3.0 RESEARCH METHODOLOGY The composition of media for the growth of different Candida species, chemicals and reagents, equipment and glassware used

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1 3.0 RESEARCH METHODOLOGY The composition of media for the growth of different Candida species, chemicals and reagents, equipment and glassware used in the study are given in Annexture-I, II and III respectively. 3.1 Collection of isolates of Candida species A total of 72 isolates of Candida species used in study were obtained from the National Culture Collection of Pathogenic Fungi at Post Graduate Institute of Medical Education and Research (P.G.I.M.E.R.) Chandigarh, India. These isolates originated from blood stream infections during the period The strains included; Candida albicans 19/72 (26.38%) and nonalbicans Candida 53/72 (73.61%). Their distribution of the latter group was as follows: C. tropicalis 20/53 (37.74%), C. guilliermondii 8 (15.09%), C. glabrata 8 (15.09%) and C. parapsilosis 17 (32.07%).The isolates were preserved in 10% glycerol at -80ºC till their use in the experiment. The research project number SUIEC/10/03 was cleared by the Institute Ethics Committee (IEC) of the Shoolini University, Solan vide letter no SUBMS/IEC/10/57-60 dated 26 th Oct, Antifungal drugs and inhibitors Discs of fluonazole (25mcg) and its powder form were purchased from Hi-Media Laboratories, Mumbai (India). Pamidronate disodium in powder form was purchased from Sigma while Alendronate sodium pharmaceutical grade (Fosamax) from Cipla Ltd. All the drugs were diluted in the suitable solvents and the dilutions for antifungal susceptibility testing were prepared in RPMI 1640 medium in final concentration ranging from 64 microgram/ml to microgram/ml as per guidelines of CLSI M27 A3 protocol. 3.3 Morphological examination on Corn meal agar Pseudohyphae and chlamydospore production by Candida species was observed by streaking a portion of the isolate on to Corn meal agar (Hi-Media) plates supplemented with 1% tween 80. Each plate was divided into 4 quadrants. Using a sterile needle or straight wire, the yeast colony was lightly touched in each quadrant and streaked. Cover glass slip was flame sterilized and placed over control part of streak after it had cooled. The plates were incubated at 25ºC for 3-5 days. The cultures were examined for various morphological features such as pseudohyphae, blastospores or chlamydospores Morphological examination on Hicrome Candida diffrential agar Cultures of all the isolates grown overnight were streaked on Hicrome Candida differential agar (Hi-Media), incubated at 25ºC for hrs and observed for the appearance of coloured colonies of different Candida species. 32

2 3.3.2 Germ tube test This germ tube test is generally used for presumptive identification of Candida albicans. It is a rapid screening test where the production of germ tube is observed within two hours. For this test, a fresh growth from a pure culture was pick up and a very light suspension of the test organisms in ml of sterile serum (pooled from human serum or calf serum) was prepared. Inoculum optimally contained 10 5 to 10 6 cells per ml. The incubation was done at 37ºC for exactly two hrs. One drop from incubated serum was transferred on a glass slide with coverslip, observed under microscope for the development of germ tubes, which is indicative of initiation of hyphal growth arising from the yeast cell. Candida albicans strain ATCC was used as control in the test. The test was considered positive only if 30 % of the cells produced germ tubes Sugar fermentation test Liquid fermentation medium consisted of peptone-1%, NaCl-%, and Andrade s indicator0.005%, which was sterilized by autoclaving at 120ºC for 15 min at 15 pound pressure. Filter sterilized sugar (2%) was added to this medium, poured in to the sterile test tubes containing sterile Durham tubes were placed. The inoculum was prepared by suspending heavy inoculums of the yeast grown on Yeast nitrogen base (YNB, Hi-Media), which is a sugar free medium. The carbohydrate broth was inoculated with approx. 0.1ml of each inoculum, incubated at 25ºC for up to one week and examined at an interval of 48-72hrs interval for the production of acid indicated by pink color and gas in Durham tubes Sugar assimilation test Suspension of the yeast cells was prepared by adding heavy inoculum from a 24-48hr old culture in 2ml of YNB (Hi-Media). The melted agar in a vol. of 18ml was cooled to 45ºC, and added to the suspension and mixed thoroughly. Entire volume was then poured into 90 mm Petri plates, agar allowed to set in the plate at room temperature. Carbohydrate impregnated sterile discs (Hi- Media) were then placed on the surface of agar plate. A drop of 10% filter sterilized sugar solution was placed on each disc. The discs were dried at 37ºC and stored at 4ºC in air tight container. The plates were incubated at 37ºC for 3-4 days. The presence of growth around the disc indicated the assimilation of the carbohydrate. 3.4 Susceptibility testing of Candida species against antifungal agents Following CLSI M 44 A2 and M27A3 protocols, the susceptibility of Candida isolates was performed by disc diffusion and broth dilution methods. 33

3 3.4.1 Disc diffusion method. The susceptibility of Candida strains to antifungal drug was performed by disc diffusion method as per CLSI M44-A2 protocol. In this method, Muller-Hinton agar supplemented with 2% glucose and µg/ml methylene blue dye (GMB) medium was used. The discs of fluconazole stored at or below 8 C, in a nonfrost-free freezer were used in the test. Inoculum of the culture was prepared by picking five distinct colonies of approximately one mm diameter from a 24-hour-old culture of Candida species, and suspending them in 5 ml of sterile mol/l saline (8.5 g/l NaCl; 0.85% saline). The suspension was vortexed for 15 seconds and its turbidity adjusted visually with McFarland standard (Annexture-2). Within 15 minutes after adjusting the turbidity of the inoculum suspension, a sterile cotton swab was dipped into it, and spread over the dried surface of a sterile Mueller-Hinton + GMB agar plates. This procedure was repeated by twice, rotating the plate approximately at an angle of 60 each time in order to evenly distribute the inoculum. In the final step, the rim of the agar was swabbed. Fluconazole discs (25 mcg) and one sterile uninocolated disc as control were place over the surface of the inoculated agar plates, Incubated in inverted position at 35 0 C (± 2 C). Each plate was examined after 20 to 24 hours for observing inhibitory activity. The diameter of each zone of inhibition was measured. Candida albicans (ATCC 90028) was used as reference strain and Candida krusei (ATCC 6258) as quality control strain (CLSI, 2009), in the test Broth dilution method. The susceptibility testing of Candida isolates to fluconazole was also performed by the micro broth dilution method as per CLSI M27-A3 protocol. Dilutions of fluconazole powder were made and RPMI 1640 medium with L glutamine and without sodium bicarbonate (Hi-Media) was used in the test. The ph was adjusted to 7.0±0.1 in MOPS buffer (Hi-Media) and I N NaOH (Table-3.2, Fig-3.2, Annexture-1). The scheme for preparation of dilution is given in Table 3.1. The inoculum was prepared by picking five colonies, one mm in diameter, and suspended in 5ml of sterile 0.145mol/L saline (8.5g/l NaCl) or sterile water. The suspension was vortexed for 15 sec. and cell density was adjusted with spectrophotometer by adding sterile saline or sterile water. A working solution was made by 1:20 dilution followed by 1:50 dilution of stock suspension with RPMI 1640 broth medium which resulted in 1 x 10 3 to 5.0x 10 3 cells per ml. The test strain and the drug dilutions were mixed in equal volumes in a microtitre plate, incubated for 24 to 48 hrs at 35 0 C (± 2 C). The amount of growth in tubes containing the agent was compared visually with the amount 34

4 of growth in positive control tubes. Candida albicans (ATCC 90028) was used as reference strain whereas Candida krusei (ATCC 6258) as quality control strain in the test. The MIC values of fluconazole were determined as per guidelines of CLSI, Calculation of minimum inhibitory concentration values for fluconazole MIC50 and MIC90 values for fluconazole were calculated by arranging the MIC data in ascending manner and selecting cumulative % nearest to 50 as MIC50 and cumulative % nearest to 90 as MIC90 respectively. 3.6 Determination of in vitro interaction between fluconazole and ergosterol inhibitors by microdilution checkerboard method The in vitro interaction between fluconazole and ergosterol pathway inhibitors (Pamidronate disodium and Alendronate sodium) was assessed for fluconazole resistant strains by broth microdilution checkerboard method. In this method the drug interaction was determined by using fractional inhibitory (FIC) index, which is defined as the sum of the MIC of each drug when used in combination divided by MIC of drug when used alone. Synergistic and antagonistic FIC indices were defined as < and >4 respectively (Canton et al., 2005). 3.7 Studies on selective virulence traits (in Vitro) Assessment of Phospholipase activity For assessing phospholipase activity of different Candida species, cell suspension of each yeast was prepared in sterile saline (0.85%), and placed on the surface of agar medium containing egg yolk in 10 microliter volume, incubated at 37ºC for 4 days. Phospholipase activity was measured by dividing the colony diameter by the precipitation zone around the colony and expressed as pz values. The scoring was done as follows a pz value of was scored as (-), as weak (+), as mild (++), as relatively strong (+++) and < 0.69 (++++) as very strongly positive. Each experiment was conducted using three replicates (Gokce et al., 2007) Assessment of Protease activity Protease activity on Skimmed milk agar The protease producing ability of Candida species was assessed following the methods as described by (Gokce et al., 2007 and El-Diasty and Salem, 2007). Inoculums were prepared in Yeast Extract Potato Dextrose agar (YEPD) medium. Ten microliters of the each inoculum was transferred on a sterile paper disc (Hi-Media) placed on the surface of skimmed milk agar plates in which skimmed milk (Nestle India Ltd) was added just before pouring the medium. The inoculated plates were incucated at 30 0 C for 6 days. Protease activity was determined by measuring 35

5 zone of inhibition around the discs. The zone measurements were recorded as negative (-) for no clearance, (+) for mild activity, (++) for strong activity. Each experiment was conducted in replicates of three Protease activity on Gelatin agar (Gelatinase assay) The gelatin assay was done for testing whether a Candida species produced proteinases. The yeast colonies picked up from SDA were streaked on the SDA plates containing 1% gelatin, incubated for 48hrs at 37 0 C. The gelatinase activity was determined by observing the zones of inhibition around the colonies. For enhancing the visibility of the zones of inhibition around the colonies, 0.1% mercuric chloride was added Determination of Hemolytic activity The hemolytic activity of Candida species was determined on blood agar as per methods described by Ramesh et al.(2011) and Yigit et al.,(2011) with slight modifications. Candida culture was inoculated on SDA medium enriched with 5% human blood. The inoculated plates were incubated at 37 0 C for hrs and observed for appearance of the zones of lysis around the colonies. The hemolytic index was determined as ratio between the colony diameter and the total diameter of the colony plus the halo. This index indicates the amount of hemolysin production by the Candida species Biofilm formation The biofilm` forming ability of fluconazole resistant, sensitive dose dependent as well as sensitive strains of Candida species was assessed by following methods: Visual detection method. A loopful of test organism from the surface of Sabouraud dextrose agar plate was inoculated into a polystyrene tube (screw capped) containing 10ml of Sabouraud s dextrose broth (SDB) supplemented with 8% glucose in it. The tubes were incubated at 37 0 C for 48 hrs. after which the broth in the tubes was aspirated gently, without disturbing the biofilms which were washed following incubation twice in distilled water twice and stained with 1% safranin for 10 min. Tubes were observed for the presence of an adherent layer with naked eye. The test was conducted in triplicate and results were expressed as negative -, weak +, moderate ++ and strong +++ (Gokce et al., 2007) Spectrophotometer detection test Candida isolates were examined qualitatively for biofilm formation. Test Organisms were grown at 37 0 C for 24 hr on SDA plates. One ml of broth containing active test isolate was dispensed into nine ml SDB supplemented with 8% glucose. Yeast cells suspended in a volume of 200ml were 36

6 inoculated into each well of microtiter plates (Genaxy) and inoculated at 37 0 C for 24 hr without agitation. The plates were washed 4 times in phosphate buffer saline and stained with 1% safranine, aspirated, and spectrophotometer readings were taken at 490 nm with a microtiter plate reader (Easys). The test were conducted in replicates of three and results expressed as negative -,weak +, moderate ++ and strong +++ (Gokce et al., 2007) Agar invasion assay The agar invasion assay was performed as per method of Kontoyiannis et.al.,(2001) with some modifications. Precisely, around 50 colonies of Candida species were streaked on Yeast Potato Dextrose agar (YPD) plates, incubated at 37ºC for 2 days, after which the colonies were counted. The plates were washed under running water, during this process, the colonies were gently touched with gloved finger so as to dislodge the colonies from the surface of the medium under running water. The plates were then allowed to dry, examined with naked eye as well as under microscope for the presence of colonies that had invaded the agar. The colonies that had invaded the agar were counted. The scoring of the invasion was done as follows: +++,> 75% of colonies in the agar, ++,> 50-70%; + > 25-50%; ± 5-25% and -,< 5%. The designation ± was used as the end-point to indicate that there was no significant invasion. The percent invasion was calculated as the ratio of number of colonies left after washing to the total number of colonies before washing Hyphal Growth on solid media and adherence assay The hypha formation is a major virulence trait of fungus. The experiment was conducted to assess the ability of Candida strains to produce hyphae. The cells were grown in RPMI 1640 medium (Hi-Media) enriched with 10% fetal calf serum (Hi-Media) and incubated for 24hrs, Following incubation, the cells were inoculated on agar plates containing % agar supplemented with 4% fetal calf serum in order to induce hypha formation. The plates were incubated for 4 days at 37ºC and the morphology of fungal colonies was recorded (Angiolella et al., 2008, Wai- Kei et al., 2012). For assessing adherence of Candida strains, the cells were grown in YPD broth, washed with sterile phosphate buffer saline (PBS) and suspended in six well polystyrene plates (Corning) which contained RPMI 1640 medium supplemented with morpholinepropanesulfonic acid (MOPS) buffer (Hi-Media). The plates were incubated for 3hrs at 37ºC followed by washing. SDA in two ml vol. was poured in to each well and allowed to solidify. The plates were incubated at 37 C for 24hrs. Colonies were counted and the results were expressed as a percentage of inoculum. The size of 37

7 inoculum for each cell suspension was confirmed by plating aliquots of the culture directly on SDA plates. 3.8 Amplification of ERG11 gene Selective strains of different Candida species were subjected to PCR amplification. These included; resistant, SDD as well as sensitive strains. Among resistant strains C. albicans (AGK-3, B-1599/09, GMC-6), C. tropicalis (FOD-8), C.guillermondii (CG-12), C. glabrata (1/018/9) and C.parapsilosis (FR-5) were selected for amplification whereas one SDD strain of C.guillermondii (B-1343/09) and one sensitive strain each of C.albicans (03/074/37) and C.tropicalis (FOD-9) were also included. The criteria for selection was the virulence and fluconazole resistance. The sensitive strains were included for comparative analysis DNA extraction The fluconazole resistant, sensitive dose dependent and sensitive isolates of different Candida species were selected for DNA isolation.yeast species were grown in yeast extract phosphate dextrose (YPD, Hi-Media) for 24 h at 30ºC.Centrifuging tubes containing 1.5ml of the culture were centrifuged at 7500 rpm for 2 min, each pellet was washed twice in sterile distilled water and centrifuge at 7500 rpm for 4 min. The pellet was suspended in 200 µl of lysis buffer, mixed thoroughly and added 200 µl equal amount of phenol: chloroform: isoamyl alcohol (25:24:1). The mixture was vortexed and cooled on ice for 30 seconds. This procedure was repeated thrice followed by addition of 200 µl of TE buffer and vortexing. The mixture was centrifuged at rpm for 7 min. The aqueous phase was taken in a sterile microfuge tubes and equal amount of phenol:chloroform:isoamyl alcohol (25:24:1) was added to it. The mixture was centrifuged at rpm for 7 min. Aqueous phase was collected and 1/10 th volume of 3 M sodium acetate and equal volume of chilled isopropanol was added to it, mixed and incubated at -20ºC for 2 hours. It was then centrifuged at rpm for 5 min. The supernatant was decant off, DNA was precipitated with o.5ml of 70% ethanol and DNA pelleted by centrifugation at rpm for 5min, the supernatant was aspirated and the pellet allowed to dry at 37ºC for one hour. The pellet was suspended in TE buffer. The quantity of DNA was assessed by its electrophoresis on 0.8 % agarose gel Polymerase Chain Reaction (PCR) amplification of ERG 11 gene The DNA extracted in the above manner was used as a template for the amplification of coding region of ERG11 gene by Polymerase Chain Reaction employing the following primers: Forward 5 --GTT GAA ACT GTC ATT GAT GG -3 and Reverse 5 -TCA GAA CAC TGA ATC GAA 38

8 AG-3. The amplification was carried out in 50µl reaction volume containing 10x PCR buffer (5µl), genomic DNA 5µl, 2mmol/L of each dntp (5µl), 10pmol/L of each primer (2.5µl) and 3U/µl Taq polymerase (2.5µl) and sterile molecular garde water (Hi-Media) was added to a final volume of 50 µl. Amplification was performed in thermal cycler (Genaxy). The reaction conditions were as follows: one cycle of initial denaturation at 92 º C for 3 min and followed by 30 cycles, each consisting of denaturation at 92 º C for one min, annealing of primers at 48 º C for one min and extension of the strand at 72 º C for one min. Final extension was carried out at 72ºC for 5 min. The PCR products were electrophored on 0.8% agarose gel (containing µg/ml of ethidium bromide) and visualized under gel documentation system to see the expected size of the amplicons (Pam et al., 2012) Sequencing of amplified products and determination of variability in ERG 11 gene of drug resistant and sensitive Candida species The PCR amplified products were sequenced by a commercial sequencing facility, Xcelris laboratories pvt Ltd Ahmadabad (India). The nucleotide sequence variability in ERG 11 gene among Candida species was detected by multiple sequence alignment of nucleotide sequences using CLUSTAL OMEGA with the homologous gene sequences obtained by BLAST analysis of the sequences from National Center for Biotechnological Information (NCBI). Table-3.1 The protocol for preparation of dilution series of water soluble antifungals for susceptibility testing by broth dilution method. Step Concentration (µg/ml) Source Volume (ml) Medium(ml) Intermediate conc. (µg/ml) Stock 1.ml Step 1 Step Step 3 Step 3 Step Step 6 Step 6 Step 6 Step 9 Step 9 Step Final concentration (µg/ml)