PJZ_58_1_129-135

INTRODUCTION

Prevalence to antimicrobial resistance has increased over the years and has been reported in different regions worldwide including developing and underdeveloped countries (Aceng et al., 2005; Eliopoulos et al., 2002). The reasons for the emergence of resistance may involve excessive use of drugs, prolonged hospitalization, irrational treatment strategies, and new emerging strains of pathogenic microbes. The frequency of fungal infections is on a rise and there is an utmost need to characterize these strains to ensure effective treatment (Fridkin and Jarvis, 1996).

Among many species of genus Candida, there are many diverse microorganisms but Candida albicans is seen in both commensal and pathogenic states. There has been increased concern about C. albicans infections over the past few years. About 80% of all the isolates belong to this candidal species (Raju and Rajappa, 2011). Identification of this species is of extreme importance, as different species of Candida differ in their infection pattern and susceptibility to different antifungal agents (Scully et al., 1994).

C. albicans is the most prevalent human pathogen causing superficial and fetal systemic infections (Soysa et al., 2008). Most of the emerging fungal infections arise as breakthrough infections in patients treated with various antifungal therapeutic agents. The contributing pathogens, prevalence rate, and severity of infection depend upon the basic mechanisms and underlying conditions. The mortality rate varies and has not been reported in many cases and currently, the fungal infection is regarded as a potential threat to global health (Pfaller et al., 1998; Kibbler et al., 2003). It is not easy to treat these infections as a variety of strains affects different tissues and organs, resistance, and scarcity of new antifungal drugs. The differences in fungal species showing different intrinsic antifungal susceptibilities reflect genetic variations that usually occur during their reproduction cycle (De Queiroz, 2007; Taylor et al., 2000).

It has been observed that C. albicans strains are becoming resistant to amphotericin B, fluconazole, polyenes, and 5-flucytosine (Goldman et al., 2004). The differences in resistance mechanisms have been observed in the molecular analysis (Kanafani and Perfect, 2008; Selmecki et al., 2006). From an evolutionary point of view, none of the resistance mechanism works alone.

The purpose of this study was to find and identify the emerging strains of C. albicans to avoid treatment and prognosis failures. We analyzed 15 clinical isolates collected from pus and urine samples. From these isolates, an antimicrobial susceptibility assay was performed against C. albicans using amphotericin B, caspofungin, fluconazole, ketoconazole, itraconazole, and voriconazole. Molecular identification and evolutionary relationship among resistant strains of C. albicans were studied.

MATERIALS AND METHODS

Sample collection and isolation

A total of 15 samples (pus and urine) were collected from various diagnostic labs. The sabouraud dextrose agar (SDA) medium plates were prepared (Goldschmidt et al., 1991) for culturing purposes. The samples were streaked onto the medium by using the standard streak plate method. Plates were incubated at 25°C for 48 h (Fromtling and Bulmer, 1978).

Identification of Candida albicans

Germ tube test (GTT) based on germ tube formation is a rapid and cost-effective screening test which is being used for years for the persuasive identification of C. albicans (Lee et al., 1999). The formation of germ tube in C. albicans is one of its major virulence factors. It is positive for C. albicans and C. dubliniensis. The bud formation was analyzed under a microscope to identify the C. albicans. The pure streaked culture was transferred onto selective BrillianceTM Candida Agar Base media (CHROM agar Candida Oxoid, CM1002A). The petri plates were placed in an incubator at 37°C and the typical color appearance of microorganisms was observed. C. albicans appear green in color on this medium while C. tropicalis indicate metallic blue color and other fungi appear white to mauve (Prato et al., 2008).

Antifungal susceptibility assay

Six anti-fungal drugs including amphotericin B, ketoconazole, voriconazole, caspofungin, itraconazole, and fluconazole were used. These drugs were prepared by serial dilution (Lalitha, 2004). Disk-diffusion testing was employed to evaluate the susceptibility of C. albicans against antimicrobial drugs. Filter paper discs impregnated with drugs to be tested were used. A pure culture of C. albicans was taken and emulsified in normal saline; it was mixed thoroughly so that no solid material from the colony was visible. A sterile culture swab was dipped in the emulsion; extra fluid was removed by gently squeezing the swab inside of the tube. With a culture swab, a lawn of growth was streaked onto the media agar plates. Sterilized forceps were used to place the individual antifungal disks on the streaked plate. The plates were inverted for 24 h at 25-30°C. The drug diffuses from the disc into the agar and the strains that were susceptible to the impregnated drug showed a zone of inhibition (Heatley, 1944). Zone of inhibition (if present) was measured, and then compared with the table of standards of each disk to decide either the strain was resistant or sensitive to that antifungal drug. The results of antifungal susceptibility testing were recorded as sensitive (S), resistant (R), and intermediate (I) based on the diameter of the zone of inhibition (Heatley, 1944).

Extraction of fungal DNA

The genomic DNA from the resistant strains of C. albicans (AZM1, AZM2, and AMZ3) was extracted using a commercial DNA extraction kit as per the manufacturer’s instructions (Promega, USA). The DNA was quantified by spectrophotometer at OD 260/280 nm by ratios 1.5-1.6. The sensitivity of the PFPRIM-F3 and PFPRIM-R4 primers was calculated by amplification through PCR for sequentially diluted concentrations (10ng-100ng) (Ilkit and Guzel, 2011).

Molecular sequencing and construction of a phylogenetic tree

The PCR reaction for the detection of the 18S rDNA gene of C. albicans was carried out. For this purpose, 25μl of reaction mixture containing 5.5μl of nuclease-free water, 12.5μl of Master mix (2X Green GoTaq), 2.5μl of 20 pmol PFPRIM-F3 primer and 2.5μl of 20 pmol PFPRIMR4 primer, and 2μl sample of genomic DNA was prepared. PCR was performed in the thermal cycler (Applied Biosystem 9902, Singapore) according to the PCR program (Embong et al., 2008). The analysis of the PCR product of the 18S rRNA gene of C. albicans was carried out on 1% agarose gel. 18S rDNA region was analyzed and sequenced by using primers NS1 5’ (GTA GTC ATA TGC TTG TCT C) 3’ and NS24 5’ (AAA CCT TGT TAC GAC TTT TA) 3’. PCR reaction was carried out by taking 20ng of genomic DNA and used as a template in 30μl reaction mixture using an EF-Taq (SolGent, Korea). A basic local alignment search tool (BLAST) at NCBI was applied to align the 18S rRNA sequences of C. albicans (AZM1, AZM2, and AMZ3) with known and related sequences in the database, and the percentage of homology was checked. C. albicans sequences with >99% similarity was considered to be of the same phylotype (Felsenstein, 2008; Saitou and Nei, 1987). Phylogenetic tree was constructed with the help of neighbor-joining method using custal omega online server (http://www.ebi.ac.uk/Tools/msa/clustalo/).

RESULTS

From 15 clinical specimens (Table I), eight C. albicans were isolated (7 urine and 1 sample of sputum). The germ tube test was positive which confirms the presence of C. albicans in these samples. The germ tube formation has been shown in the form of bud (Fig. 1A). A short hyphal (filamentous) extension arising laterally from yeast cells, with no constriction at the point of origin was observed microscopically. Germ tube was half the width and 3 to 4 times the length of the yeast cell and there was no presence of a nucleus. BrillianceTM Candida Agar Base, a selective medium was used for the isolation of C. albicans from other candidal and yeast species. In this media, C. albicans colonies appeared green in color which confirms the purified culture of this species as compared to other Candida species (Fig. 1B).

Table I. Collection of samples from diagnostic laboratories.

Sample code	Positive for C. albicans
Sputum
S1	-
S2	+
S3	-
Urine
U1	+
U2	+
U3	+
U4	-
U5	+
U6	+
U7	+
U8	-
U9	-
U 10	-
U 11	-
U 12	+

By disc diffusion and minimum inhibitory concentration method, we studied the susceptibility of eight identified strains of C. albicans against six antifungal drugs. From these 8 cultures, five candidal strains (AZM1-5) showed resistance to these drugs. Strain AZM1, AZM2, and AMZ3 of C. albicans showed significant resistance against all tested antifungal drugs (Fig. 1C) even at 1000 µg/ml as compared to AZM4 and AZM5 which were sensitive to amphotericin B, voriconazole, and caspofungin (Table II). Strain U-2, U-6, and U-7 were susceptible to most of these drugs even at 125 µg/ml (Fig. 1D).

The 18S rDNA region of resistant strains AZM1, AZM2, and AMZ3 of C. albicans was characterized to observe the novelty. The PCR product of 18S rDNA has a 600-320 bp size. According to blast outcome at NCBI server, it was confirmed that C. albicans strains AZM1, AZM2, and AMZ3 showed 100% sequence identity with our Candidal strains at the NCBI database. Phylogenetic trees were constructed by the neighbor-joining method and the evolutionary relationship was analyzed (Fig. 2). We have observed that strains AZM1, AZM2, and AMZ3 of C. albicans are evolutionarily related with a partial sequence of C. albicans with accession numbers: KX557291, KX557292, and KX557293, respectively when a blast on NCBI database.

Table II. Antibiotic susceptibility of C. albicans against selected anti-fungal drugs.

Antibiotic durg	U-1	S-2	U-3	U-5	U-12	U 2	U 6	U-7
Ketoconazole (KT) (µg/ml)
125	R	R	R	R	R	R	S	R
250	R	R	R	R	I	I	I	I
500	R	I	R	I	S	S	S	S
1000	R	I	R	S	S	S	S	S
Amphotericin B (AB) (µg/ml)
125	R	R	R	R	R	S	R	R
250	R	R	R	I	I	S	S	R
500	R	R	R	I	S	S	S	S
1000	R	I	R	S	S	S	S	S
Voriconazole (VRC) (µg/ml)
125	R	R	R	S	R	R	R	R
250	R	R	R	S	R	R	R	R
500	R	R	R	S	R	R	I	I
1000	R	R	R	S	I	I	I	S
Caspofungin (CAS) (µg/ml)
125	R	R	R	I	R	I	R	I
250	R	R	R	I	R	S	I	I
500	R	R	R	S	R	S	S	S
1000	I	R	R	S	R	S	S	S
Itraconazole (IT) (µg/ml)
125	R	R	R	R	R	R	R	S
250	R	R	R	R	R	R	R	S
500	R	R	R	R	R	S	R	S
1000	R	R	R	S	I	S	I	S
Fluconazole (FLC) (µg/ml)
125	R	R	R	R	R	S	S	I
250	R	R	R	R	I	S	S	S
500	R	I	R	I	I	S	S	S
1000	I	I	R	S	I	S	S	S

*Indications: R, resistance (<12mm); I, intermediate (13-17mm); S, sensitivity (>17mm).

DISCUSSION

The antifungal drug resistance is recognized as a threat to public health due to emerging strains, comparatively less awareness, and research. This study highlights the need for improvement in antifungal therapy to either stop or minimizes the emergence of resistance. The activities of six antifungal drugs including amphotericin B, caspofungin, itraconazole, voriconazole, fluconazole, and ketoconazole against C. albicans were analyzed. Van et al. (1989) conducted the antifungal assay using amphotericin B, itraconazole, and fluconazole against systemic C. albicans (Van et al., 1989). The antifungal assay showed the resistance of C. albicans against the azole class of compounds (Sanglard et al., 2003). Such studies contained a limited number of samples and a susceptibility test was performed only against azole drugs. A total of 8 isolates were confirmed as C. albicans among which five strains were resistant to antifungal drugs. C. albicans was primarily isolated by culturing on Sabouraud dextrose agar (SDA) and subsequently cultured on selective BrillianceTM Candida Agar Base, however, conventional techniques of identification are time-consuming and are based on extensive series of tests like carbohydrate fermentation and assimilation. While in other studies, Candida species were distinguished by using Pagano-Levin agar, which works on the reduction of triphenyl tetrazolium chloride. This medium produces colonies of C. albicans having pale color, while colonies of other Candida species show variable degrees of pink coloration. The sensitivity of Pagano-Levin agar is similar to SDA, but SDA has an advantage due to ease of availability and is standard media against fungal species (Samaranayake et al., 1987). Morphologically, C. albicans was identified by germ tube test (Lee et al., 1999).

Antifungal susceptibility was performed by the disk diffusion method (Jorgensen and Turnidge, 2015). It has been demonstrated that the disc diffusion method is more useful as compared to other antifungal susceptibility techniques (Serrano et al., 2004; Valle et al., 2004). Strain AZM1, AZM2, and AMZ3 of C. albicans showed significant resistance against all tested antifungal drugs, however, AMZ4 and AMZ5 showed resistance to ketoconazole and itraconazole only while the rest of the strains were susceptible. The prevalence and antifungal susceptibility of C. albicans showed that isolates were susceptible to amphotericin B followed by 50 % of resistance to fluconazole only (Ajenjo et al., 2011). We have observed that strain AZM1, AZM2, and AMZ3 of C. albicans exhibited resistance to voriconazole which was found effective drug against this fungus in previous studies (Krcmery and Barnes, 2002). The resistant strains of C. albicans were characterized by molecular 18 rDNA technique using NS1-NS24 primers (Aubert et al., 1996).

CONCLUSION

We identified new emerging strains of C. albicans that are resistant to available antifungal drugs. This is an alarming situation, as in most cases of antifungal therapies, the azole class of compounds is being used as a drug of choice. There is a need for developing and designing of new drug molecules to cure this pathogenic organism and this study would be helpful to improve prognosis.

Declarations

Acknowledgement

Authors acknowledge the Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University Multan for providing the lab facilities and related platform to execute this study.

Funding

The study received no external funding.

IRB approval

The study has been approved by the Institutional Ethical Committee of Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University Multan, Pakistan under notification no. IMBB-05/17.

Ethical statement

The present study was ethically approved by the Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan.

Statement of conflict of interest

The authors have declared no conflict of interest.

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