View Article][PubMed]
     [Google Scholar]
  35. Wallace IM, O'Sullivan O, Higgins DG, Notredame C. M-Coffee: combining multiple sequence alignment methods with T-Coffee. Nucleic Acids Res 2006; 34:1692–1699 [View Article][PubMed]
     [Google Scholar]
  36. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 2000; 17:540–552 [View Article]
     [Google Scholar]
  37. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 2003; 19:1572–1574 [View Article]
     [Google Scholar]
  38. Erill I. Dispersal and regulation of an adaptive mutagenesis cassette in the bacteria domain. Nucleic Acids Res 2006; 34:66–77 [View Article]
     [Google Scholar]
  39. Rambaut A, Drummond AJ, Xie D, Baele G, Suchard MA. Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Syst Biol 2018; 67:901–904 [View Article]
     [Google Scholar]
  40. Yu G, Smith DK, Zhu H, Guan Y, Lam TT‐Y. ggtree: an r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods Ecol Evol 2017; 8:28–36 [View Article]
     [Google Scholar]
  41. Pagel M, Meade A, Barker D. Bayesian estimation of ancestral character states on phylogenies. Syst Biol 2004; 53:673–684 [View Article]
     [Google Scholar]
  42. Mayola A, Irazoki O, Martínez IA, Petrov D, Menolascina F et al. RecA protein plays a role in the chemotactic response and chemoreceptor clustering of Salmonella enterica . PLoS One 2014; 9:e105578 [View Article]
     [Google Scholar]
  43. Clinical and Laboratory Standards Institute Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, Approved Standard, 6th edn. Wayne, PA: Clinical and Laboratory Standards Institute; 2003
     [Google Scholar]
  44. Fling ME, Richards C. The nucleotide sequence of the trimethoprim-resistant dihydrofolate reductase gene harbored by Tn7. Nucleic Acids Res 1983; 11:5147–5158 [View Article]
     [Google Scholar]
  45. Smith DR, Calvo JM. Nucleotide sequence of the E. coli gene coding for dihydrofolate reductase. Nucleic Acids Res 1980; 8:2255–2274 [View Article]
     [Google Scholar]
  46. Faltyn M, Alcock B, McArthur A. Evolution and nomenclature of the trimethoprim resistant dihydrofolate (dfr) reductases. Preprints 20192019050137
     [Google Scholar]
  47. van Hoek AHAM, Mevius D, Guerra B, Mullany P, Roberts AP et al. Acquired antibiotic resistance genes: an overview. Front Microbiol 2011; 2:203 [View Article][PubMed]
     [Google Scholar]
  48. Villa L, Visca P, Tosini F, Pezzella C, Carattoli A. Composite integron array generated by insertion of an ORF341-type integron within a Tn21-like element. Microb Drug Resist 2002; 8:1–8 [View Article]
     [Google Scholar]
  49. Grape M, Farra A, Kronvall G, Sundström L. Integrons and gene cassettes in clinical isolates of co-trimoxazole-resistant Gram-negative bacteria. Clin Microbiol Infect 2005; 11:185–192 [View Article]
     [Google Scholar]
  50. Ho PL, Wong RC, Chow KH, Que TL. Distribution of integron-associated trimethoprim-sulfamethoxazole resistance determinants among Escherichia coli from humans and food-producing animals. Lett Appl Microbiol 2009; 49:627–634
     [Google Scholar]
  51. Volz C, Ramoni J, Beisken S, Galata V, Keller A et al. Clinical resistome screening of 1,110 Escherichia coli isolates efficiently recovers diagnostically relevant antibiotic resistance biomarkers and potential novel resistance mechanisms. Front Microbiol 2019; 10:1671 [View Article]
     [Google Scholar]
  52. MacGowan AP, Wise R. Establishing MIC breakpoints and the interpretation of in vitro susceptibility tests. J Antimicrob Chemother 2001; 48:17–28 [View Article]
     [Google Scholar]
  53. Kumar S, Stecher G, Suleski M, Hedges SB. TimeTree: a resource for timelines, timetrees, and divergence times. Mol Biol Evol 2017; 34:1812–1819 [View Article]
     [Google Scholar]
  54. Sigala J-C, Suárez BP, Lara AR, Borgne SL, Bustos P et al. Genomic and physiological characterization of a laboratory-isolated Acinetobacter schindleri ACE strain that quickly and efficiently catabolizes acetate. Microbiology 2017; 163:1052–1064 [View Article]
     [Google Scholar]
  55. Falagas ME, Vardakas KZ, Roussos NS. Trimethoprim/sulfamethoxazole for Acinetobacter spp.: a review of current microbiological and clinical evidence. Int J Antimicrob Agents 2015; 46:231–241 [View Article]
     [Google Scholar]
  56. Pérez-Varela M, Corral J, Aranda J, Barbé J. Roles of efflux pumps from different superfamilies in the surface-associated motility and virulence of Acinetobacter baumannii ATCC 17978. Antimicrob Agents Chemother 2019; 63:e02190-18 [View Article][PubMed]
     [Google Scholar]
  57. Leelaporn A, Firth N, Byrne ME, Roper E, Skurray RA. Possible role of insertion sequence IS257 in dissemination and expression of high- and low-level trimethoprim resistance in staphylococci. Antimicrob Agents Chemother 1994; 38:2238–2244 [View Article]
     [Google Scholar]
  58. Vickers AA, Potter NJ, Fishwick CWG, Chopra I, O'Neill AJ. Analysis of mutational resistance to trimethoprim in Staphylococcus aureus by genetic and structural modelling techniques. J Antimicrob Chemother 2009; 63:1112–1117 [View Article]
     [Google Scholar]
  59. Watson M, Liu J-W, Ollis D. Directed evolution of trimethoprim resistance in Escherichia coli . FEBS J 2007; 274:2661–2671 [View Article][PubMed]
     [Google Scholar]
  60. Toprak E, Veres A, Michel J-B, Chait R, Hartl DL et al. Evolutionary paths to antibiotic resistance under dynamically sustained drug selection. Nat Genet 2012; 44:101–105 [View Article]
     [Google Scholar]
  61. Swedberg G, Fermér C, Sköld O. Point mutations in the dihydropteroate synthase gene causing sulfonamide resistance. In Ayling JE, Nair MG, Baugh CM. eds Chemistry and Biology of Pteridines and Folates Boston, MA: Springer; 1993 pp 555–558
     [Google Scholar]
  62. Griffith EC, Wallace MJ, Wu Y, Kumar G, Gajewski S et al. The structural and functional basis for recurring sulfa drug resistance mutations in Staphylococcus aureus dihydropteroate synthase. Front Microbiol 2018; 9:1369 [View Article][PubMed]
     [Google Scholar]
  63. Palmer AC, Kishony R. Opposing effects of target overexpression reveal drug mechanisms. Nat Commun 2014; 5:4296 [View Article]
     [Google Scholar]
  64. Dale GE, Broger C, Hartman PG, Langen H, Page MG et al. Characterization of the gene for the chromosomal dihydrofolate reductase (DHFR) of Staphylococcus epidermidis ATCC 14990: the origin of the trimethoprim-resistant S1 DHFR from Staphylococcus aureus?. J Bacteriol 1995; 177:2965–2970 [View Article]
     [Google Scholar]
  65. Coque TM, Singh KV, Weinstock GM, Murray BE. Characterization of dihydrofolate reductase genes from trimethoprim-susceptible and trimethoprim-resistant strains of Enterococcus faecalis . Antimicrob Agents Chemother 1999; 43:141–147 [View Article]
     [Google Scholar]
  66. Barrow EW, Bourne PC, Barrow WW. Functional cloning of Bacillus anthracis dihydrofolate reductase and confirmation of natural resistance to trimethoprim. Antimicrob Agents Chemother 2004; 48:4643–4649 [View Article]
     [Google Scholar]
  67. Parkhill J, Dougan G, James KD, Thomson NR, Pickard D et al. Complete genome sequence of a multiple drug resistant Salmonella enterica serovar Typhi CT18. Nature 2001; 413:848–852 [View Article]
     [Google Scholar]
  68. Ahmed SA, Awosika J, Baldwin C, Bishop-Lilly KA, Biswas B et al. Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including shiga toxin encoding phage stx2. PLoS One 2012; 7:e48228 [View Article]
     [Google Scholar]
  69. Kim M, Kim S, Ryu S. Complete genome sequence of bacteriophage SSU5 specific for Salmonella enterica serovar typhimurium rough strains. J Virol 2012; 86:10894 [View Article]
     [Google Scholar]
  70. Octavia S, Sara J, Lan R. Characterization of a large novel phage-like plasmid in Salmonella enterica serovar Typhimurium. FEMS Microbiol Lett 2015; 362:fnv044 [View Article][PubMed]
     [Google Scholar]
  71. Asare PT, Jeong T-Y, Ryu S, Klumpp J, Loessner MJ et al. Putative type 1 thymidylate synthase and dihydrofolate reductase as signature genes of a novel bastille-like group of phages in the subfamily Spounavirinae . BMC Genomics 2015; 16:582 [View Article]
     [Google Scholar]
  72. Muniesa M, Colomer-Lluch M, Jofre J. Could bacteriophages transfer antibiotic resistance genes from environmental bacteria to human-body associated bacterial populations?. Mob Genet Elements 2013; 3:e25847 [View Article]
     [Google Scholar]
  73. Balcazar JL. Bacteriophages as vehicles for antibiotic resistance genes in the environment. PLoS Pathog 10:e1004219 [View Article]
     [Google Scholar]
  74. Enault F, Briet A, Bouteille L, Roux S, Sullivan MB et al. Phages rarely encode antibiotic resistance genes: a cautionary tale for virome analyses. ISME J 2017; 11:237–247 [View Article]
     [Google Scholar]
  75. Bhattacharya B, Giri N, Mitra M, Gupta SKD. Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5. FEMS Microbiol Lett 2008; 280:64–72 [View Article]
     [Google Scholar]
  76. Wittmann J, Gartemann K-H, Eichenlaub R, Dreiseikelmann B. Genomic and molecular analysis of phage CMP1 from Clavibacter michiganensis subspecies michiganensis . Bacteriophage 2011; 1:6–14 [View Article]
     [Google Scholar]
  77. Huang S, Zhang S, Jiao N, Chen F. Comparative genomic and phylogenomic analyses reveal a conserved core genome shared by estuarine and oceanic Cyanopodoviruses. PLoS One 2015; 10:e0142962 [View Article]
     [Google Scholar]
  78. Kozloff LM, Lute M, Crosby LK. Bacteriophage T4 virion baseplate thymidylate synthetase and dihydrofolate reductase. J Virol 1977; 23:637–644 [View Article]
     [Google Scholar]
  79. Peters DL, McCutcheon JG, Stothard P, Dennis JJ. Novel Stenotrophomonas maltophilia temperate phage DLP4 is capable of lysogenic conversion. BMC Genomics 2019; 20:300 [View Article]
     [Google Scholar]
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Exploration into the origins and mobilization of di-hydrofolate reductase genes and the emergence of clinical resistance to trimethoprim
M Gen 6, e000440 (2020); https://doi.org/10.1099/mgen.0.000440
/content/journal/mgen/10.1099/mgen.0.000440
/content/journal/mgen/10.1099/mgen.0.000440
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