Biofilm-forming fluconazole-resistant Candida auris causing vulvovaginal candidiasis in an immunocompetent patient: A case report

Lakshmi Krishnasamy, Jayasankari Senthilganesh, Chitralekha Saikumar, Paramasivam Nithyanand✉

1Department of Microbiology, Sree Balaji Medical College and Hospital, Chennai-600044, Bharath Institute of Higher Education and Research,India

2Biofilm Biology Laboratory, Centre for Research on Infectious Diseases (CRID), School of Chemical and Biotechnology, SASTRA Deemed University,Tirumalaisamudram, Thanjavur 613 401, Tamil Nadu, India

ABSTRACT

KEYWORDS: Candida auris; Resistance; Biofilm; Scanning electron microscopy

1. Introduction

Candida (C.) auris is an emerging invasive pathogen reported from ear canal, blood, wound, respiratory tract infections, etc. C. auris was first isolated in 2009 in Japan from the external ear canal of a patient. Currently it exhibits resistance to fluconazole and shows variable susceptibility to antifungal drugs like amphotericin B and echinocandins[1]. Kumar et al. have reported a case of vulvovaginal candidiasis (VVC) caused by itraconazole resistant C. auris[2]. VVC is one of the frequent causes of vaginitis affecting women globally.Improper use of antifungal agents like incorrect dosage, prolonged hospitalization, poor immune status are some other factors for emergence of unusual species like C. auris. Due to its resistance to other conventional drugs, the pathogen has turned out to be difficult to be treated because of its altered virulence mechanism which includes drug efflux and dimorphism characteristics. The increasing occurrence as well as the varying antifungal drug susceptibility profiles of C. auris emphasizes the importance of identifying Candida to the species level in diagnostic laboratories.Understanding the antifungal susceptibility profile of various Candida species helps in developing appropriate protocols for empirical antifungal therapy in emergency cases. There is a huge need for new antifungals to treat the resistant strains.

In the present study, we report a case of 26-year-old female with vulvovaginal candidiasis caused due to C. auris, identified by both phenotypic and molecular methods. We also sought to examine the bio film forming potential of the isolate along with their antifungal susceptibility profile. A written consent has been obtained from the patient for publication and the study has been approved by the institutional ethical committee (Ref. No. 002/ SBMC/IHEC/2016/189).

2. Case report

A 26-year-old female presented to the Department of Gynaecology,Sree Balaji Medical College and hospital, Chennai with complaints of vaginal discharge, low back pain and itching sensation in the vaginal area. Two high vaginal swab samples were collected and processed for bacterial and fungal culture. Direct microscopy showed the presence of ovoid budding yeast cells. The sample was inoculated in Sabouraud Dextrose Agar (SDA) (Hi Media, India) for culturing fungi and on Nutrient agar, Blood agar and Mac Conkey agar plates (Hi Media, India) for culturing bacteria. The inoculated culture plates were incubated at 37 ℃ for 48 h.

After 48 h of incubation, bacterial culture was negative. SDA plate showed white to cream coloured smooth colonies. Gram staining showed Gram positive budding yeast cells. The colonies were sub-cultured on CHROM agar (Hi Media, India) for species identification. No characteristic colour was produced by the isolate in CHROM agar plate. Sugar assimilation and fermentation tests were indecisive. The antifungal susceptibility testing was performed as per manufacturer’s instructions using disc diffusion test as per Clinical and Laboratory Standards Institute M44A document[3],using fluconazole (25 μg), itraconazole (10 μg), nystatin (100 Unit),clotrimazole (10 μg), ketoconazole (10 μg) and amphotericin B(100 Unit) (Hi Media, Mumbai, India). The yeast cell suspension was prepared by inoculating 5 isolated yeast colonies from SDA plate in 5 mL of sterile 0.85% saline and turbidity adjusted to 0.5 McFarland standard. The isolate was found to be resistant to fluconazole, amphotericin B and clotrimazole. It showed a dose dependent susceptibility to itraconazole and was susceptible to ketoconazole and nystatin.

The strain was further processed for molecular identification and examined for its ability to form biofilms. The fungal DNA was extracted and the Internal Transcribed Spacer (ITS) region of 18S rRNA was amplified using the primers ITS1 (forward)3’TCCGTAGGTGAACCTGCGG-5’ and ITS4 (reverse)5’TCCTCCGCTTATTGATATGC-3’[4]. PCR products were purified and sequencing was carried out by the Dideoxy chain termination method (Euro fins, Bangalore). The resulting sequence was submitted in GenBank under the accession number MK108049.

The bio film forming ability of C. auris was assessed by the classical ring test[5]. A total of 1 mL of yeast peptone dextrose broth (Hi Media, India) was supplemented with 20 μL of yeast cell suspension and incubated statically at 37 ℃ for 24 h. The planktonic cells were removed and C. auris formed bio films as a ring on the walls of the test tube which was stained and observed using crystal violet (Hi Media, India) (Figure 1A). Preformed or mature bio films of C. auris were observed by adding 100 μL of C. auris cells to 1 mL of yeast peptone dextrose broth in a 24-well polystyrene plate (Tarsons,India). The plate was incubated at 37 ℃ for 48 h without agitation.After crystal violet staining, C. auris formed a thick mat, indicating a well-developed bio film which was adhered to the bottom and sides of the microtiter well (Figure 1B). The slide with the bio film cells was dried and examined under Scanning Electron Microscopy. SEM helped to visualise the bio films formed by C. auris on glass slides.The C. auris strain formed a biofilm with a thick aggregation of cells and the budding yeast cells embedded in the slime layer were also observed in scanning electron microscopy VEGA3 TESCAN(China) (Figure 1C).

在配电网中，由于受到变电站选址和通道受限的影响，往往需要对已有变电站进行升级改造，以满足长期负荷增长需求；但由于现场施工条件限制和电网安全规程要求，不得不选择全站停电改造，且改造周期较长。以某地市公司110 kV变电站为例，停电时间长达5个月，在此改造期间，配电网运行压力巨大，能否平稳度过负荷高峰时期，缺乏理论支撑和可行性论证，施工中能否安排全站停电进行升级改造缺乏有效规程参考和指导意见。

The patient was initially started on empirical treatment of single dose of oral fluconazole 150 mg and intravaginal clotrimazole 1% cream 5 g for 7 days. However, the patient did not show any improvement clinically. With the antifungal susceptibility report of the patient, the patient was started on oral ketoconazole 200 mg twice daily for 2 weeks. The patient responded well to ketoconazole.She improved and her symptoms got relieved. The patient was asymptomatic in her follow up visit after 2 weeks.

3. Discussion

C. auris has gained importance in recent years due to the emergence of drug resistant strains in this species[1]. C. auris isolate from non-blood source in a study by Larkin et al, showed decreased susceptibility to fluconazole when compared to other C. auris isolates[6]. In the present study, the isolate was resistant to fluconazole and amphotericin B in concordance to previous reports[6,7]. C. auris strains occasionally get misidentified because the current culture based diagnostic methods fail to distinguish them from other closely related species[7]. This factor stresses the need to employ molecular identification tools for appropriate diagnosis of Candidal infections rather than rely only on culture based methods.

Figure 1. Bio film formation by Candida auris which were collected from a 26-year-old female with vulvovaginal candidiasis. (A) Young bio film of Candida auris formed as a ring at the air liquid interface. (B) A thick mat of matured bio film formed at the bottom of polysterene plate well. (C) Scanning electron microscopy analysis of bio film.

C. auris has been reported causing candidaemia and wound infections[1,8]. C. auris has also been recently reported from an immunocompetent ICU patient[9]. The increasing reports of C. auris isolated from various clinical specimens clearly indicate the ability of the pathogen to colonise, invade and cause various diseases. Bio film formation by C. auris is considered as one of the virulence factors attributing to its drug resistance[6]. It was interesting to notice that the budding yeast cells were embedded in a thick slime layer. Slime is an important component of the exopolysaccharide layer of bio films formed by most of the Candida strains. This sequesters antifungal drugs, preventing them from reaching their cellular targets which leads to drug resistance. Slime also aids in the anchorage of C. auris cells on inert surfaces, making it capable to persist on nosocomial surfaces. Recent reports suggest that C. auris has successfully spread in nosocomial environments and was even detected among healthcare staff indicating the efficient human to human transmission of this strain[10]. The ability of C. auris to survive in bio film-form in patients is considered to be an important factor of its resistance to systemic antifungals and is linked to increased morbidity and mortality[10]. Candida strains have evolved effective resistance mechanisms for the commonly encountered drugs. Resistance mechanisms include modification in the target enzyme which results in point mutation in ERG11 gene, which is the target for azole class of antifungals. This may be due to different affinities of azoles that have different structures leading to differential activity. Future treatment regimens should target on destroying the biofilms of Candida sp. with antibio film agents in combinations with anti-fungal agents. Currently, very little is known about the virulence factors,transmission and mechanisms of drug resistance in C. auris. Species identification is critical for epidemiology, appropriate management as well as for implementation of infection control practices targeting C. auris bio films.

这个夜晚左小龙特别难熬，还有一只不懂事的蚊子在他的左手中指上咬了一口，那一口恰好咬在骨头断裂的位置，还不能挠，真是生不如死。有的时候疼好忍，但痒就不好忍了，忍还不能挠是最不能忍的。左小龙在这个时候想起了泥巴。他突然想，不知道这个小姑娘现在怎么样了。明天应该去找找她，告诉她自己受伤了，当然，是在见完黄莹的情况下。

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

Fellowship to J.S. in the form of Teaching Assistant (TA) by SASTRA Deemed University is thankfully acknowledged.

Funding

Financial support to PN from the Department of Biotechnology(DBT), Ministry of science and Technology, New Delhi (BT/PR/23592/MED/29/1203/2017) is gratefully acknowledged.

Authors’ contributions

L.K. collected samples and isolated the strain. J.S. performed the bio film analysis and imaging. L.K., C.S. and P.N. contributed to the final version of the manuscript. L.K. and P.N. supervised the project.