#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Characterization of the diverse plasmid pool harbored by the blaNDM-1-containing Acinetobacter bereziniae HPC229 clinical strain


Autoři: Marco Brovedan aff001;  Guillermo D. Repizo aff001;  Patricia Marchiaro aff001;  Alejandro M. Viale aff001;  Adriana Limansky aff001
Působiště autorů: Instituto de Biología Molecular y Celular de Rosario (IBR), Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, Universidad Nacional de Rosario (UNR), Rosario, Argentina aff001
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pone.0220584

Souhrn

Acinetobacter bereziniae is an environmental microorganism with increasing clinical incidence, and may thus provide a model for a bacterial species bridging the gap between the environment and the clinical setting. A. bereziniae plasmids have been poorly studied, and their characterization could offer clues on the causes underlying the leap between these two different habitats. Here we characterized the whole plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal resistance, a bacteriophage defense BREX system and four different toxin-antitoxin (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein genes, and functional analysis of their transfer regions revealed the mobilizable nature of them. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229-1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter lacking known replication functions. Comparative sequence analyses against deposited Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, although some regions were also present in other of these genomes. The transfer, replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229-9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine recombinases, thus suggesting a potential mechanism for their acquisition. The presence of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive functions including resistance to heavy metal and other environmental stressors, as well as antimicrobial resistance genes, and a high content of XerC/XerD sites among HPC229 plasmids provide evidence of substantial links with bacterial species derived from both environmental and clinical habitats.

Klíčová slova:

Sequence analysis – DNA sequence analysis – Sequence databases – Plasmid construction – Antimicrobial resistance – Plasmids – Genomic databases – Acinetobacter


Zdroje

1. Norman A, Hansen LH, and Sorensen SJ. Conjugative plasmids: vessels of the communal gene pool. Philos Trans R Soc Lond B Biol Sci. 2009; 364:2275–2289. doi: 10.1098/rstb.2009.0037 19571247

2. Fondi M, Bacci G, Brilli M, Papaleo MC, Mengoni A, Vaneechoutte M, et al. Exploring the evolutionary dynamics of plasmids: the Acinetobacter pan-plasmidome. BMC Evol Biol. 2010; 10:59. doi: 10.1186/1471-2148-10-59 20181243

3. Garcillan-Barcia MP, Alvarado A, and de la Cruz F. Identification of bacterial plasmids based on mobility and plasmid population biology. FEMS Microbiol Rev. 2011; 35:936–956. doi: 10.1111/j.1574-6976.2011.00291.x 21711366

4. Bennett PM. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. British J Pharmac. 2008; 153 Suppl 1:S347–57.

5. Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol. 2013; 303: 298–304. doi: 10.1016/j.ijmm.2013.02.001 23499304

6. Lean SS, and Yeo CC. Small, Enigmatic Plasmids of the Nosocomial Pathogen, Acinetobacter baumannii: Good, Bad, Who Knows?. Front Microbiol. 2017; 8:1547. doi: 10.3389/fmicb.2017.01547 28861061

7. Smalla K, Jechalke S, and Top EM. Plasmid Detection, Characterization, and Ecology. Microbiol Spectr. 2015; 3:PLAS-0038-2014.

8. Bonnin RA, Nordmann P, Carattoli A, and Poirel L. Comparative genomics of IncL/M-type plasmids: evolution by acquisition of resistance genes and insertion sequences. Antimicrob Agents Chemother. 2013; 57:674–676. doi: 10.1128/AAC.01086-12 23114767

9. Bellanger X, Payot S, Leblond-Bourget N, and Guedon G. Conjugative and mobilizable genomic islands in bacteria: evolution and diversity. FEMS Microbiol Rev. 2014; 38:720–760. doi: 10.1111/1574-6976.12058 24372381

10. Loftie-Eaton W, Yano H, Burleigh S, Simmons RS, Hughes JM, Rogers LM, et al. Evolutionary Paths That Expand Plasmid Host-Range: Implications for Spread of Antibiotic Resistance. Mol Biol Evol. 2016; 33:885–897. doi: 10.1093/molbev/msv339 26668183

11. Porse A, Schonning K, Munck C, and Sommer MO. Survival and Evolution of a Large Multidrug Resistance Plasmid in New Clinical Bacterial Hosts. Mol Biol Evol. 2016; 33:2860–2873. doi: 10.1093/molbev/msw163 27501945

12. Wright MS, Mountain S, Beeri K, and Adams MD. Assessment of Insertion Sequence Mobilization as an Adaptive Response to Oxidative Stress in Acinetobacter baumannii Using IS-seq. J Bacteriol. 2017; 199:9.

13. Cameranesi MM, Moran-Barrio J, Limansky AS, Repizo GD, and Viale AM. Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58 and TnaphA6-Resistance Module in Acinetobacter baumannii. Front Microbiol. 2018; 9:66. doi: 10.3389/fmicb.2018.00066 29434581

14. Carnoy C, and Roten CA. The dif/Xer recombination systems in proteobacteria. PLoS One. 2009; 4:e6531. doi: 10.1371/journal.pone.0006531 19727445

15. Poirel L, Nordmann P. Genetic structures at the origin of acquisition and expression of the carbapenem-hydrolyzing oxacillinase gene blaOXA-58 in Acinetobacter baumannii. Antimicrob Agents Chemother. 2006; 50(4):1442–8. doi: 10.1128/AAC.50.4.1442-1448.2006 16569863

16. Merino M, Acosta J, Poza M, Sanz F, Beceiro A, Chaves F, et al. OXA-24 carbapenemase gene flanked by XerC/XerD-like recombination sites in different plasmids from different Acinetobacter species isolated during a nosocomial outbreak. Antimicrob Agents Chemother. 2010; 54:2724–2727. doi: 10.1128/AAC.01674-09 20385865

17. Blackwell GA, and Hall RM. The tet39 Determinant and the msrE-mphE Genes in Acinetobacter Plasmids Are Each Part of Discrete Modules Flanked by Inversely Oriented pdif (XerC-XerD) Sites. Antimicrob Agents Chemother. 2017; 61:8.

18. Mindlin S, Petrenko A, Petrova M. Chromium resistance genetic element flanked by XerC/XerD recombination sites and its distribution in environmental and clinical Acinetobacter strains. FEMS Microbiology Letters. 2018; 365(8):fny047.

19. Brovedan M, Marchiaro PM, Moran-Barrio J, Revale S, Cameranesi M, Brambilla L, et al. Draft Genome Sequence of Acinetobacter bereziniae HPC229, a Carbapenem-Resistant Clinical Strain from Argentina Harboring blaNDM-1. Genome Announc. 2016; 4:e00117–16. doi: 10.1128/genomeA.00117-16 26966220

20. Brovedan M, Marchiaro PM, Moran-Barrio J, Cameranesi M, Cera G, Rinaudo M, et al. Complete Sequence of a blaNDM-1-Harboring Plasmid in an Acinetobacter bereziniae Clinical Strain Isolated in Argentina. Antimicrob Agents Chemother. 2015; 59:6667–6669. doi: 10.1128/AAC.00367-15 26248354

21. Hu H, Hu Y, Pan Y, Liang H, Wang H, Wang X, et al. Novel plasmid and its variant harboring both a blaNDM-1 gene and type IV secretion system in clinical isolates of Acinetobacter lwoffii. Antimicrob Agents Chemother. 2012; 56:1698–1702. doi: 10.1128/AAC.06199-11 22290961

22. Jones LS, Carvalho MJ, Toleman MA, White PL, Connor TR, Mushtaq A, et al. Characterization of plasmids in extensively drug-resistant Acinetobacter strains isolated in India and Pakistan. Antimicrob Agents Chemother. 2015; 59:923–929.

23. Nemec A, Musilek M, Sedo O, De Baere T, Maixnerova M, van der Reijden TJ, et al. Acinetobacter bereziniae sp. nov. and Acinetobacter guillouiae sp. nov., to accommodate Acinetobacter genomic species 10 and 11, respectively. Int J Syst Evol Microbiol. 2010; 60:896–903. doi: 10.1099/ijs.0.013656-0 19661501

24. Zander E, Seifert H, and Higgins PG. Insertion sequence IS18 mediates overexpression of blaOXA-257 in a carbapenem-resistant Acinetobacter bereziniae isolate. J Antimicrob Chemother. 2014; 69:270–271. doi: 10.1093/jac/dkt313 23934740

25. Grosso F, Silva L, Sousa C, Ramos H, Quinteira S, and Peixe L. Extending the reservoir of blaIMP-5: the emerging pathogen Acinetobacter bereziniae. Future Microbiol. 2015; 10:1609–1613. doi: 10.2217/fmb.15.88 26439605

26. Garcia-Garcera M, Touchon M, Brisse S, and Rocha E. Metagenomic assessment of the interplay between the environment and the genetic diversification of Acinetobacter. Environmental microbiology. 2017; 19:5010–5024. doi: 10.1111/1462-2920.13949 28967182

27. Turton JF, Shah J, Ozongwu C, and Pike R. Incidence of Acinetobacter species other than A. baumannii among clinical isolates of Acinetobacter: evidence for emerging species. J Clin Microbiol. 2010; 48:1445–1449. doi: 10.1128/JCM.02467-09 20181894

28. Lee K, Kim CK, Hong SG, Choi J, Song S, Koh E, et al. Characteristics of clinical isolates of Acinetobacter genomospecies 10 carrying two different metallo-beta-lactamases. Int J Antimicrob Agents. 2010; 36:259–263. doi: 10.1016/j.ijantimicag.2010.05.018 20599361

29. Bertini A, Poirel L, Mugnier PD, Villa L, Nordmann P, Carattoli A. Characterization and PCR-based replicon typing of resistance plasmids in Acinetobacter baumannii. Antimicrob Agents Chemother. 2010; 54(10):4168–77. doi: 10.1128/AAC.00542-10 20660691

30. Salto IP, Torres Tejerizo G, Wibberg D, Puhler A, Schluter A, and Pistorio M. Comparative genomic analysis of Acinetobacter spp. plasmids originating from clinical settings and environmental habitats. Sci Rep. 2018; 8:7783. doi: 10.1038/s41598-018-26180-3 29773850

31. Konstantinidis KT, Tiedje JM. Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci.2005; 102:2567–2572. doi: 10.1073/pnas.0409727102 15701695

32. Lee I, Kim YO, Park SC, Chun J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol. 2015; 66:1100–1103. doi: 10.1099/ijsem.0.000760 26585518

33. Chan JZ, Halachev MR, Loman NJ, Constantinidou C, & Pallen MJ. Defining bacterial species in the genomic era: insights from the genus Acinetobacter. BMC microbiology. 2012; 12:302. doi: 10.1186/1471-2180-12-302 23259572

34. Altschul SF, Gish W, Miller W, Myers EW, and Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990; 215:403–410. doi: 10.1016/S0022-2836(05)80360-2 2231712

35. Grant JR, and Stothard P. The CGView Server: a comparative genomics tool for circular genomes. Nucleic Acids Res. 2008; 36: W181–184. doi: 10.1093/nar/gkn179 18411202

36. Saier MH Jr, Reddy VS, Tsu BV, Ahmed MS, Li C, and Moreno-Hagelsieb G. The Transporter Classification Database (TCDB): recent advances. Nucleic Acids Res. 2016; 44:D372–379. doi: 10.1093/nar/gkv1103 26546518

37. Xie Y, Wei Y, Shen Y, Li X, Zhou H, Tai C, et al. TADB 2.0: an updated database of bacterial type II toxin-antitoxin loci. Nucleic Acids Res. 2018; 46:D749–D753. doi: 10.1093/nar/gkx1033 29106666

38. Sevin EW, and Barloy-Hubler F. RASTA-Bacteria: a web-based tool for identifying toxin-antitoxin loci in prokaryotes. Genome Biol. 2007; 8:R155. doi: 10.1186/gb-2007-8-8-r155 17678530

39. Siguier P, Perochon J, Lestrade L, Mahillon J, and Chandler M. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res. 2006; 34:D32–36. doi: 10.1093/nar/gkj014 16381877

40. Varani AM, Siguier P, Gourbeyre E, Charneau V, and Chandler M. ISsaga is an ensemble of web-based methods for high throughput identification and semi-automatic annotation of insertion sequences in prokaryotic genomes. Genome Biol. 2011; 12:R30. doi: 10.1186/gb-2011-12-3-r30 21443786

41. Roberts AP, Chandler M, Courvalin P, Guedon G, Mullany P, Pembroke T, et al. Revised nomenclature for transposable genetic elements. Plasmid. 2008; 60:167–173. doi: 10.1016/j.plasmid.2008.08.001 18778731

42. Benson G. Tandem repeats finder: a program to analyze DNA sequences. Nucleic acids research. 1999; 27:573–80. doi: 10.1093/nar/27.2.573 9862982

43. Grigoriev A. Analyzing genomes with cumulative skew diagrams. Nucleic Acids Res.1998; 26:2286–2290. doi: 10.1093/nar/26.10.2286 9580676

44. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, et al. CDD: NCBI's conserved domain database. Nucleic Acids Res. 2015; 43:D222–226. doi: 10.1093/nar/gku1221 25414356

45. Tamura K, Stecher G, Peterson D, Filipski A, and Kumar S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013; 30:2725–2729. doi: 10.1093/molbev/mst197 24132122

46. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. ClustalW and ClustalX version 2. Bioinformatics 2007; 23:2947–2948. doi: 10.1093/bioinformatics/btm404 17846036

47. Dorsey CW, Tomaras AP, Actis LA. Sequence and organization of pMAC, an Acinetobacter baumannii plasmid harboring genes involved in organic peroxide resistance. Plasmid. 2006;56(2):112–23. doi: 10.1016/j.plasmid.2006.01.004 16530832

48. Simon R, Priefer U, Pühler A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology. 1983;1:784–791.

49. Konieczny I, Bury K, Wawrzycka A, Wegrzyn K. Iteron plasmids. Microbiol. Spectr. 2014; 2:PLAS-0026.

50. Feng Y, Yang P, Wang X, and Zong Z. Characterization of Acinetobacter johnsonii isolate XBB1 carrying nine plasmids and encoding NDM-1, OXA-58 and PER-1 by genome sequencing. J Antimicrob Chemother. 2016; 71:71–75. doi: 10.1093/jac/dkv324 26462992

51. Haines A, Akhtar P, Stephens E, Jones K, Thomas C, Perkins C, et al. Plasmids from freshwater environments capable of IncQ retrotransfer are diverse and include pQKH54, a new IncP-1 subgroup archetype. Microbiology 2006; 52:2689–2701.

52. Fondi M, Rizzi E, Emiliani G, Orlandini V, Berna L, Papaleo MC, et al. The genome sequence of the hydrocarbon-degrading Acinetobacter venetianus VE-C3. Res Microbiol. 2013; 164:439–449. doi: 10.1016/j.resmic.2013.03.003 23528645

53. Chan WT, Espinosa M, and Yeo CC. Keeping the Wolves at Bay: Antitoxins of Prokaryotic Type II Toxin-Antitoxin Systems. Front Mol Biosci. 2016; 3:9. doi: 10.3389/fmolb.2016.00009 27047942

54. Ou HY, Kuang SN, He X, Molgora BM, Ewing PJ, Deng Z, et al. Complete genome sequence of hypervirulent and outbreak-associated Acinetobacter baumannii strain LAC-4: epidemiology, resistance genetic determinants and potential virulence factors. Sci Rep. 2015; 5:8643. doi: 10.1038/srep08643 25728466

55. Post V, and Hall RM. AbaR5, a large multiple-antibiotic resistance region found in Acinetobacter baumannii. Antimicrob Agents Chemother. 2009; 53:2667–2671. doi: 10.1128/AAC.01407-08 19364869

56. Lee YK, Chang HH, Lee HJ, Park H, Lee KH, and Joe MH. Isolation of a novel plasmid, pNi15, from Enterobacter sp. Ni15 containing a nickel resistance gene. FEMS Microbiol Lett. 2006; 257:177–181. doi: 10.1111/j.1574-6968.2006.00130.x 16553850

57. Krahn T, Gilmour C, Tilak J, Fraud S, Kerr N, Lau CH, et al. Determinants of intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2012; 56:5591–5602. doi: 10.1128/AAC.01446-12 22908149

58. Gallagher LA, Lee SA, and Manoil C. Importance of Core Genome Functions for an Extreme Antibiotic Resistance Trait. MBio. 2017; 8:6.

59. Goldfarb T, Sberro H, Weinstock E, Cohen O, Doron S, Charpak-Amikam Y, et al. BREX is a novel phage resistance system widespread in microbial genomes. EMBO J. 2015; 34:169–183. doi: 10.15252/embj.201489455 25452498

60. Farrugia DN, Elbourne LDH, Hassan KA, Eijkelkamp BA, Tetu SG, et al. The Complete Genome and Phenome of a Community-Acquired Acinetobacter baumannii. PLoS ONE. 2013; 8:e58628. doi: 10.1371/journal.pone.0058628 23527001

61. Adams MD, Bishop B, and Wright MS. Quantitative assessment of insertion sequence impact on bacterial genome architecture. Microb Genom. 2016; 2:e000062. doi: 10.1099/mgen.0.000062 28348858

62. Touchon M, Cury J, Yoon EJ, Krizova L, Cerqueira GC, Murphy C, et al. The genomic diversification of the whole Acinetobacter genus: origins, mechanisms, and consequences. Genome Biol Evol. 2014; 6:2866–2882. doi: 10.1093/gbe/evu225 25313016

63. Cladera AM, Bennasar A, Barcelo M, Lalucat J, and Garcia-Valdes E. Comparative genetic diversity of Pseudomonas stutzeri genomovars, clonal structure, and phylogeny of the species. J Bacteriol. 2004; 186:5239–5248. doi: 10.1128/JB.186.16.5239-5248.2004 15292125

64. Mugnier PD, Poirel L, and Nordmann P. Functional Analysis of Insertion Sequence ISAba1, Responsible for Genomic Plasticity of Acinetobacter baumannii. J. Bacteriol. 2009; 191:2414–2418. doi: 10.1128/JB.01258-08 19136598

65. Afshari E, Amini-Bayat Z, Hosseinkhani S, & Bakhtiari N. Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase. Avicenna journal of medical biotechnology 2017; 9:169–175. 29090065

66. Jurenaite M, Markuckas A, and Suziedeliene E. Identification and characterization of type II toxin-antitoxin systems in the opportunistic pathogen Acinetobacter baumannii. J Bacteriol. 2013; 195:3165–3172. doi: 10.1128/JB.00237-13 23667234

67. Francia MV, Varsaki A, Garcillan-Barcia MP, Latorre A, Drainas C, and de la Cruz F. A classification scheme for mobilization regions of bacterial plasmids. FEMS Microbiol Rev. 2004; 28:79–100. doi: 10.1016/j.femsre.2003.09.001 14975531

68. del Solar G, Giraldo R, Ruiz-Echevarria MJ, Espinosa M, Diaz-Orejas R. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. 1998; 62:434–464. 9618448

69. Camps M. Modulation of ColE1-like plasmid replication for recombinant gene expression. Recent Pat DNA Gene Seq. 2010; 4:58–73. doi: 10.2174/187221510790410822 20218961

70. Rajewska M, Wegrzyn K, and Konieczny I. AT-rich region and repeated sequences—the essential elements of replication origins of bacterial replicons. FEMS Microbiol Rev. 2012; 36:408–434. doi: 10.1111/j.1574-6976.2011.00300.x 22092310

71. Cameranesi MM, Limansky AS, Morán-Barrio J, Repizo GD, Viale AM. Three Novel Acinetobacter baumannii Plasmid Replicase-Homology Groups Inferred from the Analysis of a Multidrug-Resistant Clinical Strain Isolated in Argentina. J Infect Dis Epidemiol. 2017; 3:046.

72. Midonet C, and Barre FX. Xer Site-Specific Recombination: Promoting Vertical and Horizontal Transmission of Genetic Information. Microbiol Spectr.2014; 2: 6.

73. Tran T, Sherratt DJ, Tolmasky ME. fpr, a deficient Xer recombination site from a Salmonella plasmid, fails to confer stability by dimer resolution: comparative studies with the pJHCMW1 mwr site. J Bacteriol. 2010; 192(3):883–887. doi: 10.1128/JB.01082-09 19966005

74. Gardner MN, Deane SM, and Rawlings DE. Isolation of a new broad-host-range IncQ-like plasmid, pTC-F14, from the acidophilic bacterium Acidithiobacillus caldus and analysis of the plasmid replicon. J Bacteriol. 2001; 183:3303–3309. doi: 10.1128/JB.183.11.3303-3309.2001 11344137

75. Meyer R. Replication and conjugative mobilization of broad host-range IncQ plasmids. Plasmid. 2009; 62:57–70. doi: 10.1016/j.plasmid.2009.05.001 19465049

76. Antunes LC, Visca P, and Towner KJ. Acinetobacter baumannii: evolution of a global pathogen. Pathog Dis. 2014; 71: 292–301. doi: 10.1111/2049-632X.12125 24376225

77. Cho GS, Li B, Rostalsky A, Fiedler G, Rosch N, Igbinosa E., et al. Diversity and Antibiotic Susceptibility of Acinetobacter Strains From Milk Powder Produced in Germany. Front Microbiol. 2018; 9:536. doi: 10.3389/fmicb.2018.00536 29636733

78. Li AD, Li LG, and Zhang T. Exploring antibiotic resistance genes and metal resistance genes in plasmid metagenomes from wastewater treatment plants. Front Microbiol. 2015; 6:1025. doi: 10.3389/fmicb.2015.01025 26441947

79. Van Melderen L, and Saavedra De Bast M. Bacterial Toxin–Antitoxin Systems: More Than Selfish Entities? PLoS Genetics. 2009; 5:e1000437. doi: 10.1371/journal.pgen.1000437 19325885

80. Di Cello F, Pepi M, Baldi F, and Fani R. Molecular characterization of an n-alkane-degrading bacterial community and identification of a new species, Acinetobacter venetianus. Res Microbiol. 1997; 148:237–249. doi: 10.1016/S0923-2508(97)85244-8 9765804


Článok vyšiel v časopise

PLOS One


2019 Číslo 11
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#