The emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare–associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro

Borman, Andrew

Johnson, Elizabeth M.

Kean, Ryan

Ramage, Gordon

Rautemaa-Richardson, Riina

Richardson, Malcolm D.

Sherry, Leighann

Emerging Infectious Diseases

English

PubMed

Enlighten

328 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017DISPATCHESLeighann Sherry, Gordon Ramage, Ryan Kean, Andrew Borman, Elizabeth M. Johnson,  Malcolm D. Richardson,  Riina Rautemaa-RichardsonThe emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare–associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro.The yeast pathogen Candida auris was first detected in 2009 from an ear canal infection in Japan (1). This spe-cies initially attracted attention because of its reduced sus-ceptibility to azoles and amphotericin B, combined with the lack of reliable culture-based methods for its identification (2). More recently, C. auris has been associated globally with life-threatening invasive diseases, such as bloodstream and wound infections. C. auris has also caused hospital outbreaks across Asia and South America, as highlighted in a 2016 clinical alert (3). In addition, in a UK intensive care unit, candidemia developed in 20% of patients colo-nized with C. auris (4). Although the mode of transmission within hospitals is unknown, C. auris may substantially contaminate rooms of colonized or infected patients (5). Phospholipase and proteinase activity have been identi-fied as virulence factors (6); however, because previously used assessment techniques were rudimentary, this patho-gen’s ability to form biofilm remains under question (7). The draft genome identifying various proteins involved in biofilm formation (8), coupled with recent descriptions of aggregative and nonaggregative phenotypes, the latter of which are more virulent in vivo (9), indicate the possibility of heterogeneous C. auris biofilm formation, as described for C. albicans (10). We sought to examine these aggrega-tive and nonaggregative C. auris phenotypes in the context of biofilm-forming capacity, investigate their susceptibil-ity to a panel of antifungal agents and the skin disinfectant chlorhexidine, and investigate their virulence in vivo.The StudyThroughout this study, we used C. albicans SC5314 and Candida glabrata WT2001 as comparators for C. auris nonaggregative strains NCPF 8971 (strain 10) and NCPF 8973 (strain 12) and aggregative strains NCPF 8977 (strain 2) and NCPF 8978 (strain 6), as previously described (9). Strains were propagated in YPD broth (Sigma-Aldrich, Dorset, UK), incubated overnight at 30°C, and adjusted to 106 cells/mL in RPMI 1640 medium (11). On 3 separate occasions, 8 biofilms of each Candida species were grown in flat-bottomed, 96-well polystyrene microtiter plates and incubated for 24 h at 37°C, after which biomass was as-sessed by crystal violet assay (12). C. albicans displayed the greatest biofilm mass (Figure 1, panel A), consistent with previous findings  (10). Compared with C. albicans, all C. auris strains formed significantly reduced biofilms (p<0.0001): biomass for nonaggregative C. auris strains 10 and 12 was 2.4 and 1.5 times less, respectively, than those for C. albicans, and biomass for aggregative C. auris strains 2 and 6 was 3.0 and 3.1 times less, respectively. However, these strains formed significantly greater bio-films (p<0.0001) than those formed by C. glabrata (3.8, 6.0, 3.0, and 2.9 times more for strains 10, 12, 2, and 6, respectively). We confirmed these findings for each species by scanning electron microscopy after growing the strains on Thermanox Coverslips (Thermo Fisher Scientific, Pais-ley, UK) for 24 h, as previously described (12). C. albi-cans biofilms were typically densely packed with hyphae (Figure 1, panel B), whereas C. glabrata formed a sparse biofilm consisting of yeast cells only, without extracellular matrix (Figure 1, panel C). C. auris strain 10 biofilm for-mation was intermediate to the C. albicans and C. glabrata phenotypes, showing predominately budding yeast and oc-casional pseudohyphae (Figure 1, panel D). In agreement with previous findings (9), all tested C. auris strains dis-played the same phenotype.To determine MICs for planktonic and sessile cells of the C. auris strains, we performed antifungal susceptibility testing using standardized Clinical Laboratory Standards Institute M27-A3 broth microdilution (visual inspection) and standardized candidal biofilm testing (metabolic vi-ability) with fluconazole, voriconazole, caspofungin, mi-cafungin, liposomal amphotericin B, amphotericin B, and chlorhexidine (13,14). Antifungal agents were tested in se-rial 2-fold dilutions (0.06–32.0 mg/L) for planktonic and sessile cells. Fluconazole was ineffective (MICs of >32 mg/L) against planktonic and sessile communities, whereas Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida aurisAuthor affiliations: University of Glasgow, Glasgow, Scotland, UK (L. Sherry, G. Ramage, R. Kean); Public Health England, Bristol, UK (A. Borman, E.M. Johnson); The University of Manchester, Manchester, UK (M.D. Richardson, R. Rautemaa-Richardson); University Hospital of South Manchester, Manchester  (M.D. Richardson, R. Rautemaa-Richardson)DOI: http://dx.doi.org/10.3201/eid2302.161320 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 329voriconazole displayed minimal activity against planktonic cells (Table 1, 2). Although liposomal amphotericin B was active against planktonic C. auris at 0.25–1.0 mg/L, up to 16 mg/L was required to reduce biofilm metabolic viability by 90%. Amphotericin B was more effective, requiring 4 mg/L to kill biofilms. Micafungin was the most active echi-nocandin, requiring <0.5 mg/L to inhibit planktonic cells, compared with 2–32 mg/L for caspofungin. However, these 2 antifungal agents were ineffective against biofilms, requir-ing >32 mg/L to inhibit sessile cells. Of note, chlorhexidine exhibited the greatest activity, requiring <0.02% to effec-tively inhibit planktonic and sessile cells across all strains tested. All strains showed similar sensitivity profiles, with the exception of strain 10, for which voriconazole was re-quired in higher concentrations and caspofungin in lower concentrations to effectively inhibit planktonic growth.Killing assays in Galleria mellonella were performed, as previously described (12), to assess the pathogenicity of each Candida species. Ten G. mellonella larvae (Livefoods Direct Ltd, Sheffield, UK) with bodyweights of ≈300 mg were used for each test group. Standardized inoculums of 106 and 105 and to 5 × 105 and 5 × 104 cells/larvae (Figure 2) in PBS, were injected into the hemocoel, as previously described (9). We assessed pathogenicity using a Kaplan-Meier plot, monitoring the percent survival over 5 days. Survival data for 5 × 105 cells/larvae showed a significant difference in the killing of larvae by C. glabrata and the other Candida species (p<0.0001) (Figure 2, panel B). Al-though C. albicans and C. auris had similar kill kinetics in this model, infection with nonaggregative C. auris strain 10 achieved a 100% death rate within 48 h, compared with a rate of ≈87% with C. albicans (p = 0.3076). Moreover, Figure 1. Biofilm formation on Candida auris, C. albicans, and C. glabrata yeast strains. A) Biomass quantities were determined spectrophotometrically for 4 strains of C. auris and 1 each of C. albicans and C. glabrata. Isolates were standardized to 106 cells/mL in RPMI-1640 and grown in flat-bottomed 96-well microtiter plates for 24 h at 37°C. Biofilms were then washed, stained with crystal violet solution, and quantified. Data represent the mean ± SD of experiments performed on 3 separate occasions, using 8 replicates for each strain. Results show that C. auris can form heterogeneous intermediate biofilms. B–D) C. albicans (B), C. glabrata (C) and C. auris (D) were also grown on Thermanox coverslips (Thermo Fisher Scientific, Paisley, UK) for 24 h at 37°C. Biofilms were then processed and viewed on a JEOL (Tokyo, Japan) JSM-6400 scanning electron microscope; images were assembled using Photoshop software (http://www.photoshop.com/products). Arrow indicates pseudohyphae in C. auris biofilm (D). Scale bars represent 20 μm (original magnification ×1,000). OD, optical density.   Table 1. Planktonic susceptibility profiles of 7 antifungals against Candida auris yeast Drug Planktonic MIC* Strain 2 Strain 6 Strain 10 Strain 12 Fluconazole >32 >32 >32 >32 Voriconazole 8 8 32 1 Caspofungin 32 32 2 >32 Micafungin 0.5 <0.0625 <0.06 <0.0625 Liposomal amphotericin B 0.25 0.25 0.5 1 Amphotericin B 0.25 0.25 0.5 0.5 Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 *Values are mg/L except as indicated. All MIC tests were performed on 3 independent occasions, showing identical results each time.  Biofilm-Forming Capability of Candida aurisDISPATCHES330 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017nonaggregative C. auris was significantly more pathogenic than C. albicans when a lower inoculum of 105 (p<0.05) and 5 × 104 cells/larvae (p<0.01) was administered. These data, along with those of Borman et al. (9), suggest that the nonaggregative C. auris phenotype has the capacity to form biofilms with enhanced virulence capacity.ConclusionsBiofilm formation is a key driver of C. albicans patho-genicity and is associated with patient death (10,15). We show that C. auris can differentially adhere to polymeric surfaces, form biofilms, and resist antifungal agents that are active against its planktonic counterparts. Of particular interest, caspofungin was predominately inactive against C. auris biofilms; this finding was unexpected because caspofungin is normally highly effective against Candida biofilms. These features contribute not only to C. auris vir-ulence but also to its survival in hospital environments, in-creasing its ability to cause outbreaks (5). The results of the in vivo model used in this study are in line with our clinical experience and validated by findings in other in vivo stud-ies (9), affirming that C. auris is highly virulent or more virulent than C. albicans.Although unable to form biofilms equivalent to C. al-bicans, C. auris has a noteworthy virulence capacity that merits further exploration, particularly given the apparent heterogeneity associated with aggregative capacity. These factors, together with the innate resistance of C. auris to most antifungal agents, may explain why it is an emerg-ing pathogen. Our findings suggest it is improbable that the spread and prevalence of C. auris can be controlled with antifungal stewardship approaches alone. We showed that chlorhexidine is effective against C. auris planktonic and sessile communities. Thus, use of this disinfectant can be advocated for topical control of C. auris at standard con-centrations used for skin and wound cleansing and dis-infection (0.05%–4.0%). Infection-prevention measures targeting C. auris biofilms in patients, on medical devices (e.g., equipment in contact with patients), and in the hospi-tal environment will be required.   Table 2. Sessile susceptibility profiles of 7 antifungals against Candida auris yeast Drug Sessile MIC* Strain 2 Strain 6 Strain 10 Strain 12 Fluconazole >32 >32 >32 >32 Voriconazole >32 >32 >32 >32 Caspofungin >32 >32 >32 >32 Micafungin >32 >32 0.25 >32 Liposomal amphotericin B 2 8 16 16 Amphotericin B 2 4 2 4 Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 *Values indicate mg/L except as indicated. Sessile MICs are defined as a 90% inhibition of the metabolic dye XTT, 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (Sigma-Aldrich, Dorset, UK) compared with the untreated control; MIC tests were performed on 3 independent occasions and showed identical results each time.  Figure 2. Pathogenicity of Candida species yeast infections in vivo. Galleria mellonella larvae were infected with 106 (A), 5 × 105 (B), 105 (C), and 5 × 104 (D) cells/larvae of C. albicans, C. glabrata, and 4 C. auris strains, and larvae survival measured every 12 h over 5 d. Ten samples of each yeast were used, and experiments were performed on 3 independent occasions. Data represents the mean percentage survival, as determined using a Kaplan-Meier plot. PBS and controls, which were pierced only, were also included and had no effect on larvae survival. Results show that C. auris and C. albicans infection exhibit similar pathogenicity. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 331Biofilm-Forming Capability of Candida aurisDr. Sherry works as a medical mycologist at the University of Glasgow, Glasgow, UK. Her specific interest is fungal biofilms of clinical significance.References  1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K,  Yamaguchi H. Candida auris sp. nov., a novel ascomycetous  yeast isolated from the external ear canal of an inpatient in a  Japanese hospital. Microbiol Immunol. 2009;53:41–4.  http://dx.doi.org/10.1111/j.1348-0421.2008.00083.x  2. Kathuria S, Singh PK, 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, CLSI broth microdilution, and Etest method. J Clin Microbiol. 2015;53:1823–30. http://dx.doi.org/10.1128/JCM.00367-15  3. Centers for Disease Control and Prevention. Clinical alert to US healthcare facilities—June 2016. Global emergence of invasive infections caused by the multidrug-resistant yeast Candida auris. 2016 [cited 2016 Jun 24]. http://www.cdc.gov/fungal/diseases/candidiasis/candida-auris-alert.html  4. Public Health England. Candida auris identified in England. 2016 [cited 2016 Jul 1]. https://www.gov.uk/government/publications/candida-auris-emergence-in-england/candida-auris-identified-in-england  5. Calvo B, Melo AS, Perozo-Mena A, Hernandez M, Francisco EC, Hagen F, et al. First report of Candida auris in America: Clinical and microbiological aspects of 18 episodes of candidemia. J Infect. 2016;73:369–74. http://dx.doi.org/10.1016/j.jinf.2016.07.008  6. Kumar D, Banerjee T, Pratap CB, Tilak R. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin  activity from a case of vulvovaginitis. J Infect Dev Ctries. 2015;9:435–7. http://dx.doi.org/10.3855/jidc.4582  7. Oh BJ, Shin JH, Kim MN, Sung H, Lee K, Joo MY, et al.  Biofilm formation and genotyping of Candida haemulonii,  Candida pseudohaemulonii, and a proposed new species (Candida auris) isolates from Korea. Med Mycol. 2011;49:98–102.  http://dx.doi.org/10.3109/13693786.2010.4935638. Chatterjee S, Alampalli SV, Nageshan RK, Chettiar ST, Joshi S, Tatu US. Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris. BMC Genomics. 2015;16:686. http://dx.doi.org/10.1186/s12864-015-1863-z9. Borman AM, Szekely A, Johnson EM. Comparative pathogenicity of United Kingdom isolates of the emerging pathogen Candida auris and other key pathogenic Candida species. mSphere. 2016;1:pii:e00189-16.10. Rajendran R, Sherry L, Nile CJ, Sherriff A, Johnson EM,  Hanson MF, et al. Biofilm formation is a risk factor for mortality  in patients with Candida albicans bloodstream infection- Scotland, 2012-2013. Clin Microbiol Infect. 2016;22:87–93.  http://dx.doi.org/10.1016/j.cmi.2015.09.01811. Ramage G, Vande Walle K, Wickes BL, López-Ribot JL.  Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother. 2001;45:2475–9. http://dx.doi.org/10.1128/AAC.45.9.2475-2479.200112. Sherry L, Rajendran R, Lappin DF, Borghi E, Perdoni F, Falleni M,  et al. Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity. BMC Microbiol. 2014;14:182. http://dx.doi.org/10.1186/1471-2180-14-18213. Clinical Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts.  Approved standard M27–A3, 3rd ed. Wayne, PA: The Institute; 2008.14. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G, et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc. 2008;3:1494–500. http://dx.doi.org/10.1038/nprot.2008.14115. Rajendran R, Sherry L, Deshpande A, Johnson EM, Hanson MF, Williams C, et al. A prospective surveillance study of  candidaemia: epidemiology, risk factors, antifungal treatment and outcome in hospitalized patients. Front Microbiol. 2016;7:915. http://dx.doi.org/10.3389/fmicb.2016.00915Address for correspondence: Riina Rautemaa-Richardson, Education and Research Centre, Wythenshawe Hospital, Southmoor Rd, Manchester M23 9LT, UK; email: riina.richardson@manchester.ac.ukIn 1974, Lewis Thomas (1913–1993), physician, professor, and dean, published The Lives of A Cell, the first of 2 books subtitled Notes of a Biology Watcher. The phrase “lives of a cell” refers to the independent yet interrelated parts of a human cell—including mitochondria, centrioles, and basal bodies—that once led independent lives. Without these previously independent lives working together, we would not have the capacity for thought, communication, and movement. Dr. Thomas wrote, “Our membranes hold against equilibrium, maintain imbalance, bank against entropy…. We are shared, rent-ed and occupied.”Our human lives do not depend just on the lives in our individual cells. Our lives depend fully on the earth, including the atmosphere, and the many other human and nonhuman lives that occupy it. In explaining this complex interdependence, Dr. Thomas observed that the earth is “most like a cell.” This second interpretation of lives of a cell refers to the many interrelated earthly entities, such as plants, whales, humans, and even viruses, that “dart rather like bees from organism to organism, from plant to insect to mammal to me and back again,” all protected by the sky—a membrane that “works, and for what it is designed to accomplish it is as infallible as anything in nature.”EID Podcast: Lives of a Cell:  40 Years Later, A Third InterpretationVisit our website to listen: http://www2c.cdc.gov/podcasts/player.asp?f=8637494

Biofilm-forming capability of highly virulent, multidrug-resistant Candida auris

The emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare-associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro.</p

Johnson, Elizabeth M

Richardson, Malcolm D

The University of Manchester - Institutional Repository

The University of Manchester ResearchBiofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida aurisDOI:10.3201/eid2302.161320Document VersionFinal published versionLink to publication record in Manchester Research ExplorerCitation for published version (APA):Sherry, L., Ramage, G., Kean, R., Borman, A., Johnson, E. M., Richardson, M. D., & Rautemaa-Richardson, R.(2017). Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida auris. Emerging infectiousdiseases, 23(2), 328-331. https://doi.org/10.3201/eid2302.161320Published in:Emerging infectious diseasesCiting this paperPlease note that where the full-text provided on Manchester Research Explorer is the Author Accepted Manuscriptor Proof version this may differ from the final Published version. If citing, it is advised that you check and use thepublisher's definitive version.General rightsCopyright and moral rights for the publications made accessible in the Research Explorer are retained by theauthors and/or other copyright owners and it is a condition of accessing publications that users recognise andabide by the legal requirements associated with these rights.Takedown policyIf you believe that this document breaches copyright please refer to the University of Manchester’s TakedownProcedures [http://man.ac.uk/04Y6Bo] or contact uml.scholarlycommunications@manchester.ac.uk providingrelevant details, so we can investigate your claim.Download date:12. Nov. 2022328 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017DISPATCHESLeighann Sherry, Gordon Ramage, Ryan Kean, Andrew Borman, Elizabeth M. Johnson,  Malcolm D. Richardson,  Riina Rautemaa-RichardsonThe emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare–associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro.The yeast pathogen Candida auris was first detected in 2009 from an ear canal infection in Japan (1). This spe-cies initially attracted attention because of its reduced sus-ceptibility to azoles and amphotericin B, combined with the lack of reliable culture-based methods for its identification (2). More recently, C. auris has been associated globally with life-threatening invasive diseases, such as bloodstream and wound infections. C. auris has also caused hospital outbreaks across Asia and South America, as highlighted in a 2016 clinical alert (3). In addition, in a UK intensive care unit, candidemia developed in 20% of patients colo-nized with C. auris (4). Although the mode of transmission within hospitals is unknown, C. auris may substantially contaminate rooms of colonized or infected patients (5). Phospholipase and proteinase activity have been identi-fied as virulence factors (6); however, because previously used assessment techniques were rudimentary, this patho-gen’s ability to form biofilm remains under question (7). The draft genome identifying various proteins involved in biofilm formation (8), coupled with recent descriptions of aggregative and nonaggregative phenotypes, the latter of which are more virulent in vivo (9), indicate the possibility of heterogeneous C. auris biofilm formation, as described for C. albicans (10). We sought to examine these aggrega-tive and nonaggregative C. auris phenotypes in the context of biofilm-forming capacity, investigate their susceptibil-ity to a panel of antifungal agents and the skin disinfectant chlorhexidine, and investigate their virulence in vivo.The StudyThroughout this study, we used C. albicans SC5314 and Candida glabrata WT2001 as comparators for C. auris nonaggregative strains NCPF 8971 (strain 10) and NCPF 8973 (strain 12) and aggregative strains NCPF 8977 (strain 2) and NCPF 8978 (strain 6), as previously described (9). Strains were propagated in YPD broth (Sigma-Aldrich, Dorset, UK), incubated overnight at 30°C, and adjusted to 106 cells/mL in RPMI 1640 medium (11). On 3 separate occasions, 8 biofilms of each Candida species were grown in flat-bottomed, 96-well polystyrene microtiter plates and incubated for 24 h at 37°C, after which biomass was as-sessed by crystal violet assay (12). C. albicans displayed the greatest biofilm mass (Figure 1, panel A), consistent with previous findings  (10). Compared with C. albicans, all C. auris strains formed significantly reduced biofilms (p<0.0001): biomass for nonaggregative C. auris strains 10 and 12 was 2.4 and 1.5 times less, respectively, than those for C. albicans, and biomass for aggregative C. auris strains 2 and 6 was 3.0 and 3.1 times less, respectively. However, these strains formed significantly greater bio-films (p<0.0001) than those formed by C. glabrata (3.8, 6.0, 3.0, and 2.9 times more for strains 10, 12, 2, and 6, respectively). We confirmed these findings for each species by scanning electron microscopy after growing the strains on Thermanox Coverslips (Thermo Fisher Scientific, Pais-ley, UK) for 24 h, as previously described (12). C. albi-cans biofilms were typically densely packed with hyphae (Figure 1, panel B), whereas C. glabrata formed a sparse biofilm consisting of yeast cells only, without extracellular matrix (Figure 1, panel C). C. auris strain 10 biofilm for-mation was intermediate to the C. albicans and C. glabrata phenotypes, showing predominately budding yeast and oc-casional pseudohyphae (Figure 1, panel D). In agreement with previous findings (9), all tested C. auris strains dis-played the same phenotype.To determine MICs for planktonic and sessile cells of the C. auris strains, we performed antifungal susceptibility testing using standardized Clinical Laboratory Standards Institute M27-A3 broth microdilution (visual inspection) and standardized candidal biofilm testing (metabolic vi-ability) with fluconazole, voriconazole, caspofungin, mi-cafungin, liposomal amphotericin B, amphotericin B, and chlorhexidine (13,14). Antifungal agents were tested in se-rial 2-fold dilutions (0.06–32.0 mg/L) for planktonic and sessile cells. Fluconazole was ineffective (MICs of >32 mg/L) against planktonic and sessile communities, whereas Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida aurisAuthor affiliations: University of Glasgow, Glasgow, Scotland, UK (L. Sherry, G. Ramage, R. Kean); Public Health England, Bristol, UK (A. Borman, E.M. Johnson); The University of Manchester, Manchester, UK (M.D. Richardson, R. Rautemaa-Richardson); University Hospital of South Manchester, Manchester  (M.D. Richardson, R. Rautemaa-Richardson)DOI: http://dx.doi.org/10.3201/eid2302.161320 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 329voriconazole displayed minimal activity against planktonic cells (Table 1, 2). Although liposomal amphotericin B was active against planktonic C. auris at 0.25–1.0 mg/L, up to 16 mg/L was required to reduce biofilm metabolic viability by 90%. Amphotericin B was more effective, requiring 4 mg/L to kill biofilms. Micafungin was the most active echi-nocandin, requiring <0.5 mg/L to inhibit planktonic cells, compared with 2–32 mg/L for caspofungin. However, these 2 antifungal agents were ineffective against biofilms, requir-ing >32 mg/L to inhibit sessile cells. Of note, chlorhexidine exhibited the greatest activity, requiring <0.02% to effec-tively inhibit planktonic and sessile cells across all strains tested. All strains showed similar sensitivity profiles, with the exception of strain 10, for which voriconazole was re-quired in higher concentrations and caspofungin in lower concentrations to effectively inhibit planktonic growth.Killing assays in Galleria mellonella were performed, as previously described (12), to assess the pathogenicity of each Candida species. Ten G. mellonella larvae (Livefoods Direct Ltd, Sheffield, UK) with bodyweights of ≈300 mg were used for each test group. Standardized inoculums of 106 and 105 and to 5 × 105 and 5 × 104 cells/larvae (Figure 2) in PBS, were injected into the hemocoel, as previously described (9). We assessed pathogenicity using a Kaplan-Meier plot, monitoring the percent survival over 5 days. Survival data for 5 × 105 cells/larvae showed a significant difference in the killing of larvae by C. glabrata and the other Candida species (p<0.0001) (Figure 2, panel B). Al-though C. albicans and C. auris had similar kill kinetics in this model, infection with nonaggregative C. auris strain 10 achieved a 100% death rate within 48 h, compared with a rate of ≈87% with C. albicans (p = 0.3076). Moreover, Figure 1. Biofilm formation on Candida auris, C. albicans, and C. glabrata yeast strains. A) Biomass quantities were determined spectrophotometrically for 4 strains of C. auris and 1 each of C. albicans and C. glabrata. Isolates were standardized to 106 cells/mL in RPMI-1640 and grown in flat-bottomed 96-well microtiter plates for 24 h at 37°C. Biofilms were then washed, stained with crystal violet solution, and quantified. Data represent the mean ± SD of experiments performed on 3 separate occasions, using 8 replicates for each strain. Results show that C. auris can form heterogeneous intermediate biofilms. B–D) C. albicans (B), C. glabrata (C) and C. auris (D) were also grown on Thermanox coverslips (Thermo Fisher Scientific, Paisley, UK) for 24 h at 37°C. Biofilms were then processed and viewed on a JEOL (Tokyo, Japan) JSM-6400 scanning electron microscope; images were assembled using Photoshop software (http://www.photoshop.com/products). Arrow indicates pseudohyphae in C. auris biofilm (D). Scale bars represent 20 μm (original magnification ×1,000). OD, optical density.   Table 1. Planktonic susceptibility profiles of 7 antifungals against Candida auris yeast Drug Planktonic MIC* Strain 2 Strain 6 Strain 10 Strain 12 Fluconazole >32 >32 >32 >32 Voriconazole 8 8 32 1 Caspofungin 32 32 2 >32 Micafungin 0.5 <0.0625 <0.06 <0.0625 Liposomal amphotericin B 0.25 0.25 0.5 1 Amphotericin B 0.25 0.25 0.5 0.5 Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 *Values are mg/L except as indicated. All MIC tests were performed on 3 independent occasions, showing identical results each time.  Biofilm-Forming Capability of Candida aurisDISPATCHES330 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017nonaggregative C. auris was significantly more pathogenic than C. albicans when a lower inoculum of 105 (p<0.05) and 5 × 104 cells/larvae (p<0.01) was administered. These data, along with those of Borman et al. (9), suggest that the nonaggregative C. auris phenotype has the capacity to form biofilms with enhanced virulence capacity.ConclusionsBiofilm formation is a key driver of C. albicans patho-genicity and is associated with patient death (10,15). We show that C. auris can differentially adhere to polymeric surfaces, form biofilms, and resist antifungal agents that are active against its planktonic counterparts. Of particular interest, caspofungin was predominately inactive against C. auris biofilms; this finding was unexpected because caspofungin is normally highly effective against Candida biofilms. These features contribute not only to C. auris vir-ulence but also to its survival in hospital environments, in-creasing its ability to cause outbreaks (5). The results of the in vivo model used in this study are in line with our clinical experience and validated by findings in other in vivo stud-ies (9), affirming that C. auris is highly virulent or more virulent than C. albicans.Although unable to form biofilms equivalent to C. al-bicans, C. auris has a noteworthy virulence capacity that merits further exploration, particularly given the apparent heterogeneity associated with aggregative capacity. These factors, together with the innate resistance of C. auris to most antifungal agents, may explain why it is an emerg-ing pathogen. Our findings suggest it is improbable that the spread and prevalence of C. auris can be controlled with antifungal stewardship approaches alone. We showed that chlorhexidine is effective against C. auris planktonic and sessile communities. Thus, use of this disinfectant can be advocated for topical control of C. auris at standard con-centrations used for skin and wound cleansing and dis-infection (0.05%–4.0%). Infection-prevention measures targeting C. auris biofilms in patients, on medical devices (e.g., equipment in contact with patients), and in the hospi-tal environment will be required.   Table 2. Sessile susceptibility profiles of 7 antifungals against Candida auris yeast Drug Sessile MIC* Strain 2 Strain 6 Strain 10 Strain 12 Fluconazole >32 >32 >32 >32 Voriconazole >32 >32 >32 >32 Caspofungin >32 >32 >32 >32 Micafungin >32 >32 0.25 >32 Liposomal amphotericin B 2 8 16 16 Amphotericin B 2 4 2 4 Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 *Values indicate mg/L except as indicated. Sessile MICs are defined as a 90% inhibition of the metabolic dye XTT, 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (Sigma-Aldrich, Dorset, UK) compared with the untreated control; MIC tests were performed on 3 independent occasions and showed identical results each time.  Figure 2. Pathogenicity of Candida species yeast infections in vivo. Galleria mellonella larvae were infected with 106 (A), 5 × 105 (B), 105 (C), and 5 × 104 (D) cells/larvae of C. albicans, C. glabrata, and 4 C. auris strains, and larvae survival measured every 12 h over 5 d. Ten samples of each yeast were used, and experiments were performed on 3 independent occasions. Data represents the mean percentage survival, as determined using a Kaplan-Meier plot. PBS and controls, which were pierced only, were also included and had no effect on larvae survival. Results show that C. auris and C. albicans infection exhibit similar pathogenicity. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 331Biofilm-Forming Capability of Candida aurisDr. Sherry works as a medical mycologist at the University of Glasgow, Glasgow, UK. Her specific interest is fungal biofilms of clinical significance.References  1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K,  Yamaguchi H. Candida auris sp. nov., a novel ascomycetous  yeast isolated from the external ear canal of an inpatient in a  Japanese hospital. Microbiol Immunol. 2009;53:41–4.  http://dx.doi.org/10.1111/j.1348-0421.2008.00083.x  2. Kathuria S, Singh PK, 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, CLSI broth microdilution, and Etest method. J Clin Microbiol. 2015;53:1823–30. http://dx.doi.org/10.1128/JCM.00367-15  3. Centers for Disease Control and Prevention. Clinical alert to US healthcare facilities—June 2016. Global emergence of invasive infections caused by the multidrug-resistant yeast Candida auris. 2016 [cited 2016 Jun 24]. http://www.cdc.gov/fungal/diseases/candidiasis/candida-auris-alert.html  4. Public Health England. Candida auris identified in England. 2016 [cited 2016 Jul 1]. https://www.gov.uk/government/publications/candida-auris-emergence-in-england/candida-auris-identified-in-england  5. Calvo B, Melo AS, Perozo-Mena A, Hernandez M, Francisco EC, Hagen F, et al. First report of Candida auris in America: Clinical and microbiological aspects of 18 episodes of candidemia. J Infect. 2016;73:369–74. http://dx.doi.org/10.1016/j.jinf.2016.07.008  6. Kumar D, Banerjee T, Pratap CB, Tilak R. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin  activity from a case of vulvovaginitis. J Infect Dev Ctries. 2015;9:435–7. http://dx.doi.org/10.3855/jidc.4582  7. Oh BJ, Shin JH, Kim MN, Sung H, Lee K, Joo MY, et al.  Biofilm formation and genotyping of Candida haemulonii,  Candida pseudohaemulonii, and a proposed new species (Candida auris) isolates from Korea. Med Mycol. 2011;49:98–102.  http://dx.doi.org/10.3109/13693786.2010.4935638. Chatterjee S, Alampalli SV, Nageshan RK, Chettiar ST, Joshi S, Tatu US. Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris. BMC Genomics. 2015;16:686. http://dx.doi.org/10.1186/s12864-015-1863-z9. Borman AM, Szekely A, Johnson EM. Comparative pathogenicity of United Kingdom isolates of the emerging pathogen Candida auris and other key pathogenic Candida species. mSphere. 2016;1:pii:e00189-16.10. Rajendran R, Sherry L, Nile CJ, Sherriff A, Johnson EM,  Hanson MF, et al. Biofilm formation is a risk factor for mortality  in patients with Candida albicans bloodstream infection- Scotland, 2012-2013. Clin Microbiol Infect. 2016;22:87–93.  http://dx.doi.org/10.1016/j.cmi.2015.09.01811. Ramage G, Vande Walle K, Wickes BL, López-Ribot JL.  Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother. 2001;45:2475–9. http://dx.doi.org/10.1128/AAC.45.9.2475-2479.200112. Sherry L, Rajendran R, Lappin DF, Borghi E, Perdoni F, Falleni M,  et al. Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity. BMC Microbiol. 2014;14:182. http://dx.doi.org/10.1186/1471-2180-14-18213. Clinical Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts.  Approved standard M27–A3, 3rd ed. Wayne, PA: The Institute; 2008.14. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G, et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc. 2008;3:1494–500. http://dx.doi.org/10.1038/nprot.2008.14115. Rajendran R, Sherry L, Deshpande A, Johnson EM, Hanson MF, Williams C, et al. A prospective surveillance study of  candidaemia: epidemiology, risk factors, antifungal treatment and outcome in hospitalized patients. Front Microbiol. 2016;7:915. http://dx.doi.org/10.3389/fmicb.2016.00915Address for correspondence: Riina Rautemaa-Richardson, Education and Research Centre, Wythenshawe Hospital, Southmoor Rd, Manchester M23 9LT, UK; email: riina.richardson@manchester.ac.ukIn 1974, Lewis Thomas (1913–1993), physician, professor, and dean, published The Lives of A Cell, the first of 2 books subtitled Notes of a Biology Watcher. The phrase “lives of a cell” refers to the independent yet interrelated parts of a human cell—including mitochondria, centrioles, and basal bodies—that once led independent lives. Without these previously independent lives working together, we would not have the capacity for thought, communication, and movement. Dr. Thomas wrote, “Our membranes hold against equilibrium, maintain imbalance, bank against entropy…. We are shared, rent-ed and occupied.”Our human lives do not depend just on the lives in our individual cells. Our lives depend fully on the earth, including the atmosphere, and the many other human and nonhuman lives that occupy it. In explaining this complex interdependence, Dr. Thomas observed that the earth is “most like a cell.” This second interpretation of lives of a cell refers to the many interrelated earthly entities, such as plants, whales, humans, and even viruses, that “dart rather like bees from organism to organism, from plant to insect to mammal to me and back again,” all protected by the sky—a membrane that “works, and for what it is designed to accomplish it is as infallible as anything in nature.”EID Podcast: Lives of a Cell:  40 Years Later, A Third InterpretationVisit our website to listen: http://www2c.cdc.gov/podcasts/player.asp?f=8637494

Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida auris

Leighann Sherry

Gordon Ramage

Ryan Kean

Andrew Borman

Elizabeth M. Johnson

Malcolm D. Richardson

Riina Rautemaa-Richardson

Crossref

Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant<i>Candida auris</i>

Enlighten: Publications

328 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017
DISPATCHES
Leighann Sherry, Gordon Ramage, Ryan Kean, 
Andrew Borman, Elizabeth M. Johnson,  
Malcolm D. Richardson,  
Riina Rautemaa-Richardson
The emerging multidrug-resistant yeast pathogen Candida 
auris has attracted considerable attention as a source of 
healthcare–associated infections. We report that this highly 
virulent yeast has the capacity to form antifungal resistant 
biofilms sensitive to the disinfectant chlorhexidine in vitro.
The yeast pathogen Candida auris was first detected in 2009 from an ear canal infection in Japan (1). This spe-
cies initially attracted attention because of its reduced sus-
ceptibility to azoles and amphotericin B, combined with the 
lack of reliable culture-based methods for its identification 
(2). More recently, C. auris has been associated globally 
with life-threatening invasive diseases, such as bloodstream 
and wound infections. C. auris has also caused hospital 
outbreaks across Asia and South America, as highlighted 
in a 2016 clinical alert (3). In addition, in a UK intensive 
care unit, candidemia developed in 20% of patients colo-
nized with C. auris (4). Although the mode of transmission 
within hospitals is unknown, C. auris may substantially 
contaminate rooms of colonized or infected patients (5). 
Phospholipase and proteinase activity have been identi-
fied as virulence factors (6); however, because previously 
used assessment techniques were rudimentary, this patho-
gen’s ability to form biofilm remains under question (7). 
The draft genome identifying various proteins involved in 
biofilm formation (8), coupled with recent descriptions of 
aggregative and nonaggregative phenotypes, the latter of 
which are more virulent in vivo (9), indicate the possibility 
of heterogeneous C. auris biofilm formation, as described 
for C. albicans (10). We sought to examine these aggrega-
tive and nonaggregative C. auris phenotypes in the context 
of biofilm-forming capacity, investigate their susceptibil-
ity to a panel of antifungal agents and the skin disinfectant 
chlorhexidine, and investigate their virulence in vivo.
The Study
Throughout this study, we used C. albicans SC5314 and 
Candida glabrata WT2001 as comparators for C. auris 
nonaggregative strains NCPF 8971 (strain 10) and NCPF 
8973 (strain 12) and aggregative strains NCPF 8977 (strain 
2) and NCPF 8978 (strain 6), as previously described (9). 
Strains were propagated in YPD broth (Sigma-Aldrich, 
Dorset, UK), incubated overnight at 30°C, and adjusted to 
106 cells/mL in RPMI 1640 medium (11). On 3 separate 
occasions, 8 biofilms of each Candida species were grown 
in flat-bottomed, 96-well polystyrene microtiter plates and 
incubated for 24 h at 37°C, after which biomass was as-
sessed by crystal violet assay (12). C. albicans displayed 
the greatest biofilm mass (Figure 1, panel A), consistent 
with previous findings  (10). Compared with C. albicans, 
all C. auris strains formed significantly reduced biofilms 
(p<0.0001): biomass for nonaggregative C. auris strains 
10 and 12 was 2.4 and 1.5 times less, respectively, than 
those for C. albicans, and biomass for aggregative C. auris 
strains 2 and 6 was 3.0 and 3.1 times less, respectively. 
However, these strains formed significantly greater bio-
films (p<0.0001) than those formed by C. glabrata (3.8, 
6.0, 3.0, and 2.9 times more for strains 10, 12, 2, and 6, 
respectively). We confirmed these findings for each species 
by scanning electron microscopy after growing the strains 
on Thermanox Coverslips (Thermo Fisher Scientific, Pais-
ley, UK) for 24 h, as previously described (12). C. albi-
cans biofilms were typically densely packed with hyphae 
(Figure 1, panel B), whereas C. glabrata formed a sparse 
biofilm consisting of yeast cells only, without extracellular 
matrix (Figure 1, panel C). C. auris strain 10 biofilm for-
mation was intermediate to the C. albicans and C. glabrata 
phenotypes, showing predominately budding yeast and oc-
casional pseudohyphae (Figure 1, panel D). In agreement 
with previous findings (9), all tested C. auris strains dis-
played the same phenotype.
To determine MICs for planktonic and sessile cells of 
the C. auris strains, we performed antifungal susceptibility 
testing using standardized Clinical Laboratory Standards 
Institute M27-A3 broth microdilution (visual inspection) 
and standardized candidal biofilm testing (metabolic vi-
ability) with fluconazole, voriconazole, caspofungin, mi-
cafungin, liposomal amphotericin B, amphotericin B, and 
chlorhexidine (13,14). Antifungal agents were tested in se-
rial 2-fold dilutions (0.06–32.0 mg/L) for planktonic and 
sessile cells. Fluconazole was ineffective (MICs of >32 
mg/L) against planktonic and sessile communities, whereas 
Biofilm-Forming Capability of Highly Virulent, 
Multidrug-Resistant Candida auris
Author affiliations: University of Glasgow, Glasgow, Scotland, UK 
(L. Sherry, G. Ramage, R. Kean); Public Health England, Bristol, 
UK (A. Borman, E.M. Johnson); The University of Manchester, 
Manchester, UK (M.D. Richardson, R. Rautemaa-Richardson); 
University Hospital of South Manchester, Manchester  
(M.D. Richardson, R. Rautemaa-Richardson)
DOI: http://dx.doi.org/10.3201/eid2302.161320
 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 329
voriconazole displayed minimal activity against planktonic 
cells (Table 1, 2). Although liposomal amphotericin B was 
active against planktonic C. auris at 0.25–1.0 mg/L, up to 
16 mg/L was required to reduce biofilm metabolic viability 
by 90%. Amphotericin B was more effective, requiring 4 
mg/L to kill biofilms. Micafungin was the most active echi-
nocandin, requiring <0.5 mg/L to inhibit planktonic cells, 
compared with 2–32 mg/L for caspofungin. However, these 
2 antifungal agents were ineffective against biofilms, requir-
ing >32 mg/L to inhibit sessile cells. Of note, chlorhexidine 
exhibited the greatest activity, requiring <0.02% to effec-
tively inhibit planktonic and sessile cells across all strains 
tested. All strains showed similar sensitivity profiles, with 
the exception of strain 10, for which voriconazole was re-
quired in higher concentrations and caspofungin in lower 
concentrations to effectively inhibit planktonic growth.
Killing assays in Galleria mellonella were performed, 
as previously described (12), to assess the pathogenicity of 
each Candida species. Ten G. mellonella larvae (Livefoods 
Direct Ltd, Sheffield, UK) with bodyweights of ≈300 mg 
were used for each test group. Standardized inoculums of 
106 and 105 and to 5 × 105 and 5 × 104 cells/larvae (Figure 
2) in PBS, were injected into the hemocoel, as previously 
described (9). We assessed pathogenicity using a Kaplan-
Meier plot, monitoring the percent survival over 5 days. 
Survival data for 5 × 105 cells/larvae showed a significant 
difference in the killing of larvae by C. glabrata and the 
other Candida species (p<0.0001) (Figure 2, panel B). Al-
though C. albicans and C. auris had similar kill kinetics in 
this model, infection with nonaggregative C. auris strain 
10 achieved a 100% death rate within 48 h, compared with 
a rate of ≈87% with C. albicans (p = 0.3076). Moreover, 
Figure 1. Biofilm formation 
on Candida auris, C. 
albicans, and C. glabrata 
yeast strains. A) Biomass 
quantities were determined 
spectrophotometrically for 
4 strains of C. auris and 
1 each of C. albicans and 
C. glabrata. Isolates were 
standardized to 106 cells/
mL in RPMI-1640 and grown 
in flat-bottomed 96-well 
microtiter plates for 24 h at 
37°C. Biofilms were then 
washed, stained with crystal 
violet solution, and quantified. 
Data represent the mean ± 
SD of experiments performed 
on 3 separate occasions, 
using 8 replicates for each 
strain. Results show that C. 
auris can form heterogeneous 
intermediate biofilms. B–D) 
C. albicans (B), C. glabrata 
(C) and C. auris (D) were 
also grown on Thermanox 
coverslips (Thermo Fisher 
Scientific, Paisley, UK) for 24 
h at 37°C. Biofilms were then processed and viewed on a JEOL (Tokyo, Japan) JSM-6400 scanning electron microscope; images were 
assembled using Photoshop software (http://www.photoshop.com/products). Arrow indicates pseudohyphae in C. auris biofilm (D). Scale 
bars represent 20 μm (original magnification ×1,000). OD, optical density.
 
 
 
Table 1. Planktonic susceptibility profiles of 7 antifungals against Candida auris yeast 
Drug 
Planktonic MIC* 
Strain 2 Strain 6 Strain 10 Strain 12 
Fluconazole >32 >32 >32 >32 
Voriconazole 8 8 32 1 
Caspofungin 32 32 2 >32 
Micafungin 0.5 <0.0625 <0.06 <0.0625 
Liposomal amphotericin B 0.25 0.25 0.5 1 
Amphotericin B 0.25 0.25 0.5 0.5 
Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 
*Values are mg/L except as indicated. All MIC tests were performed on 3 independent occasions, showing identical results each time. 
 
Biofilm-Forming Capability of Candida auris
DISPATCHES
330 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017
nonaggregative C. auris was significantly more pathogenic 
than C. albicans when a lower inoculum of 105 (p<0.05) 
and 5 × 104 cells/larvae (p<0.01) was administered. These 
data, along with those of Borman et al. (9), suggest that the 
nonaggregative C. auris phenotype has the capacity to form 
biofilms with enhanced virulence capacity.
Conclusions
Biofilm formation is a key driver of C. albicans patho-
genicity and is associated with patient death (10,15). We 
show that C. auris can differentially adhere to polymeric 
surfaces, form biofilms, and resist antifungal agents that 
are active against its planktonic counterparts. Of particular 
interest, caspofungin was predominately inactive against 
C. auris biofilms; this finding was unexpected because 
caspofungin is normally highly effective against Candida 
biofilms. These features contribute not only to C. auris vir-
ulence but also to its survival in hospital environments, in-
creasing its ability to cause outbreaks (5). The results of the 
in vivo model used in this study are in line with our clinical 
experience and validated by findings in other in vivo stud-
ies (9), affirming that C. auris is highly virulent or more 
virulent than C. albicans.
Although unable to form biofilms equivalent to C. al-
bicans, C. auris has a noteworthy virulence capacity that 
merits further exploration, particularly given the apparent 
heterogeneity associated with aggregative capacity. These 
factors, together with the innate resistance of C. auris to 
most antifungal agents, may explain why it is an emerg-
ing pathogen. Our findings suggest it is improbable that the 
spread and prevalence of C. auris can be controlled with 
antifungal stewardship approaches alone. We showed that 
chlorhexidine is effective against C. auris planktonic and 
sessile communities. Thus, use of this disinfectant can be 
advocated for topical control of C. auris at standard con-
centrations used for skin and wound cleansing and dis-
infection (0.05%–4.0%). Infection-prevention measures 
targeting C. auris biofilms in patients, on medical devices 
(e.g., equipment in contact with patients), and in the hospi-
tal environment will be required.
 
 
 
Table 2. Sessile susceptibility profiles of 7 antifungals against Candida auris yeast 
Drug 
Sessile MIC* 
Strain 2 Strain 6 Strain 10 Strain 12 
Fluconazole >32 >32 >32 >32 
Voriconazole >32 >32 >32 >32 
Caspofungin >32 >32 >32 >32 
Micafungin >32 >32 0.25 >32 
Liposomal amphotericin B 2 8 16 16 
Amphotericin B 2 4 2 4 
Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 
*Values indicate mg/L except as indicated. Sessile MICs are defined as a 90% inhibition of the metabolic dye XTT, 2,3-Bis(2-methoxy-4-nitro-5-
sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (Sigma-Aldrich, Dorset, UK) compared with the untreated control; MIC tests were performed on 3 
independent occasions and showed identical results each time. 
 
Figure 2. Pathogenicity of 
Candida species yeast infections 
in vivo. Galleria mellonella larvae 
were infected with 106 (A), 5 × 105 
(B), 105 (C), and 5 × 104 (D) cells/
larvae of C. albicans, C. glabrata, 
and 4 C. auris strains, and larvae 
survival measured every 12 h 
over 5 d. Ten samples of each 
yeast were used, and experiments 
were performed on 3 independent 
occasions. Data represents the 
mean percentage survival, as 
determined using a Kaplan-Meier 
plot. PBS and controls, which 
were pierced only, were also 
included and had no effect on 
larvae survival. Results show that 
C. auris and C. albicans infection 
exhibit similar pathogenicity.
 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 331
Biofilm-Forming Capability of Candida auris
Dr. Sherry works as a medical mycologist at the University of 
Glasgow, Glasgow, UK. Her specific interest is fungal biofilms 
of clinical significance.
References
  1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K,  
Yamaguchi H. Candida auris sp. nov., a novel ascomycetous  
yeast isolated from the external ear canal of an inpatient in a  
Japanese hospital. Microbiol Immunol. 2009;53:41–4.  
http://dx.doi.org/10.1111/j.1348-0421.2008.00083.x
  2. Kathuria S, Singh PK, 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, 
CLSI broth microdilution, and Etest method. J Clin Microbiol. 
2015;53:1823–30. http://dx.doi.org/10.1128/JCM.00367-15
  3. Centers for Disease Control and Prevention. Clinical alert to US 
healthcare facilities—June 2016. Global emergence of invasive 
infections caused by the multidrug-resistant yeast Candida auris. 
2016 [cited 2016 Jun 24]. http://www.cdc.gov/fungal/diseases/
candidiasis/candida-auris-alert.html
  4. Public Health England. Candida auris identified in England. 2016 
[cited 2016 Jul 1]. https://www.gov.uk/government/publications/
candida-auris-emergence-in-england/candida-auris-identified-in-
england
  5. Calvo B, Melo AS, Perozo-Mena A, Hernandez M, Francisco EC, 
Hagen F, et al. First report of Candida auris in America: Clinical 
and microbiological aspects of 18 episodes of candidemia. J Infect. 
2016;73:369–74. http://dx.doi.org/10.1016/j.jinf.2016.07.008
  6. Kumar D, Banerjee T, Pratap CB, Tilak R. Itraconazole-resistant 
Candida auris with phospholipase, proteinase and hemolysin  
activity from a case of vulvovaginitis. J Infect Dev Ctries. 
2015;9:435–7. http://dx.doi.org/10.3855/jidc.4582
  7. Oh BJ, Shin JH, Kim MN, Sung H, Lee K, Joo MY, et al.  
Biofilm formation and genotyping of Candida haemulonii,  
Candida pseudohaemulonii, and a proposed new species (Candida 
auris) isolates from Korea. Med Mycol. 2011;49:98–102.  
http://dx.doi.org/10.3109/13693786.2010.493563
8. Chatterjee S, Alampalli SV, Nageshan RK, Chettiar ST, Joshi S, 
Tatu US. Draft genome of a commonly misdiagnosed multidrug 
resistant pathogen Candida auris. BMC Genomics. 2015;16:686. 
http://dx.doi.org/10.1186/s12864-015-1863-z
9. Borman AM, Szekely A, Johnson EM. Comparative pathogenicity 
of United Kingdom isolates of the emerging pathogen Candida 
auris and other key pathogenic Candida species. mSphere. 
2016;1:pii:e00189-16.
10. Rajendran R, Sherry L, Nile CJ, Sherriff A, Johnson EM,  
Hanson MF, et al. Biofilm formation is a risk factor for mortality  
in patients with Candida albicans bloodstream infection- 
Scotland, 2012-2013. Clin Microbiol Infect. 2016;22:87–93.  
http://dx.doi.org/10.1016/j.cmi.2015.09.018
11. Ramage G, Vande Walle K, Wickes BL, López-Ribot JL.  
Standardized method for in vitro antifungal susceptibility testing 
of Candida albicans biofilms. Antimicrob Agents Chemother. 
2001;45:2475–9. http://dx.doi.org/10.1128/AAC.45.9.2475-
2479.2001
12. Sherry L, Rajendran R, Lappin DF, Borghi E, Perdoni F, Falleni M,  
et al. Biofilms formed by Candida albicans bloodstream isolates 
display phenotypic and transcriptional heterogeneity that are 
associated with resistance and pathogenicity. BMC Microbiol. 
2014;14:182. http://dx.doi.org/10.1186/1471-2180-14-182
13. Clinical Laboratory Standards Institute (CLSI). Reference method 
for broth dilution antifungal susceptibility testing of yeasts.  
Approved standard M27–A3, 3rd ed. Wayne, PA: The Institute; 2008.
14. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, 
Ramage G, et al. A simple and reproducible 96-well plate-based 
method for the formation of fungal biofilms and its application to 
antifungal susceptibility testing. Nat Protoc. 2008;3:1494–500. 
http://dx.doi.org/10.1038/nprot.2008.141
15. Rajendran R, Sherry L, Deshpande A, Johnson EM, Hanson MF, 
Williams C, et al. A prospective surveillance study of  
candidaemia: epidemiology, risk factors, antifungal treatment and 
outcome in hospitalized patients. Front Microbiol. 2016;7:915. 
http://dx.doi.org/10.3389/fmicb.2016.00915
Address for correspondence: Riina Rautemaa-Richardson, Education and 
Research Centre, Wythenshawe Hospital, Southmoor Rd, Manchester 
M23 9LT, UK; email: riina.richardson@manchester.ac.uk
In 1974, Lewis Thomas (1913–1993), physician, professor, and dean, published The Lives of A 
Cell, the first of 2 books subtitled Notes of a Biology Watcher. The phrase “lives of a cell” refers to 
the independent yet interrelated parts of a human cell—including mitochondria, centrioles, and basal 
bodies—that once led independent lives. Without these previously independent lives working together, 
we would not have the capacity for thought, communication, and movement. Dr. Thomas wrote, “Our 
membranes hold against equilibrium, maintain imbalance, bank against entropy…. We are shared, rent-
ed and occupied.”
Our human lives do not depend just on the lives in our individual cells. Our lives depend fully on the 
earth, including the atmosphere, and the many other human and nonhuman lives that occupy it. In 
explaining this complex interdependence, Dr. Thomas observed that the earth is “most like a cell.” This 
second interpretation of lives of a cell refers to the many interrelated earthly entities, such as plants, 
whales, humans, and even viruses, that “dart rather like bees from organism to organism, from plant 
to insect to mammal to me and back again,” all protected by the sky—a membrane that “works, and 
for what it is designed to accomplish it is as infallible as anything in nature.”
EID Podcast: Lives of a Cell:  
40 Years Later, A Third Interpretation
Visit our website to listen: 
http://www2c.cdc.gov/podcasts/player.asp?f=8637494


Directory of Open Access Journals

The University of Manchester ResearchBiofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida aurisDOI:10.3201/eid2302.161320Document VersionFinal published versionLink to publication record in Manchester Research ExplorerCitation for published version (APA):Sherry, L., Ramage, G., Kean, R., Borman, A., Johnson, E. M., Richardson, M. D., & Rautemaa-Richardson, R.(2017). Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida auris. Emerging infectiousdiseases, 23(2), 328-331. https://doi.org/10.3201/eid2302.161320Published in:Emerging infectious diseasesCiting this paperPlease note that where the full-text provided on Manchester Research Explorer is the Author Accepted Manuscriptor Proof version this may differ from the final Published version. If citing, it is advised that you check and use thepublisher's definitive version.General rightsCopyright and moral rights for the publications made accessible in the Research Explorer are retained by theauthors and/or other copyright owners and it is a condition of accessing publications that users recognise andabide by the legal requirements associated with these rights.Takedown policyIf you believe that this document breaches copyright please refer to the University of Manchester’s TakedownProcedures [http://man.ac.uk/04Y6Bo] or contact openresearch@manchester.ac.uk providing relevant details, sowe can investigate your claim.Download date:15. Oct. 2025328 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017DISPATCHESLeighann Sherry, Gordon Ramage, Ryan Kean, Andrew Borman, Elizabeth M. Johnson,  Malcolm D. Richardson,  Riina Rautemaa-RichardsonThe emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare–associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro.The yeast pathogen Candida auris was first detected in 2009 from an ear canal infection in Japan (1). This spe-cies initially attracted attention because of its reduced sus-ceptibility to azoles and amphotericin B, combined with the lack of reliable culture-based methods for its identification (2). More recently, C. auris has been associated globally with life-threatening invasive diseases, such as bloodstream and wound infections. C. auris has also caused hospital outbreaks across Asia and South America, as highlighted in a 2016 clinical alert (3). In addition, in a UK intensive care unit, candidemia developed in 20% of patients colo-nized with C. auris (4). Although the mode of transmission within hospitals is unknown, C. auris may substantially contaminate rooms of colonized or infected patients (5). Phospholipase and proteinase activity have been identi-fied as virulence factors (6); however, because previously used assessment techniques were rudimentary, this patho-gen’s ability to form biofilm remains under question (7). The draft genome identifying various proteins involved in biofilm formation (8), coupled with recent descriptions of aggregative and nonaggregative phenotypes, the latter of which are more virulent in vivo (9), indicate the possibility of heterogeneous C. auris biofilm formation, as described for C. albicans (10). We sought to examine these aggrega-tive and nonaggregative C. auris phenotypes in the context of biofilm-forming capacity, investigate their susceptibil-ity to a panel of antifungal agents and the skin disinfectant chlorhexidine, and investigate their virulence in vivo.The StudyThroughout this study, we used C. albicans SC5314 and Candida glabrata WT2001 as comparators for C. auris nonaggregative strains NCPF 8971 (strain 10) and NCPF 8973 (strain 12) and aggregative strains NCPF 8977 (strain 2) and NCPF 8978 (strain 6), as previously described (9). Strains were propagated in YPD broth (Sigma-Aldrich, Dorset, UK), incubated overnight at 30°C, and adjusted to 106 cells/mL in RPMI 1640 medium (11). On 3 separate occasions, 8 biofilms of each Candida species were grown in flat-bottomed, 96-well polystyrene microtiter plates and incubated for 24 h at 37°C, after which biomass was as-sessed by crystal violet assay (12). C. albicans displayed the greatest biofilm mass (Figure 1, panel A), consistent with previous findings  (10). Compared with C. albicans, all C. auris strains formed significantly reduced biofilms (p<0.0001): biomass for nonaggregative C. auris strains 10 and 12 was 2.4 and 1.5 times less, respectively, than those for C. albicans, and biomass for aggregative C. auris strains 2 and 6 was 3.0 and 3.1 times less, respectively. However, these strains formed significantly greater bio-films (p<0.0001) than those formed by C. glabrata (3.8, 6.0, 3.0, and 2.9 times more for strains 10, 12, 2, and 6, respectively). We confirmed these findings for each species by scanning electron microscopy after growing the strains on Thermanox Coverslips (Thermo Fisher Scientific, Pais-ley, UK) for 24 h, as previously described (12). C. albi-cans biofilms were typically densely packed with hyphae (Figure 1, panel B), whereas C. glabrata formed a sparse biofilm consisting of yeast cells only, without extracellular matrix (Figure 1, panel C). C. auris strain 10 biofilm for-mation was intermediate to the C. albicans and C. glabrata phenotypes, showing predominately budding yeast and oc-casional pseudohyphae (Figure 1, panel D). In agreement with previous findings (9), all tested C. auris strains dis-played the same phenotype.To determine MICs for planktonic and sessile cells of the C. auris strains, we performed antifungal susceptibility testing using standardized Clinical Laboratory Standards Institute M27-A3 broth microdilution (visual inspection) and standardized candidal biofilm testing (metabolic vi-ability) with fluconazole, voriconazole, caspofungin, mi-cafungin, liposomal amphotericin B, amphotericin B, and chlorhexidine (13,14). Antifungal agents were tested in se-rial 2-fold dilutions (0.06–32.0 mg/L) for planktonic and sessile cells. Fluconazole was ineffective (MICs of >32 mg/L) against planktonic and sessile communities, whereas Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida aurisAuthor affiliations: University of Glasgow, Glasgow, Scotland, UK (L. Sherry, G. Ramage, R. Kean); Public Health England, Bristol, UK (A. Borman, E.M. Johnson); The University of Manchester, Manchester, UK (M.D. Richardson, R. Rautemaa-Richardson); University Hospital of South Manchester, Manchester  (M.D. Richardson, R. Rautemaa-Richardson)DOI: http://dx.doi.org/10.3201/eid2302.161320 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 329voriconazole displayed minimal activity against planktonic cells (Table 1, 2). Although liposomal amphotericin B was active against planktonic C. auris at 0.25–1.0 mg/L, up to 16 mg/L was required to reduce biofilm metabolic viability by 90%. Amphotericin B was more effective, requiring 4 mg/L to kill biofilms. Micafungin was the most active echi-nocandin, requiring <0.5 mg/L to inhibit planktonic cells, compared with 2–32 mg/L for caspofungin. However, these 2 antifungal agents were ineffective against biofilms, requir-ing >32 mg/L to inhibit sessile cells. Of note, chlorhexidine exhibited the greatest activity, requiring <0.02% to effec-tively inhibit planktonic and sessile cells across all strains tested. All strains showed similar sensitivity profiles, with the exception of strain 10, for which voriconazole was re-quired in higher concentrations and caspofungin in lower concentrations to effectively inhibit planktonic growth.Killing assays in Galleria mellonella were performed, as previously described (12), to assess the pathogenicity of each Candida species. Ten G. mellonella larvae (Livefoods Direct Ltd, Sheffield, UK) with bodyweights of ≈300 mg were used for each test group. Standardized inoculums of 106 and 105 and to 5 × 105 and 5 × 104 cells/larvae (Figure 2) in PBS, were injected into the hemocoel, as previously described (9). We assessed pathogenicity using a Kaplan-Meier plot, monitoring the percent survival over 5 days. Survival data for 5 × 105 cells/larvae showed a significant difference in the killing of larvae by C. glabrata and the other Candida species (p<0.0001) (Figure 2, panel B). Al-though C. albicans and C. auris had similar kill kinetics in this model, infection with nonaggregative C. auris strain 10 achieved a 100% death rate within 48 h, compared with a rate of ≈87% with C. albicans (p = 0.3076). Moreover, Figure 1. Biofilm formation on Candida auris, C. albicans, and C. glabrata yeast strains. A) Biomass quantities were determined spectrophotometrically for 4 strains of C. auris and 1 each of C. albicans and C. glabrata. Isolates were standardized to 106 cells/mL in RPMI-1640 and grown in flat-bottomed 96-well microtiter plates for 24 h at 37°C. Biofilms were then washed, stained with crystal violet solution, and quantified. Data represent the mean ± SD of experiments performed on 3 separate occasions, using 8 replicates for each strain. Results show that C. auris can form heterogeneous intermediate biofilms. B–D) C. albicans (B), C. glabrata (C) and C. auris (D) were also grown on Thermanox coverslips (Thermo Fisher Scientific, Paisley, UK) for 24 h at 37°C. Biofilms were then processed and viewed on a JEOL (Tokyo, Japan) JSM-6400 scanning electron microscope; images were assembled using Photoshop software (http://www.photoshop.com/products). Arrow indicates pseudohyphae in C. auris biofilm (D). Scale bars represent 20 μm (original magnification ×1,000). OD, optical density.   Table 1. Planktonic susceptibility profiles of 7 antifungals against Candida auris yeast Drug Planktonic MIC* Strain 2 Strain 6 Strain 10 Strain 12 Fluconazole >32 >32 >32 >32 Voriconazole 8 8 32 1 Caspofungin 32 32 2 >32 Micafungin 0.5 <0.0625 <0.06 <0.0625 Liposomal amphotericin B 0.25 0.25 0.5 1 Amphotericin B 0.25 0.25 0.5 0.5 Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 *Values are mg/L except as indicated. All MIC tests were performed on 3 independent occasions, showing identical results each time.  Biofilm-Forming Capability of Candida aurisDISPATCHES330 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017nonaggregative C. auris was significantly more pathogenic than C. albicans when a lower inoculum of 105 (p<0.05) and 5 × 104 cells/larvae (p<0.01) was administered. These data, along with those of Borman et al. (9), suggest that the nonaggregative C. auris phenotype has the capacity to form biofilms with enhanced virulence capacity.ConclusionsBiofilm formation is a key driver of C. albicans patho-genicity and is associated with patient death (10,15). We show that C. auris can differentially adhere to polymeric surfaces, form biofilms, and resist antifungal agents that are active against its planktonic counterparts. Of particular interest, caspofungin was predominately inactive against C. auris biofilms; this finding was unexpected because caspofungin is normally highly effective against Candida biofilms. These features contribute not only to C. auris vir-ulence but also to its survival in hospital environments, in-creasing its ability to cause outbreaks (5). The results of the in vivo model used in this study are in line with our clinical experience and validated by findings in other in vivo stud-ies (9), affirming that C. auris is highly virulent or more virulent than C. albicans.Although unable to form biofilms equivalent to C. al-bicans, C. auris has a noteworthy virulence capacity that merits further exploration, particularly given the apparent heterogeneity associated with aggregative capacity. These factors, together with the innate resistance of C. auris to most antifungal agents, may explain why it is an emerg-ing pathogen. Our findings suggest it is improbable that the spread and prevalence of C. auris can be controlled with antifungal stewardship approaches alone. We showed that chlorhexidine is effective against C. auris planktonic and sessile communities. Thus, use of this disinfectant can be advocated for topical control of C. auris at standard con-centrations used for skin and wound cleansing and dis-infection (0.05%–4.0%). Infection-prevention measures targeting C. auris biofilms in patients, on medical devices (e.g., equipment in contact with patients), and in the hospi-tal environment will be required.   Table 2. Sessile susceptibility profiles of 7 antifungals against Candida auris yeast Drug Sessile MIC* Strain 2 Strain 6 Strain 10 Strain 12 Fluconazole >32 >32 >32 >32 Voriconazole >32 >32 >32 >32 Caspofungin >32 >32 >32 >32 Micafungin >32 >32 0.25 >32 Liposomal amphotericin B 2 8 16 16 Amphotericin B 2 4 2 4 Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 *Values indicate mg/L except as indicated. Sessile MICs are defined as a 90% inhibition of the metabolic dye XTT, 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (Sigma-Aldrich, Dorset, UK) compared with the untreated control; MIC tests were performed on 3 independent occasions and showed identical results each time.  Figure 2. Pathogenicity of Candida species yeast infections in vivo. Galleria mellonella larvae were infected with 106 (A), 5 × 105 (B), 105 (C), and 5 × 104 (D) cells/larvae of C. albicans, C. glabrata, and 4 C. auris strains, and larvae survival measured every 12 h over 5 d. Ten samples of each yeast were used, and experiments were performed on 3 independent occasions. Data represents the mean percentage survival, as determined using a Kaplan-Meier plot. PBS and controls, which were pierced only, were also included and had no effect on larvae survival. Results show that C. auris and C. albicans infection exhibit similar pathogenicity. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 331Biofilm-Forming Capability of Candida aurisDr. Sherry works as a medical mycologist at the University of Glasgow, Glasgow, UK. Her specific interest is fungal biofilms of clinical significance.References  1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K,  Yamaguchi H. Candida auris sp. nov., a novel ascomycetous  yeast isolated from the external ear canal of an inpatient in a  Japanese hospital. Microbiol Immunol. 2009;53:41–4.  http://dx.doi.org/10.1111/j.1348-0421.2008.00083.x  2. Kathuria S, Singh PK, 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, CLSI broth microdilution, and Etest method. J Clin Microbiol. 2015;53:1823–30. http://dx.doi.org/10.1128/JCM.00367-15  3. Centers for Disease Control and Prevention. Clinical alert to US healthcare facilities—June 2016. Global emergence of invasive infections caused by the multidrug-resistant yeast Candida auris. 2016 [cited 2016 Jun 24]. http://www.cdc.gov/fungal/diseases/candidiasis/candida-auris-alert.html  4. Public Health England. Candida auris identified in England. 2016 [cited 2016 Jul 1]. https://www.gov.uk/government/publications/candida-auris-emergence-in-england/candida-auris-identified-in-england  5. Calvo B, Melo AS, Perozo-Mena A, Hernandez M, Francisco EC, Hagen F, et al. First report of Candida auris in America: Clinical and microbiological aspects of 18 episodes of candidemia. J Infect. 2016;73:369–74. http://dx.doi.org/10.1016/j.jinf.2016.07.008  6. Kumar D, Banerjee T, Pratap CB, Tilak R. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin  activity from a case of vulvovaginitis. J Infect Dev Ctries. 2015;9:435–7. http://dx.doi.org/10.3855/jidc.4582  7. Oh BJ, Shin JH, Kim MN, Sung H, Lee K, Joo MY, et al.  Biofilm formation and genotyping of Candida haemulonii,  Candida pseudohaemulonii, and a proposed new species (Candida auris) isolates from Korea. Med Mycol. 2011;49:98–102.  http://dx.doi.org/10.3109/13693786.2010.4935638. Chatterjee S, Alampalli SV, Nageshan RK, Chettiar ST, Joshi S, Tatu US. Draft genome of a commonly misdiagnosed multidrug resistant pathogen Candida auris. BMC Genomics. 2015;16:686. http://dx.doi.org/10.1186/s12864-015-1863-z9. Borman AM, Szekely A, Johnson EM. Comparative pathogenicity of United Kingdom isolates of the emerging pathogen Candida auris and other key pathogenic Candida species. mSphere. 2016;1:pii:e00189-16.10. Rajendran R, Sherry L, Nile CJ, Sherriff A, Johnson EM,  Hanson MF, et al. Biofilm formation is a risk factor for mortality  in patients with Candida albicans bloodstream infection- Scotland, 2012-2013. Clin Microbiol Infect. 2016;22:87–93.  http://dx.doi.org/10.1016/j.cmi.2015.09.01811. Ramage G, Vande Walle K, Wickes BL, López-Ribot JL.  Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother. 2001;45:2475–9. http://dx.doi.org/10.1128/AAC.45.9.2475-2479.200112. Sherry L, Rajendran R, Lappin DF, Borghi E, Perdoni F, Falleni M,  et al. Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity. BMC Microbiol. 2014;14:182. http://dx.doi.org/10.1186/1471-2180-14-18213. Clinical Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts.  Approved standard M27–A3, 3rd ed. Wayne, PA: The Institute; 2008.14. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G, et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc. 2008;3:1494–500. http://dx.doi.org/10.1038/nprot.2008.14115. Rajendran R, Sherry L, Deshpande A, Johnson EM, Hanson MF, Williams C, et al. A prospective surveillance study of  candidaemia: epidemiology, risk factors, antifungal treatment and outcome in hospitalized patients. Front Microbiol. 2016;7:915. http://dx.doi.org/10.3389/fmicb.2016.00915Address for correspondence: Riina Rautemaa-Richardson, Education and Research Centre, Wythenshawe Hospital, Southmoor Rd, Manchester M23 9LT, UK; email: riina.richardson@manchester.ac.ukIn 1974, Lewis Thomas (1913–1993), physician, professor, and dean, published The Lives of A Cell, the first of 2 books subtitled Notes of a Biology Watcher. The phrase “lives of a cell” refers to the independent yet interrelated parts of a human cell—including mitochondria, centrioles, and basal bodies—that once led independent lives. Without these previously independent lives working together, we would not have the capacity for thought, communication, and movement. Dr. Thomas wrote, “Our membranes hold against equilibrium, maintain imbalance, bank against entropy…. We are shared, rent-ed and occupied.”Our human lives do not depend just on the lives in our individual cells. Our lives depend fully on the earth, including the atmosphere, and the many other human and nonhuman lives that occupy it. In explaining this complex interdependence, Dr. Thomas observed that the earth is “most like a cell.” This second interpretation of lives of a cell refers to the many interrelated earthly entities, such as plants, whales, humans, and even viruses, that “dart rather like bees from organism to organism, from plant to insect to mammal to me and back again,” all protected by the sky—a membrane that “works, and for what it is designed to accomplish it is as infallible as anything in nature.”EID Podcast: Lives of a Cell:  40 Years Later, A Third InterpretationVisit our website to listen: http://www2c.cdc.gov/podcasts/player.asp?f=8637494

The University of Manchester ResearchBiofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida aurisDOI:10.3201/eid2302.161320Document VersionFinal published versionLink to publication record in Manchester Research ExplorerCitation for published version (APA):Sherry, L., Ramage, G., Kean, R., Borman, A., Johnson, E. M., Richardson, M. D., & Rautemaa-Richardson, R.(2017). Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida auris. Emerging infectiousdiseases, 23(2), 328-331. https://doi.org/10.3201/eid2302.161320Published in:Emerging infectious diseasesCiting this paperPlease note that where the full-text provided on Manchester Research Explorer is the Author Accepted Manuscriptor Proof version this may differ from the final Published version. If citing, it is advised that you check and use thepublisher's definitive version.General rightsCopyright and moral rights for the publications made accessible in the Research Explorer are retained by theauthors and/or other copyright owners and it is a condition of accessing publications that users recognise andabide by the legal requirements associated with these rights.Takedown policyIf you believe that this document breaches copyright please refer to the University of Manchester’s TakedownProcedures [http://man.ac.uk/04Y6Bo] or contact openresearch@manchester.ac.uk providing relevant details, sowe can investigate your claim.Download date:26. Dec. 2025328 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017DISPATCHESLeighann Sherry, Gordon Ramage, Ryan Kean, Andrew Borman, Elizabeth M. Johnson,  Malcolm D. Richardson,  Riina Rautemaa-RichardsonThe emerging multidrug-resistant yeast pathogen Candida auris has attracted considerable attention as a source of healthcare–associated infections. We report that this highly virulent yeast has the capacity to form antifungal resistant biofilms sensitive to the disinfectant chlorhexidine in vitro.The yeast pathogen Candida auris was first detected in 2009 from an ear canal infection in Japan (1). This spe-cies initially attracted attention because of its reduced sus-ceptibility to azoles and amphotericin B, combined with the lack of reliable culture-based methods for its identification (2). More recently, C. auris has been associated globally with life-threatening invasive diseases, such as bloodstream and wound infections. C. auris has also caused hospital outbreaks across Asia and South America, as highlighted in a 2016 clinical alert (3). In addition, in a UK intensive care unit, candidemia developed in 20% of patients colo-nized with C. auris (4). Although the mode of transmission within hospitals is unknown, C. auris may substantially contaminate rooms of colonized or infected patients (5). Phospholipase and proteinase activity have been identi-fied as virulence factors (6); however, because previously used assessment techniques were rudimentary, this patho-gen’s ability to form biofilm remains under question (7). The draft genome identifying various proteins involved in biofilm formation (8), coupled with recent descriptions of aggregative and nonaggregative phenotypes, the latter of which are more virulent in vivo (9), indicate the possibility of heterogeneous C. auris biofilm formation, as described for C. albicans (10). We sought to examine these aggrega-tive and nonaggregative C. auris phenotypes in the context of biofilm-forming capacity, investigate their susceptibil-ity to a panel of antifungal agents and the skin disinfectant chlorhexidine, and investigate their virulence in vivo.The StudyThroughout this study, we used C. albicans SC5314 and Candida glabrata WT2001 as comparators for C. auris nonaggregative strains NCPF 8971 (strain 10) and NCPF 8973 (strain 12) and aggregative strains NCPF 8977 (strain 2) and NCPF 8978 (strain 6), as previously described (9). Strains were propagated in YPD broth (Sigma-Aldrich, Dorset, UK), incubated overnight at 30°C, and adjusted to 106 cells/mL in RPMI 1640 medium (11). On 3 separate occasions, 8 biofilms of each Candida species were grown in flat-bottomed, 96-well polystyrene microtiter plates and incubated for 24 h at 37°C, after which biomass was as-sessed by crystal violet assay (12). C. albicans displayed the greatest biofilm mass (Figure 1, panel A), consistent with previous findings  (10). Compared with C. albicans, all C. auris strains formed significantly reduced biofilms (p<0.0001): biomass for nonaggregative C. auris strains 10 and 12 was 2.4 and 1.5 times less, respectively, than those for C. albicans, and biomass for aggregative C. auris strains 2 and 6 was 3.0 and 3.1 times less, respectively. However, these strains formed significantly greater bio-films (p<0.0001) than those formed by C. glabrata (3.8, 6.0, 3.0, and 2.9 times more for strains 10, 12, 2, and 6, respectively). We confirmed these findings for each species by scanning electron microscopy after growing the strains on Thermanox Coverslips (Thermo Fisher Scientific, Pais-ley, UK) for 24 h, as previously described (12). C. albi-cans biofilms were typically densely packed with hyphae (Figure 1, panel B), whereas C. glabrata formed a sparse biofilm consisting of yeast cells only, without extracellular matrix (Figure 1, panel C). C. auris strain 10 biofilm for-mation was intermediate to the C. albicans and C. glabrata phenotypes, showing predominately budding yeast and oc-casional pseudohyphae (Figure 1, panel D). In agreement with previous findings (9), all tested C. auris strains dis-played the same phenotype.To determine MICs for planktonic and sessile cells of the C. auris strains, we performed antifungal susceptibility testing using standardized Clinical Laboratory Standards Institute M27-A3 broth microdilution (visual inspection) and standardized candidal biofilm testing (metabolic vi-ability) with fluconazole, voriconazole, caspofungin, mi-cafungin, liposomal amphotericin B, amphotericin B, and chlorhexidine (13,14). Antifungal agents were tested in se-rial 2-fold dilutions (0.06–32.0 mg/L) for planktonic and sessile cells. Fluconazole was ineffective (MICs of >32 mg/L) against planktonic and sessile communities, whereas Biofilm-Forming Capability of Highly Virulent, Multidrug-Resistant Candida aurisAuthor affiliations: University of Glasgow, Glasgow, Scotland, UK (L. Sherry, G. Ramage, R. Kean); Public Health England, Bristol, UK (A. Borman, E.M. Johnson); The University of Manchester, Manchester, UK (M.D. Richardson, R. Rautemaa-Richardson); University Hospital of South Manchester, Manchester  (M.D. Richardson, R. Rautemaa-Richardson)DOI: http://dx.doi.org/10.3201/eid2302.161320 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 329voriconazole displayed minimal activity against planktonic cells (Table 1, 2). Although liposomal amphotericin B was active against planktonic C. auris at 0.25–1.0 mg/L, up to 16 mg/L was required to reduce biofilm metabolic viability by 90%. Amphotericin B was more effective, requiring 4 mg/L to kill biofilms. Micafungin was the most active echi-nocandin, requiring <0.5 mg/L to inhibit planktonic cells, compared with 2–32 mg/L for caspofungin. However, these 2 antifungal agents were ineffective against biofilms, requir-ing >32 mg/L to inhibit sessile cells. Of note, chlorhexidine exhibited the greatest activity, requiring <0.02% to effec-tively inhibit planktonic and sessile cells across all strains tested. All strains showed similar sensitivity profiles, with the exception of strain 10, for which voriconazole was re-quired in higher concentrations and caspofungin in lower concentrations to effectively inhibit planktonic growth.Killing assays in Galleria mellonella were performed, as previously described (12), to assess the pathogenicity of each Candida species. Ten G. mellonella larvae (Livefoods Direct Ltd, Sheffield, UK) with bodyweights of ≈300 mg were used for each test group. Standardized inoculums of 106 and 105 and to 5 × 105 and 5 × 104 cells/larvae (Figure 2) in PBS, were injected into the hemocoel, as previously described (9). We assessed pathogenicity using a Kaplan-Meier plot, monitoring the percent survival over 5 days. Survival data for 5 × 105 cells/larvae showed a significant difference in the killing of larvae by C. glabrata and the other Candida species (p<0.0001) (Figure 2, panel B). Al-though C. albicans and C. auris had similar kill kinetics in this model, infection with nonaggregative C. auris strain 10 achieved a 100% death rate within 48 h, compared with a rate of ≈87% with C. albicans (p = 0.3076). Moreover, Figure 1. Biofilm formation on Candida auris, C. albicans, and C. glabrata yeast strains. A) Biomass quantities were determined spectrophotometrically for 4 strains of C. auris and 1 each of C. albicans and C. glabrata. Isolates were standardized to 106 cells/mL in RPMI-1640 and grown in flat-bottomed 96-well microtiter plates for 24 h at 37°C. Biofilms were then washed, stained with crystal violet solution, and quantified. Data represent the mean ± SD of experiments performed on 3 separate occasions, using 8 replicates for each strain. Results show that C. auris can form heterogeneous intermediate biofilms. B–D) C. albicans (B), C. glabrata (C) and C. auris (D) were also grown on Thermanox coverslips (Thermo Fisher Scientific, Paisley, UK) for 24 h at 37°C. Biofilms were then processed and viewed on a JEOL (Tokyo, Japan) JSM-6400 scanning electron microscope; images were assembled using Photoshop software (http://www.photoshop.com/products). Arrow indicates pseudohyphae in C. auris biofilm (D). Scale bars represent 20 μm (original magnification ×1,000). OD, optical density.   Table 1. Planktonic susceptibility profiles of 7 antifungals against Candida auris yeast Drug Planktonic MIC* Strain 2 Strain 6 Strain 10 Strain 12 Fluconazole >32 >32 >32 >32 Voriconazole 8 8 32 1 Caspofungin 32 32 2 >32 Micafungin 0.5 <0.0625 <0.06 <0.0625 Liposomal amphotericin B 0.25 0.25 0.5 1 Amphotericin B 0.25 0.25 0.5 0.5 Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 *Values are mg/L except as indicated. All MIC tests were performed on 3 independent occasions, showing identical results each time.  Biofilm-Forming Capability of Candida aurisDISPATCHES330 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017nonaggregative C. auris was significantly more pathogenic than C. albicans when a lower inoculum of 105 (p<0.05) and 5 × 104 cells/larvae (p<0.01) was administered. These data, along with those of Borman et al. (9), suggest that the nonaggregative C. auris phenotype has the capacity to form biofilms with enhanced virulence capacity.ConclusionsBiofilm formation is a key driver of C. albicans patho-genicity and is associated with patient death (10,15). We show that C. auris can differentially adhere to polymeric surfaces, form biofilms, and resist antifungal agents that are active against its planktonic counterparts. Of particular interest, caspofungin was predominately inactive against C. auris biofilms; this finding was unexpected because caspofungin is normally highly effective against Candida biofilms. These features contribute not only to C. auris vir-ulence but also to its survival in hospital environments, in-creasing its ability to cause outbreaks (5). The results of the in vivo model used in this study are in line with our clinical experience and validated by findings in other in vivo stud-ies (9), affirming that C. auris is highly virulent or more virulent than C. albicans.Although unable to form biofilms equivalent to C. al-bicans, C. auris has a noteworthy virulence capacity that merits further exploration, particularly given the apparent heterogeneity associated with aggregative capacity. These factors, together with the innate resistance of C. auris to most antifungal agents, may explain why it is an emerg-ing pathogen. Our findings suggest it is improbable that the spread and prevalence of C. auris can be controlled with antifungal stewardship approaches alone. We showed that chlorhexidine is effective against C. auris planktonic and sessile communities. Thus, use of this disinfectant can be advocated for topical control of C. auris at standard con-centrations used for skin and wound cleansing and dis-infection (0.05%–4.0%). Infection-prevention measures targeting C. auris biofilms in patients, on medical devices (e.g., equipment in contact with patients), and in the hospi-tal environment will be required.   Table 2. Sessile susceptibility profiles of 7 antifungals against Candida auris yeast Drug Sessile MIC* Strain 2 Strain 6 Strain 10 Strain 12 Fluconazole >32 >32 >32 >32 Voriconazole >32 >32 >32 >32 Caspofungin >32 >32 >32 >32 Micafungin >32 >32 0.25 >32 Liposomal amphotericin B 2 8 16 16 Amphotericin B 2 4 2 4 Chlorhexidine, % <0.02 <0.02 <0.02 <0.02 *Values indicate mg/L except as indicated. Sessile MICs are defined as a 90% inhibition of the metabolic dye XTT, 2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (Sigma-Aldrich, Dorset, UK) compared with the untreated control; MIC tests were performed on 3 independent occasions and showed identical results each time.  Figure 2. Pathogenicity of Candida species yeast infections in vivo. Galleria mellonella larvae were infected with 106 (A), 5 × 105 (B), 105 (C), and 5 × 104 (D) cells/larvae of C. albicans, C. glabrata, and 4 C. auris strains, and larvae survival measured every 12 h over 5 d. Ten samples of each yeast were used, and experiments were performed on 3 independent occasions. Data represents the mean percentage survival, as determined using a Kaplan-Meier plot. PBS and controls, which were pierced only, were also included and had no effect on larvae survival. Results show that C. auris and C. albicans infection exhibit similar pathogenicity. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 2, February 2017 331Biofilm-Forming Capability of Candida aurisDr. Sherry works as a medical mycologist at the University of Glasgow, Glasgow, UK. Her specific interest is fungal biofilms of clinical significance.References  1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K,  Yamaguchi H. Candida auris sp. nov., a novel ascomycetous  yeast isolated from the external ear canal of an inpatient in a  Japanese hospital. Microbiol Immunol. 2009;53:41–4.  http://dx.doi.org/10.1111/j.1348-0421.2008.00083.x  2. Kathuria S, Singh PK, 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, CLSI broth microdilution, and Etest method. J Clin Microbiol. 2015;53:1823–30. http://dx.doi.org/10.1128/JCM.00367-15  3. Centers for Disease Control and Prevention. Clinical alert to US healthcare facilities—June 2016. Global emergence of invasive infections caused by the multidrug-resistant yeast Candida auris. 2016 [cited 2016 Jun 24]. http://www.cdc.gov/fungal/diseases/candidiasis/candida-auris-alert.html  4. Public Health England. Candida auris identified in England. 2016 [cited 2016 Jul 1]. https://www.gov.uk/government/publications/candida-auris-emergence-in-england/candida-auris-identified-in-england  5. Calvo B, Melo AS, Perozo-Mena A, Hernandez M, Francisco EC, Hagen F, et al. First report of Candida auris in America: Clinical and microbiological aspects of 18 episodes of candidemia. J Infect. 2016;73:369–74. http://dx.doi.org/10.1016/j.jinf.2016.07.008  6. Kumar D, Banerjee T, Pratap CB, Tilak R. Itraconazole-resistant Candida auris with phospholipase, proteinase and hemolysin  activity from a case of vulvovaginitis. 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Clin Microbiol Infect. 2016;22:87–93.  http://dx.doi.org/10.1016/j.cmi.2015.09.01811. Ramage G, Vande Walle K, Wickes BL, López-Ribot JL.  Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother. 2001;45:2475–9. http://dx.doi.org/10.1128/AAC.45.9.2475-2479.200112. Sherry L, Rajendran R, Lappin DF, Borghi E, Perdoni F, Falleni M,  et al. Biofilms formed by Candida albicans bloodstream isolates display phenotypic and transcriptional heterogeneity that are associated with resistance and pathogenicity. BMC Microbiol. 2014;14:182. http://dx.doi.org/10.1186/1471-2180-14-18213. Clinical Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of yeasts.  Approved standard M27–A3, 3rd ed. Wayne, PA: The Institute; 2008.14. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G, et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc. 2008;3:1494–500. http://dx.doi.org/10.1038/nprot.2008.14115. Rajendran R, Sherry L, Deshpande A, Johnson EM, Hanson MF, Williams C, et al. A prospective surveillance study of  candidaemia: epidemiology, risk factors, antifungal treatment and outcome in hospitalized patients. Front Microbiol. 2016;7:915. http://dx.doi.org/10.3389/fmicb.2016.00915Address for correspondence: Riina Rautemaa-Richardson, Education and Research Centre, Wythenshawe Hospital, Southmoor Rd, Manchester M23 9LT, UK; email: riina.richardson@manchester.ac.ukIn 1974, Lewis Thomas (1913–1993), physician, professor, and dean, published The Lives of A Cell, the first of 2 books subtitled Notes of a Biology Watcher. The phrase “lives of a cell” refers to the independent yet interrelated parts of a human cell—including mitochondria, centrioles, and basal bodies—that once led independent lives. Without these previously independent lives working together, we would not have the capacity for thought, communication, and movement. Dr. Thomas wrote, “Our membranes hold against equilibrium, maintain imbalance, bank against entropy…. We are shared, rent-ed and occupied.”Our human lives do not depend just on the lives in our individual cells. Our lives depend fully on the earth, including the atmosphere, and the many other human and nonhuman lives that occupy it. In explaining this complex interdependence, Dr. Thomas observed that the earth is “most like a cell.” This second interpretation of lives of a cell refers to the many interrelated earthly entities, such as plants, whales, humans, and even viruses, that “dart rather like bees from organism to organism, from plant to insect to mammal to me and back again,” all protected by the sky—a membrane that “works, and for what it is designed to accomplish it is as infallible as anything in nature.”EID Podcast: Lives of a Cell:  40 Years Later, A Third InterpretationVisit our website to listen: http://www2c.cdc.gov/podcasts/player.asp?f=8637494

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Biofilm-forming capability of highly virulent, multidrug-resistant Candida auris

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