Identification of pathogenic yeasts from different clinical samples

-By Kranti Karande

Yeasts have always been a part of human microbiota. Some of the species belonging to the yeast family are opportunistic pathogens leading to infections of cutaneous, mucosal, bloodstream or deep-seated organs known as Candidiasis. These infections have become a major threat to humans. Different species of the Candida genus lead to different medical conditions and demonstrate varied sensitivity towards anti fungal agents used in practice. Failure to commence accurate anti-Candida therapy at an appropriate time has led to an increase in fatal cases of Candidiasis. Hence, it is important to correctly identify the Candida species to start appropriate and timely treatment. There are very few studies of yeast infections in Indian context. Thus this study is important as it is a study of a large number of Indian clinical samples for yeast infection reporting opportunistic and emerging pathogens. The study conducted on 176 clinical samples collected from Bharati Hospitals, Pune, Maharashtra was aimed at identifying the pathogenic species of yeast, understanding their anti fungal susceptibility and cell invasion capabilities.

Existing techniques for species identification of Candida species may be time consuming and even resulting in non-authentic identification if carried out by classical usage of blood culture technique. Recently developed molecular tests provide real time PCR assays and rapid results. Although these methods are specific, they do not improve the diagnostic sensitivity of Candidaemia and Candidiasis. Nonetheless DNA sequencing of amplified PCR products continues to be the most reliable method for authentic fungal species identification. A comparison of three different techniques for yeasts species identification was carried out along with characterization of these species for antifungal susceptibility and cellular invasion capabilities.

The clinical samples obtained were chemically treated as per standard protocol before being microscopically examined to find out the presence of unicellular yeast cells and other yeast organs. Samples with probable yeast were further treated to obtain isolated colonies and consequently obtain pure cultures which were deposited at NCMR- NCCS Pune, India. The colonies were further characterized biochemically using different identification tests. 

PCR amplification, DNA sequencing and phylogenetic analysis:
Genomic DNA from each isolate was extracted using lithium acetate. The precipitated DNA was used for PCR amplification (increasing the copies of DNA) using suitable primers. After confirmation of amplification, the PCR product was sequenced to identify different species present in the samples. A phylogenetic tree was constructed individually for each strain to confirm its species identification using known sequences of different Candida species available in GenBank at NCBI. 

MALDI-TOF/MS biotyper and chromogenic media method:
Actively growing pure cultures were used for protein extraction for MALDI-TOF MS analysis. The extracted protein samples were analyzed using a 60 Hz Nitrogen Laser and Flex Control software. Chromogenic media screening of yeasts was also performed and identification of colonies was done using colony color and appearance. Chromogenic media is used commonly in clinical laboratories for rapid identification of Candida strains. It is a fast method but not entirely reliable. Test results showed that chromogenic media successfully identified only 64 out of the 75 strains identified as C. albicans by sequencing. Seven strains were misidentified and four remained unidentified. Hence, it is safe to say that chromogenic agar technique is not a reliable method for yeast species identification. 

A total of 176 isolates were analyzed using MALDI-TOF/MS and the results were compared with results of DNA sequencing. MALDI-TOF MS could identify 157 of yeast isolates correctly. Overall, the correct identification rates of the 176 yeast isolates to species levels by the Bruker MALDI Biotyper systems was 89.2. This study confirms that MALDI-TOF MS presents an effective alternative to the sequencing for the correct identification of the emerging yeast pathogens however, there is a need to improve the database by regularly adding newer yeasts species from clinical and environmental scenarios.

Invasive fungal infections are becoming common and its rapidity of invasiveness by pathogens demands early arrest of infection by antifungal agents. There are immense changes in the host factors, infecting fungi, and antifungal agents and hence there is a need to accomplish antifungal susceptibility tests. The study conducted involved characterization of yeast species in terms of their antifungal susceptibility. A total of 157 isolates were tested for their susceptibility to antifungal drugs. Clinical yeasts showed increased resistance to fluconazole (55%) which is the most common antifungal used for treatment of candidiasis. Some emerging pathogens were found to be sensitive to most of the tested antifungal agents. It has been observed that strains of C. glabrata have highest resistance among various species of Candida. Many researchers have also isolated multi-drug resistant strains of C. glabrata

In vitro cell invasion assay involved investigation of the ability of various yeasts isolates to invade epithelial cells (HeLa Cells were used for this study). Since the invasion of host cells by pathogenic yeast cells is one of the important virulence factors in invasive candidiasis, this study is significant for the purpose of understanding potential threats from each Candida species. A total of 88 yeast isolates tested were isolated from invasive Candidiasis patients and only few could show invasion in vitro. Although the results of invasion capabilities study for different species like C. albicans, C. glabrata, C. parapsilosis showed varied results, it was observed that yeast cells invade host cells with the help of pseudo-hyphae or hyphal structures. Some studies have also shown that the transformation of C. albicans into hyphal form increases its interaction with the tissue cells, thus increasing its ability to adhere to human cells and potent invasion.

MALDI-TOF MS is an important tool in clinical set up for microbial identification as it is fast, low-cost, simple to use and wide spectrum applications in the identification of bacteria, archaea, and fungi. However, there is an urgent need to identify the species directly from the sample since it will greatly save on time of treatment which is crucial in serious cases like invasive Candidiasis. At present, both the methods which are considered reliable require pure culture, i.e. DNA sequencing and MALDI-TOF MS. The antifungal susceptibility tests showed that a large number of isolates showed resistance to all antifungals tested. A molecular analysis of all these strains is essential to substantiate the cause and mechanism of resistance. In the present study, nystatin was found to be the most effective antifungal agent. Many new types of yeast were reported in the study as probable human pathogens which should be included in diagnostic protocols.


Meet Dr. Rohit Sharma: Mycologist at NCMR, NCCS Pune

Dr. Rohit Sharma is a fungal taxonomist working at NCMR, NCCS Pune. He has been working in this field from last 15 years. He has identified 2 novel genera and 11 novel species of fungi. It was a great pleasure to interact with Dr. Sharma and find out more about him and his work.

Kranti: What motivated you to enter into the research field of fungal taxonomy?
Dr. Sharma:
I think it all started quite early when I used to go for sampling with my father, a Plant Pathologist at J.N. Agriculture University, Jabalpur. I used to go on collection trips with him during my school and college days to collect fungi infecting crops and wild plants and initial interest developed from there. During this period, I also got an opportunity to meet Dr. Kalman Vanky, expert in smut fungi and got opportunity to see his dedication towards the field of smut taxonomy. However, the actual involvement started when I began my doctoral work under Dr. Akhilesh K . Pandey and Dr. Ram C. Rajak whose lab was known to work on diversity and taxonomy of fungi. Here, I could get experience on entomopathogenic fungi, pathogenic and saprophytic micro fungi, mushrooms and yeasts. I developed a lot of insight in the lab under their guidance. Regular interactions helped to understand basics and the lab was rich in literature (monographs, journals and manuals) which usually is a bottle neck in the morpho-taxonomy of fungi. In the present organization as a curator of fungi, I got an opportunity to handle many fungi (yeasts, mycelial fungi and mushrooms) that helped in the development of the expertise in fungal taxonomy and systematics including polyphasic taxonomy.

Kranti : Why it is important to study fungal taxonomy?
Dr. Sharma: Fungi are hyper-diverse group of organisms. So far, many have been discovered but many are still to be discovered. Till now, about 1,20,000 fungi are known and researchers are discovering many more with an annual rate of approx. 1000 fungi. Based on the environmental sequence/ metagnomic data, it is now estimated that 22-38 lakhs fungi are yet to be discovered which makes it important to study the fungal diversity and taxonomy. Moreover, since they play an important role in environment and industrial biotechnology, it is important to explore, identify and subsequently study for their bio prospecting. Moreover, human and plant pathogens are increasing day by day and many environmental opportunistic pathogens are causing infections. Hence, it becomes important to have their authentic identification and proper classification for better management. The fungal taxonomy is complex and includes species specific morphological and physiological characters and intraspecific variation. The DNA-based taxonomic studies have helped to resolve many taxonomic problems and describe several cryptic species and are considered more stable than morphological characters.

Kranti: How many fungi you have collected and identified till now?
Dr. Sharma: So far we have collected and identified more than one thousand fungi from Lonar lake-Maharashtra, Famlong Lho-Sikkim, Achanakmar Sanctuary- Chhattisgarh, and other sites of India. They have been isolated from soil, litter, insect gut, sediments, water and as plant endophytes. We at NCMR-NCCS have described 2 novel genera (Matsushimamyces and Alanomyces) and 12 novel species, viz., Naganishia indica, Coniochaeta dendrobiicola, Leucosporidium himalayensis, Aureobasidium tremulum, Matsushimamyces bohaniensis, Alanomyces indica, Nothophoma raii, Curvularia lonarensis, Arthrinium gutiae, Pyrenochaeta telephoniae, Chaetomium jatrophae and Arthrinium jatrophae.

Kranti: What is your contributing role in services at NCMR?
Dr. Sharma: At present, I have been working as Curator-Fungi and looking after more than 15,000 fungal cultures preserved at our collection. My earlier work mostly was related to development of fungal culture collection at NCCS-NCMR, development of protocols, training human resource and undertaking basic research. So far, we have processed more than 2500 fungal cultures deposited by researchers from academia and from industry and accessioned approximately one thousand of them. We also provide identification services by conventional (morphology and API kit) as well molecular method, customized services and contractual research to industries. I along with my technician and project staff have been able to deposit more than 500 cultures to the NCMR-NCCS culture collection and screened them for various potential industrial applications viz., enzymes, bio active metabolites, waste degradation, etc. During the period, I have handled 4 research projects focusing on fungal diversity and their bio prospecting which in turn contributed to enrich the culture collection and preserve fungal wealth of our country.

Kranti: How has your journey been from being a PhD student to a Scientist at prestigious national institute?
Dr. Sharma: It has been a nice learning process, from working on ectomycorrhizal mushrooms during doctoral thesis to working with micro-fungi and yeasts at NCMR-NCCS. It has state of the art facilities and I can now use advanced techniques for my work. Work culture is very nice in our institute and  freedom to work and pursue my research interest in the area of diversity and taxonomy.

Kranti: What are the challenges you face during a field trip?
Dr. Sharma: During my doctoral thesis, I have went for sampling in the forests of Madhya Pradesh and Chhattisgarh for collection of ectomycorrhizal mushrooms. There are several challenges faced due to lack of communication, remoteness of the forests, etc. Moreover, we used to collect mushrooms from 8 am in the morning to 5 pm in the evening with a small packed lunch. In the evening after arriving at the forest guest house, we used to complete the mushroom data sheets and then around 7-pm we used to start culturing the mushrooms before putting them for drying. Sometimes it used to get 2-3 am in the morning because the mushrooms might decompose as the time passes after their harvest from the field.

Kranti: Which is your favorite fungus?
Dr. Sharma: The whole group of fungi are fascinating and one cannot pinpoint a single fungus. These are morphologically diverse and each one is unique in their features viz., colony color, shape, conidial shape, size, etc. The sexual and asexual stage found in these groups of organism makes them more interesting. From microscopic mycelial fungi to multi-cellular mushrooms to single celled yeasts, all have uniqueness of their own. From exogenous spores to endogenous spores, from pycnidia to cleistothecia, all have different morphological structures.

Kranti: What are the storage methods used at NCMR?
Dr. Sharma: We at NCMR-NCCS preserve the culture by five methods viz., cryopreservation at -80oC and at -196oC (liquid nitrogen) and by freeze drying. Apart from this, we also preserve the fungal cultures in distilled water and mineral oil (at 4oC). 

Kranti: What is your support system?
Dr. Sharma: My family and friends are my support system at individual level and colleagues and my team working with me at the professional level.

Kranti: What would you have liked to become if not a scientist?
Dr. Sharma: I would have been involved in doing agriculture, preferably doing scientific and organic agriculture with little bit of teaching at some academic organization so that I can interact and dissipate knowledge to students.

Kranti: What are the current and future projects in your group?
Dr. Sharma: Currently, we are working on collecting fungi from various sites and bio prospecting them for various applications like antimicrobial activity and treatment of industrial effluent. In future, we would like to focus more on the industrial applications of the fungal resource preserved in our collection and develop biotechnological usage of them.

Kranti: What are you hobbies?
Dr. Sharma: I like to read general books. Gardening is one my favorite hobbies and like to grow flowering as well as vegetable plants.

Kranti: Does research sometimes becomes stressful? What do you do for relaxation?
Dr. Sharma: Yes, research sometimes is stressful as sometimes multiple things gets lined up at the same time. To relax, I listen to classical music or go for trekking at nearby Pashan Tekri.

Kranti: What are your thoughts on Science communication in India? How can scientist contribute for better Science communication?
Dr. Sharma: I remember during my childhood I used to read a magazine ‘Science Reporter’, it used to explain scientific discoveries in simple language. Science communication in India is developing over the years by means of audio-visual and writing medium. Scientists can contribute for better science communication by conducting science talks for undergraduates explaining their research in a simplified manner. It is better now than it used to be. There are more communication channels available now with online platforms like blogs and online articles as compared to earlier days.