Molecular basis of antifungal drug resistance in yeasts

International Journal of Antimicrobial Agents

Volumn 50, Issue 5, 2017, Pages 599-606

  Morio, Florent ^a,b   Jensen, Rasmus Hare ^c   Le Pape, Patrice ^a,b   Arendrup, Maiken Cavling ^c,d,e  

^a NANTES UNIVERSITY HOSPITAL   (France)

^b UNIVERSITÉ DE NANTES   (France)

^c STATENS SERUM INSTITUT   (Denmark)

^d University of Copenhagen   (Denmark)

^e UNIVERSITY OF COPENHAGEN   (Denmark)

Author keywords

Antifungal resistance; Clinical failure; Molecular mechanisms; Multidrug resistance; Review; Yeast

Indexed keywords

AMPHOTERICIN B; ANIDULAFUNGIN; CASPOFUNGIN; ECHINOCANDIN; FLUCONAZOLE; FLUCYTOSINE; ITRACONAZOLE; MICAFUNGIN; MULTIDRUG RESISTANCE PROTEIN; POSACONAZOLE; STEROL 14ALPHA DEMETHYLASE; VORICONAZOLE; ANTIFUNGAL AGENT;

ANTIFUNGAL RESISTANCE; ANTIFUNGAL SUSCEPTIBILITY; ARTICLE; BIOFILM; CANDIDA; CANDIDA AURIS; CANDIDA GLABRATA; CANDIDA INCONSPICUA; CANDIDA PALMIOLEOPHILA; CANDIDA RUGOSA; GENE MUTATION; GENETIC ASSOCIATION; IN VITRO STUDY; MEYEROZYMA GUILLIERMONDII; MINIMUM INHIBITORY CONCENTRATION; MOLECULAR MECHANICS; MULTIDRUG RESISTANCE; NONHUMAN; PHENOTYPE; PICHIA KUDRIAVZEVII; PICHIA NORVEGENSIS; PRIORITY JOURNAL; YARROWIA LIPOLYTICA; DRUG EFFECT; FUNGAL GENE; GENETICS; HUMAN; MICROBIAL SENSITIVITY TEST; MUTATION; PROCEDURES;

ANTIFUNGAL AGENTS; CANDIDA; DRUG RESISTANCE, FUNGAL; GENES, FUNGAL; HUMANS; MICROBIAL SENSITIVITY TESTS; MUTATION;

EID: 85029425755     PISSN: 09248579     EISSN: 18727913     Source Type: Journal    
DOI: 10.1016/j.ijantimicag.2017.05.012     Document Type: Article

References (78)

1. Sanglard, D., Emerging threats in antifungal-resistant fungal pathogens. Front Med, 3, 2016, 11.
2. Pinhati, H.M.S., Casulari, L.A., Souza, A.C.R., Siqueira, R.A., Damasceno, C.M.G., Colombo, A.L., Outbreak of candidemia caused by fluconazole resistant Candida parapsilosis strains in an intensive care unit. BMC Infect Dis, 16, 2016, 433.
3. Mansfield, B.E., Oltean, H.N., Oliver, B.G., Hoot, S.J., Leyde, S.E., Hedstrom, L., et al. Azole drugs are imported by facilitated diffusion in Candida albicans and other pathogenic fungi. PLoS Pathog, 6, 2010, e1001126.
4. Lockhart, S.R., Etienne, K.A., Vallabhaneni, S., Farooqi, J., Chowdhary, A., Govender, N.P., et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin Infect Dis 64 (2017), 134–140.
5. Lopez-Ribot, J.L., McAtee, R.K., Lee, L.N., Kirkpatrick, W.R., White, T.C., Sanglard, D., et al. Distinct patterns of gene expression associated with development of fluconazole resistance in serial Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob Agents Chemother 42 (1998), 2932–2937.
6. Trouvé, C., Blot, S., Hayette, M.-P., Jonckheere, S., Patteet, S., Rodriguez-Villalobos, H., et al. Epidemiology and reporting of candidaemia in Belgium: a multi-centre study. Eur J Clin Microbiol Infect Dis 36 (2017), 649–655.
7. Morio, F., Pagniez, F., Besse, M., Gay-Andrieu, F., Miegeville, M., Le Pape, P., Deciphering azole resistance mechanisms with a focus on transcription factor-encoding genes TAC1, MRR1 and UPC2 in a set of fluconazole-resistant clinical isolates of Candida albicans. Int J Antimicrob Agents 42 (2013), 410–415.
8. Morio, F., Loge, C., Besse, B., Hennequin, C., Le Pape, P., Screening for amino acid substitutions in the Candida albicans Erg11 protein of azole-susceptible and azole-resistant clinical isolates: new substitutions and a review of the literature. Diagn Microbiol Infect Dis 66 (2010), 373–384.
9. Flowers, S.A., Colón, B., Whaley, S.G., Schuler, M.A., David Rogers, P., Contribution of clinically derived mutations in ERG11 to azole resistance in Candida albicans. Antimicrob Agents Chemother 59 (2015), 450–460.
10. Sagatova, A.A., Keniya, M.V., Wilson, R.K., Monk, B.C., Tyndall, J.D.A., Structural insights into binding of the antifungal drug fluconazole to Saccharomyces cerevisiae lanosterol 14α-demethylase. Antimicrob Agents Chemother 59 (2015), 4982–4989.
11. Alvarez-Rueda, N., Fleury, A., Morio, F., Pagniez, F., Gastinel, L., Le Pape, P., Amino acid substitutions at the major insertion loop of Candida albicans sterol 14alpha-demethylase are involved in fluconazole resistance. PLoS ONE, 6, 2011 e21239.
12. Berkow, E.L., Manigaba, K., Parker, J.E., Barker, K.S., Kelly, S.L., Rogers, P.D., Multidrug transporters and alterations in sterol biosynthesis contribute to azole antifungal resistance in Candida parapsilosis. Antimicrob Agents Chemother 59 (2015), 5942–5950.
13. Xisto, M.I., Caramalho, R.D., Rocha, D.A., Ferreira-Pereira, A., Sartori, B., Barreto-Bergter, E., et al. Pan-azole-resistant Candida tropicalis carrying homozygous erg11 mutations at position K143R: a new emerging superbug?. J Antimicrob Chemother 72 (2017), 988–992.
14. Sionov, E., Chang, Y.C., Garraffo, H.M., Dolan, M.A., Ghannoum, M.A., Kwon-Chung, K.J., Identification of a Cryptococcus neoformans cytochrome P450 lanosterol 14α-demethylase (Erg11) residue critical for differential susceptibility between fluconazole/voriconazole and itraconazole/posaconazole. Antimicrob Agents Chemother 56 (2012), 1162–1169.
15. Flowers, S.A., Barker, K.S., Berkow, E.L., Toner, G., Chadwick, S.G., Gygax, S.E., et al. Gain-of-function mutations in UPC2 are a frequent cause of ERG11 upregulation in azole-resistant clinical isolates of Candida albicans. Eukaryot Cell 11 (2012), 1289–1299.
16. Coleman, J.J., Mylonakis, E., Agrios, G., Edmond, M., Wallace, S., McClish, D., et al. Efflux in fungi: la pièce de résistance. PLoS Pathog, 5, 2009, e1000486.
17. Coste, A., Turner, V., Ischer, F., Morschhäuser, J., Forche, A., Selmecki, A., et al. A mutation in Tac1p, a transcription factor regulating CDR1 and CDR2, is coupled with loss of heterozygosity at chromosome 5 to mediate antifungal resistance in Candida albicans. Genetics 172 (2006), 2139–2156.
18. Dunkel, N., Blass, J., Rogers, P.D., Morschhäuser, J., Mutations in the multi-drug resistance regulator MRR1, followed by loss of heterozygosity, are the main cause of MDR1 overexpression in fluconazole-resistant Candida albicans strains. Mol Microbiol 69 (2008), 827–840.
19. Basso, L.R., Gast, C.E., Bruzual, I., Wong, B., Identification and properties of plasma membrane azole efflux pumps from the pathogenic fungi Cryptococcus gattii and Cryptococcus neoformans. J Antimicrob Chemother 70 (2015), 1396–1407.
20. 5,6-desaturase (Erg3p) confer azole resistance: characterization of two novel mutants with impaired virulence. J Antimicrob Chemother 67 (2012), 2131–2138.
21. Martel, C.M., Parker, J.E., Bader, O., Weig, M., Gross, U., Warrilow, A.G.S., et al. Identification and characterization of four azole-resistant erg3 mutants of Candida albicans. Antimicrob Agents Chemother 54 (2010), 4527–4533.
22. Hull, C.M., Bader, O., Parker, J.E., Weig, M., Gross, U., Warrilow, A.G.S., et al. Two clinical isolates of Candida glabrata exhibiting reduced sensitivity to amphotericin B both harbor mutations in ERG2. Antimicrob Agents Chemother 56 (2012), 6417–6421.
23. Martel, C.M., Parker, J.E., Bader, O., Weig, M., Gross, U., Warrilow, A.G., et al. A clinical isolate of Candida albicans with mutations in ERG11 (encoding sterol 14alpha-demethylase) and ERG5 (encoding C22 desaturase) is cross resistant to azoles and amphotericin B. Antimicrob Agents Chemother 54 (2010), 3578–3583.
24. Sanglard, D., Ischer, F., Parkinson, T., Falconer, D., Bille, J., Candida albicans mutations in the ergosterol biosynthetic pathway and resistance to several antifungal agents. Antimicrob Agents Chemother 47 (2003), 2404–2412.
25. Selmecki, A., Forche, A., Berman, J., Genomic plasticity of the human fungal pathogen Candida albicans. Eukaryot Cell 9 (2010), 991–1008.
26. Arendrup, M.C., Perlin, D.S., Echinocandin resistance: an emerging clinical problem?. Curr Opin Infect Dis 27 (2014), 484–492.
27. Garcia-Effron, G., Katiyar, S.K., Park, S., Edlind, T.D., Perlin, D.S., A naturally occurring proline-to-alanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis accounts for reduced echinocandin susceptibility. Antimicrob Agents Chemother 52 (2008), 2305–2312.
28. Jensen, R.H., Resistance in human pathogenic yeasts and filamentous fungi: prevalence, underlying molecular mechanisms and link to the use of antifungals in humans and the environment. Dan Med J 63 (2016), 1–34.
29. Ben-Ami, R., Garcia-Effron, G., Lewis, R.E., Gamarra, S., Leventakos, K., Perlin, D.S., et al. Fitness and virulence costs of Candida albicans FKS1 Hot spot mutations associated with echinocandin resistance. J Infect Dis 204 (2011), 626–635.
30. Singh-Babak, S.D., Babak, T., Diezmann, S., Hill, J.A., Xie, J.L., Chen, Y.-L., et al. Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog, 8, 2012, e1002718.
31. Arendrup, M.C., Boekhout, T., Akova, M., Meis, J.F., Cornely, O.A., Lortholary, O., et al. ESCMID and ECMM joint clinical guidelines for the diagnosis and management of rare invasive yeast infections. Clin Microbiol Infect 20 (2014), 76–98.
32. Johnson, M.E., Katiyar, S.K., Edlind, T.D., New Fks hot spot for acquired echinocandin resistance in Saccharomyces cerevisiae and its contribution to intrinsic resistance of Scedosporium species. Antimicrob Agents Chemother 55 (2011), 3774–3781.
33. Huang, W., Liao, G., Baker, G.M., Wang, Y., Lau, R., Paderu, P., et al. Lipid flippase subunit Cdc50 mediates drug resistance and virulence in Cryptococcus neoformans. MBio 7 (2016), 1–13.
34. Jensen, R.H., Johansen, H.K., Søes, L.M., Lemming, L.E., Rosenvinge, F.S., Nielsen, L., et al. Posttreatment antifungal resistance among colonizing Candida isolates in Candidemia patients: results from a systematic multicenter study. Antimicrob Agents Chemother 60 (2016), 1500–1508.
35. Shields, R.K., Nguyen, M.H., Press, E.G., Clancy, C.J., Abdominal candidiasis is a hidden reservoir of echinocandin resistance. Antimicrob Agents Chemother 58 (2014), 7601–7605.
36. Mesa-Arango, A.C., Scorzoni, L., Zaragoza, O., It only takes one to do many jobs: Amphotericin B as antifungal and immunomodulatory drug. Front Microbiol 3 (2012), 1–10.
37. Jensen, R.H., Astvad, K.M.T., Silva, L.V., Sanglard, D., Jørgensen, R., Nielsen, K.F., et al. Stepwise emergence of azole, echinocandin and amphotericin B multidrug resistance in vivo in Candida albicans orchestrated by multiple genetic alterations. J Antimicrob Chemother 70 (2015), 2551–2555.
38. Vandeputte, P., Tronchin, G., Larcher, G., Ernoult, E., Bergès, T., Chabasse, D., et al. A nonsense mutation in the ERG6 gene leads to reduced susceptibility to polyenes in a clinical isolate of Candida glabrata. Antimicrob Agents Chemother 52 (2008), 3701–3709.
39. Vermes, A., Guchelaar, H.J., Dankert, J., Flucytosine: a review of its pharmacology, clinical indications, pharmacokinetics, toxicity and drug interactions. J Antimicrob Chemother 46 (2000), 171–179.
40. Florent, M., Noël, T., Ruprich-Robert, G., Da Silva, B., Fitton-Ouhabi, V., Chastin, C., et al. Nonsense and missense mutations in FCY2 and FCY1 genes are responsible for flucytosine resistance and flucytosine-fluconazole cross-resistance in clinical isolates of Candida lusitaniae. Antimicrob Agents Chemother 53 (2009), 2982–2990.
41. Ostrosky-Zeichner, L., Candida glabrata and FKS mutations: witnessing the emergence of the true multidrug-resistant Candida. Clin Infect Dis 56 (2013), 1733–1734.
42. Chapeland-Leclerc, F., Hennequin, C., Papon, N., Noël, T., Girard, A., Socié, G., et al. Acquisition of flucytosine, azole, and caspofungin resistance in Candida glabrata bloodstream isolates serially obtained from a hematopoietic stem cell transplant recipient. Antimicrob Agents Chemother 54 (2010), 1360–1362.
43. Cho, E.J., Shin, J.H., Kim, S.H., Kim, H.K., Park, J.S., Sung, H., et al. Emergence of multiple resistance profiles involving azoles, echinocandins and amphotericin B in Candida glabrata isolates from a neutropenia patient with prolonged fungaemia. J Antimicrob Chemother 70 (2015), 1268–1270.
44. Alexander, B.D., Johnson, M.D., Pfeiffer, C.D., Jiménez-Ortigosa, C., Catania, J., Booker, R., et al. Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations. Clin Infect Dis 56 (2013), 1724–1732.
45. Ben-Ami, R., Zimmerman, O., Finn, T., Amit, S., Novikov, A., Wertheimer, N., et al. Heteroresistance to fluconazole is a continuously distributed phenotype among Candida glabrata clinical strains associated with in vivo persistence. MBio, 7, 2016 e00655–16.
46. Jensen, R.H., Justesen, U.S., Rewes, A., Perlin, D.S., Arendrup, M.C., Echinocandin failure case due to a yet unreported FKS1 mutation in Candida krusei. Antimicrob Agents Chemother 58 (2014), 3550–3552.
47. Hull, C.M., Parker, J.E., Bader, O., Weig, M., Gross, U., Warrilow, A.G.S., et al. Facultative sterol uptake in an ergosterol-deficient clinical isolate of Candida glabrata harboring a missense mutation in ERG11 and exhibiting cross-resistance to azoles and amphotericin B. Antimicrob Agents Chemother 56 (2012), 4223–4232.
48. Nett, J.E., Crawford, K., Marchillo, K., Andes, D.R., Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene. Antimicrob Agents Chemother 54 (2010), 3505–3508.
49. Healey, K.R., Zhao, Y., Perez, W.B., Lockhart, S.R., Sobel, J.D., Farmakiotis, D., et al. Prevalent mutator genotype identified in fungal pathogen Candida glabrata promotes multi-drug resistance. Nat Commun, 7, 2016, 11128.
50. Cowen, L.E., Lindquist, S., Hsp90 potentiates the rapid evolution of new traits: drug resistance in diverse fungi. Science 309 (2005), 2185–2189.
51. Hill, J.A., Ammar, R., Torti, D., Nislow, C., Cowen, L.E., Genetic and genomic architecture of the evolution of resistance to antifungal drug combinations. PLoS Genet, 9, 2013, e1003390.
52. Vallabhaneni, S., Cleveland, A.A., Farley, M.M., Harrison, L.H., Schaffner, W., Beldavs, Z.G., et al. Epidemiology and risk factors for echinocandin nonsusceptible Candida glabrata bloodstream infections: data from a large multisite population-based Candidemia surveillance program, 2008–2014. Open Forum Infect Dis, 2, 2015 ofv163.
53. Dellière, S., Healey, K., Gits-Muselli, M., Carrara, B., Barbaro, A., Guigue, N., et al. Fluconazole and echinocandin resistance of Candida glabrata correlates better with antifungal drug exposure rather than with MSH2 mutator genotype in a French cohort of patients harboring low rates of resistance. Front Microbiol, 7, 2016, 2038.
54. Rajendran, R., Sherry, L., Nile, C.J., Sherriff, A., Johnson, E.M., Hanson, M.F., et al. Biofilm formation is a risk factor for mortality in patients with Candida albicans bloodstream infection-Scotland, 2012–2013. Clin Microbiol Infect 22 (2016), 87–93.
55. Finkel, J.S., Mitchell, A.P., Genetic control of Candida albicans biofilm development. Nat Rev Microbiol 9 (2011), 109–118.
56. Ramage, G., Rajendran, R., Sherry, L., Williams, C., Fungal biofilm resistance. Int J Microbiol, 2012, 2012 528521.
57. Kuhn, D.M., George, T., Chandra, J., Mukherjee, P.K., Ghannoum, M.A., Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother 46 (2002), 1773–1780.
58. Uppuluri, P., Srinivasan, A., Ramasubramanian, A., Lopez-Ribot, J.L., Effects of fluconazole, amphotericin B, and caspofungin on Candida albicans biofilms under conditions of flow and on biofilm dispersion. Antimicrob Agents Chemother 55 (2011), 3591–3593.
59. Cornely, O.A., Bassetti, M., Calandra, T., Garbino, J., Kullberg, B.J., Lortholary, O., et al. ESCMID* guideline for the diagnosis and management of Candida diseases 2012: non-neutropenic adult patients. Clin Microbiol Infect 18 (2012), 19–37.
60. Andes, D.R., Safdar, N., Baddley, J.W., Playford, G., Reboli, A.C., Rex, J.H., et al. Impact of treatment strategy on outcomes in patients with candidemia and other forms of invasive candidiasis: a patient-level quantitative review of randomized trials. Clin Infect Dis 54 (2012), 1110–1122.
61. Pappas, P.G., Kauffman, C.A., Andes, D.R., Clancy, C.J., Marr, K.A., Ostrosky-Zeichner, L., et al. Clinical practice guideline for the management of candidiasis: 2016 update by the infectious diseases society of America. Clin Infect Dis 62 (2016), e1–50.
62. Arendrup, M.C., Meletiadis, J., Mouton, J.W., Guinea, J., Cuenca-Estrella, M., Lagrou, K., et al. EUCAST technical note on isavuconazole breakpoints for Aspergillus, itraconazole breakpoints for Candida and updates for the antifungal susceptibility testing method documents. Clin Microbiol Infect, 22, 2016, 571 e1-4.
63. CLSI, Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Third Edition. CLSI document M27-A3. Wayne, Pennsylvania: Wayne, PA: Clinical and Laboratory Standards Institute, 2008.
64. Clinical and Laboratory Standards Institute (CLSI), Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Fourth Informational Supplement. CLSI document M27-S4, 2012.
65. Arendrup, M.C., Cuenca-Estrella, M., Lass-Flörl, C., Hope, W.W., Breakpoints for antifungal agents: an update from EUCAST focussing on echinocandins against Candida spp. and triazoles against Aspergillus spp. Drug Resist Updat 16 (2013), 1–15.
66. Arendrup, M.C., Cuenca-Estrella, M., Lass-Flörl, C., Hope, W.W., EUCAST technical note on Candida and micafungin, anidulafungin and fluconazole. Mycoses 57 (2014), 377–379.
67. Arendrup, M.C., Pfaller, M.A., Danish Fungaemia Study Group. Caspofungin Etest susceptibility testing of Candida species: risk of misclassification of susceptible isolates of C. glabrata and C. krusei when adopting the revised CLSI caspofungin breakpoints. Antimicrob Agents Chemother 56 (2012), 3965–3968.
68. Kathuria, S., Singh, P.K., Sharma, C., Prakash, A., Masih, A., Kumar, A., et al. Multidrug-resistant Candida auris misidentified as Candida haemulonii: characterization by matrix-assisted laser desorption ionization-time of flight mass spectrometry and DNA sequencing and its antifungal susceptibility profile variability by Vitek 2, CL. J Clin Microbiol 53 (2015), 1823–1830.
69. Astvad, K.M., Perlin, D.S., Johansen, H.K., Jensen, R.H., Arendrup, M.C., Evaluation of caspofungin susceptibility testing by the new Vitek 2 AST-YS06 yeast card using a unique collection of FKS wild-type and hot spot mutant isolates, including the five most common Candida species. Antimicrob Agents Chemother 57 (2013), 177–182.
70. Espinel-Ingroff, A., Arendrup, M., Cantón, E., Cordoba, S., Dannaoui, E., García-Rodríguez, J., et al. Multicenter study of method-dependent epidemiological cutoff values for detection of resistance in Candida spp. and Aspergillus spp. to amphotericin b and echinocandins for the Etest agar diffusion method. Antimicrob Agents Chemother, 61, 2017 e01792–16.
71. Espinel-Ingroff, A., Alvarez-Fernandez, M., Cantón, E., Carver, P.L., Chen, S.C.-A., Eschenauer, G., et al. Multicenter study of epidemiological cutoff values and detection of resistance in Candida spp. to anidulafungin, caspofungin, and micafungin using the sensititre YeastOne colorimetric method. Antimicrob Agents Chemother 59 (2015), 6725–6732.
72. Posteraro, B., Vella, A., De Carolis, E., Sanguinetti, M., Molecular detection of resistance to echinocandins. Methods Mol Biol 1508 (2017), 413–421.
73. Wiederhold, N.P., Grabinski, J.L., Garcia-Effron, G., Perlin, D.S., Lee, S.A., Pyrosequencing to detect mutations in FKS1 that confer reduced echinocandin susceptibility in Candida albicans. Antimicrob Agents Chemother 52 (2008), 4145–4148.
74. Balashov, S. V, Park, S., Perlin, D.S., Assessing resistance to the echinocandin antifungal drug caspofungin in Candida albicans by profiling mutations in FKS1. Antimicrob Agents Chemother 50 (2006), 2058–2063.
75. Dudiuk, C., Gamarra, S., Jimenez-Ortigosa, C., Leonardelli, F., Macedo, D., Perlin, D.S., et al. Quick detection of FKS1 mutations responsible for clinical echinocandin resistance in Candida albicans. J Clin Microbiol 53 (2015), 2037–2041.
76. Zhao, Y., Nagasaki, Y., Kordalewska, M., Press, E.G., Shields, R.K., Nguyen, M.H., et al. Rapid detection of FKS-associated echinocandin resistance in Candida glabrata. Antimicrob Agents Chemother 60 (2016), 6573–6577.
77. Pham, C.D., Bolden, C.B., Kuykendall, R.J., Lockhart, S.R., Development of a Luminex-based multiplex assay for detection of mutations conferring resistance to echinocandins in Candida glabrata. J Clin Microbiol 52 (2014), 790–795.
78. Garnaud, C., Botterel, F., Sertour, N., Bougnoux, M.-E., Dannaoui, E., Larrat, S., et al. Next-generation sequencing offers new insights into the resistance of Candida spp. to echinocandins and azoles. J Antimicrob Chemother 70 (2015), 2556–2565.