Happy Final Thesis

Species identification, antifungal susceptibility testing
and genetic variability among Candida species

isolated from clinical samples
A Research Project Report

Submitted in partial fulfillment of the requirement
For the award of the degree of
MASTER OF SCIENCE
IN
MICROBIOLOGY
(Year- 2012)
Submitted by
Surjeet Singh
Department of Microbiology
SHOOLINI INSTITUTE OF LIFE SCIENCES &
BUSSINESS MANAGEMENT
Anand Campus, The Mall, Solan (H.P.)-173212
(Affiliated to H.P. University, Shimla)

SHOOLINI INSTITUTE OF LIFE SCIENCES AND
BUSINESS
MANAGEMENT
The Mall, Solan (H.P)
CERTIFICATE
This is to certified that the Research Project Report entitled Species
identification, antifungal susceptibility testing and genetic variability
among Candida species isolated from clinical samples submitted in
partial fulfillment of the requirements for the award of the degree of
Master of Science in the discipline of Microbiology of SILB Solan, is a
bonafide research work carried out by Surjeet Singh S/O Sh. Madan
Singh under my supervision and that no part of this research report has
been submitted for any other degree or diploma.
The assistant and help received during the course of this

investigation have been fully acknowledged.
Prof. M.P. Vohra Dr. Ajay Kumar Mr. Amit Kumar

Coordinator and Dean (Head) (Guide)
(Life Sciences)
DEDICATED
TO
MY PARENTS
ACKNOWLEDGEMENT
Every action is motivated by an ambition;
And all ambition are backed by strength given by God.

Successful completion of my project is the result of a lot of determination and laborious
work but no work can complete without the grace of God and hard work. So first I would
like to thanks almighty, having you believe in me, made it possible for me to believe in
myself, thats why I will always be thankful for confidence you have given me.
I am highly obliged and thankful to my augsted guide Mr. Amit Kumar Assistant
Professor of Microbiology, who helped me to correct my mistakes and to make my
project presentable and best.I am also thankful to Prof M.P Vohra Coordinator and
Dean of life sciences SILB and Dr. Ajay Kumar Head of department of Microbiology
who had observed my work I have a learned a lot from their knowledge.
Anyone can leave you alone in life but there is only a one greatful power which is
always with you and never let you down and that is parents. I am having my heartiest
thanks to my parents Sh. Madan Singh and Smt. Pushpa Devi who always
encouraged me and never let me down at any moment. I have no words to show my
regards towards my parents.
I owe my regardful thanks to Mr. Babu Ram and Mr. Sunil Kumar lab attendant of
Microbiology Department SILB Solan for the immense help offered to me.
I am also heartiest thankful to my friends who proved to be supportive at any good or

bad moment of my life. They always keep my mind free so that I can do my work
properly. I enjoyed their company a lot and I will always miss their company especially
Vandana,Rohit, Shivneet and Surabhi.
Surjeet Singh
ABBREVIATION
SDA - SABOURAUD DEXTROSE AGAR
SDB- SABOURAUD DEXTROSE BROTH
YNB- YEAST NITROGEN BASE
YPD- YEAST PHOSPHATE DEXTROSE
CMA-CORN MEAL AGAR
KOH- POTASSIUM HYDROXIDE
PCR-POLYMERSE CHAIN REACTION
TE BUFFER- TRIS ETHYLENE DIAMINE TETRACETIC ACID
dNTP DEOXYNUCLEOTIDE TRIPHOSPHATE
RFLP- RESTRICTION FRAGMENT LENGTH POLYMORPHISM
CONTENTS
SR. NO. TITLE PAGE NO.
1. INTRODUCTION 1-2
2. AIM AND OBJECTIVES 3
3. REVIEW OF LITERATURE 4-17
4. MATERIAL AND METHOD 18-29
5. RESULTS 30-49
6. DISCUSSION 50-51
7. SUMMARY 52-53
8. BIBLIOGRAPHY 55-64
INTRODUCTION
INTRODUCTION
Opportunistic fungal infections are widespread in immunosuppressed individuals and
are a serious concern for the management of such individuals. In the past two decades,
the frequency of invasive fungal infections and mortality has increased due to invasive
mycoses (Rapp RP 2004). Numerous yeasts and moulds such as Candida spp. and
Aspergillus spp. causes the most of the clinical diseases and common life-threatening
infections. Candida species especially Candida albicans are the part of commensal
flora (Cannon et al., 1995). The most common infecting species is C. albicans where as
other non albicans species are C. glabrata, C. parapsilosis, C. tropicalis and C. krusei
also causes various clinical diseases . These all approximately contribute 99% of all
human clinical cases (Pfaller MA et al., 2007). Candidemia is the fourth leading cause
of bloodstream infections and carries a 3555% mortality (Wisplinghoff H et al.,2004).
As these organisms show a range of susceptibilities to existing antifungal drugs,
distinguishing them is important for the selection of antifungal therapy. Despite highly
active antifungal drugs, mortality remains high at 5070% (Upton A et al.,2007).
C. albicans has been considered the predominant etiologic agent of oral candidiasis
(Stenderup, A 1998). Candida albicans is common and widespread opportunistic yeast
Pathogen, it causes an increasing number of human cutaneous infections , oral
candidiasis and vaginal candidiasis in recent years ( McCullough MJ et al., 1999). Over
the last decade, an increase in the incidence of candidiasis cases has been reported in
immunocompromised individuals associated with non albicans species, such as C.
glabrata, C. krusei, C. tropicalis and C. parapsilosis (Carrillo-Muoz et al .,2001). In
1995, C. dubliniensis, which is a species very closely related to C. albicans, was
isolated from cases of oral candidiasis in HIV-infected individuals ( Sullivan D et
al .,1995). There is great heterogeneity reported among the individuals of the Candida
isolates, C. albicans strains have been subdivided into different biological groups based
upon genetic subtypes (Tamura M et al .,2001). Several studies are known to support
the origin of genotypic differences among C. albicans isolates. The variation among
different species might be correlated with their mechanism of virulence and
pathogenicity (Lian CH et al.,2004). Polymerase chain reaction (PCR) amplification and
restriction enzyme digestion analysis are two of the most frequently used techniques in
establishing the genotyping of Candida species (Iwata T et al.,2006). The present study
is designed to examine the phenotypic traits and to observe genetic variability in drug
resistant Candida species.
In the present study, we selected restriction fragment length polymorphism (RFLP) as
our typing method to compare the genotype of drug resistant Candida species isolated
from different clinical cases. The study will help in characterization and to understood
extent of genotypic variability among drug resistant Candida species.
AIM
AND
OBJECTIVES
AIM AND OBJECTIVES
AIM:
Species identification, antifungal susceptibility testing and genetic variability among
Candida species isolated from clinical samples.
OBJECTIVES:
1. Isolation of Candida spp from various clinical samples.
2. Characterization and antifungal susceptibility testing of Candida species.
3. To check genetic variability among drug resistant Candida species.

REVIEW
OF
LITERATURE
REVIEW OF LITERATURE
CANDIDA
Candida albicans is an opportunistic pathogen that usually lives as commensal in the

healthy human host(Ryan KJ et al.,2004).Alteration in the balance between commensal
and the host ,like those that occur in the immunocompromised patient, may trigger the
infection of the mucosal epithelia, followed via the dissemination of the blood stream
and colonization of the internal organs. Candida albicans is the major fungal pathogen
of humans (Odds FC et al., 1988).Multiple strains of the candida albicans are found in
the female genital tract and HIV positive patient.
CLASSIFICATION OF Candida
Kingdom - Fungi
Phylum - Ascomycota
Subphylum - Saccharomycotina
Class - Saccharomycetes
Family - Saccharomycetacerae
The study of C.albicans biology has been hinderd because its obligates diploid nature.
However the recent studies have been indicated that organism have some type of
sexual cycle. There is the reversible conversion of C.albicans from yeast cell to
filamentous form during its adherence to the mucosal epithelia of human tissue,
invasion and disease progression. Hence, numerous molecular structure of C.albicans
has focused on genes required for filamentation; many of these genes are required for
its virulence. Candida species shows phenotypic characterstics such as formation of
germ tube, production of chlamydospore, biochemical pattern and some coloured
colonies on different agar such as CHROM agar and corn meal agar.
HISTORY OF CANDIDA
An oral Candida infection was described for the first time in the eighteenth century.
Candida albicans, was identified in the nineteenth century, but most research on its
biology was done, starting from the second half of the twentieth century. It had many
different names like Oidium albicans, Syringospora robinii, Mycoderma vini,
Saccharomyces albicans and Monilia albicans. The name Monilia albicans was used for
many years in medical publications but it was Christine Berkhout, in 1923, who invented
the name Candida albicans that is used until today. This name has been authorized for
use by the International Botanical Congress (IBC) (Barnett JA 2008).
The genus Candida includes about 150 different species, but only a few are known to
cause human infections. C. albicans is the most significant pathogenic species. Other
human pathogenic Candida species include C. tropicalis, C. glabrata, C. krusei, C.
parapsilosis, C. stellatoidea and C. kefyr (McCullough MJ et al., 1994).
A COMMENSAL YEAST
Candida albicans is a commensal yeast that in most people lives in their gastro-
intestinal tract, mouth or vagina. The anatomical site where Candida albicans occurs is
very important. When it appears at another place than its normal location, it becomes a
dangerous microorganism. As opportunistic microorganism, it causes infections in
immunocompromised people, for example in transplant receivers, intensive-care,
surgical and cancer patients and HIV infected persons. Also the unrestricted use of
antibiotics can induce infections with C. albicans by causing its overgrowth (Douglas,
2002; Seneviratne et al., 2008). Pathogenic fungi of the Candida genus are among the
main causes of hospital-acquired infections. (Chandra et al., 2001; Douglas, 2003;
Seneviratne et al., 2008). Over the period 19801990, hospital data reported a steady
increase in the rate of nosocomial fungal infections including Candida albicans
infections from 2.0 to 3.8 per 1000 discharges (De Rosa et al., 2009; Ha et al., 2010).
The increase is likely multi-factorial, including changes in clinical practice such as
increased use of long term venous catheters, use of broad-spectrum antibacterial
agents and improved laboratory techniques for identification of unusual Candida
species.
There are several types of Candida infections, for example invasive
candidiasis, including candidemia and disseminated candidiasis with deep organ
involvement, candiduria, Candida infection of gastro-intestinal tract and Candida
infection of the respiratory tract and throat. Candidemia is a bloodstream infection with
Candida. When the Candida species spread throughout the body after entering via the
bloodstream, we call it invasive candidiasis (Ha et al., 2010).
CANDIDEMIA
According to Ha et al., 2010, candidemia has become an increasingly important

infection. The mortality rate of candidemia is higher than 50% (range 1390%). In most
cases candidemia has an endogenous origin, which is caused by yeasts originated from
the human microflora. Hereby the main entry is the gastro-intestinal tract (Bouza et al.,
2008; Singhe et al., 2009). The main risk factors for candidemia are serious alteration in
cutaneous and mucous barriers (because of surgery wounds, intubation or vascular
catheters) and colonization of these barriers due to the use of broad spectrum
antibiotics (Mensa et al., 2008; Picazo et al., 2008).
INVASIVE CANDIDIASIS
Invasive candidiasis is more severe than any other nosocomial bacteraemia. It is the
fourth cause of nosocomial infection in the United States, with an appearance higher
than some other common bacterial infections. Mortality rates lay between 40 and 60%,
even with adequate treatment (Douglas, 2002; Li et al., 2007; Seneviratne et al., 2008).
Three components determine the intensity of Candida albicans translocation and so the
interaction in the pathogenesis of invasive candidiasis:
Increase in fungal colonization. This is the most important factor in the pathogenesis of
invasive candidiasis. The density and colonized surface area are determining for the
intensity of the infection. Breakdown of normal mucosal, epithelial or skin barriers, often
the result of the number of invasive procedures like recent surgery, severe burns or use
of intravascular devices. The mucosal integrity and permeability are determining for the
infection. Loss of immune mechanisms (correlated to the activity of neutrophils)
responsible for prevention of access and dissemination of Candida albicans in deep
tissues and organs. (Mensa et al., 2008; Singhi et al., 2009; Ha et al., 2010).
The main risk factors for invasive candidiasis include prolonged stays in
intensive care units, improvements in intensive care strategies like central venous
catheters, mechanical ventilation and hyper-alimentation and development of more
aggressive surgical techniques (Bedini et al., 2005).
OTHER CANDIDA INFECTIONS

URINARY CANDIDIASIS
Urinary candidiasis is one of the most confusing forms of candidiasis since the
differentiation between colonization and real infection is difficult to make. Isolation of
Candida albicans in urine is believed to represent colonization or contamination.
Candiduria can also be a sign of candidemia or invasive renal candidiasis. It can cause
candidemia during invasive urologic procedures (Hollenbach 2008; Singhi et al., 2009).
GASTROINTESTINAL CANDIDIASIS
Gastrointestinal candidiasis can occur in children and adults with immune deficiency
disorders, cancer and after surgery, in other words impaired persons. Gastrointestinal
candidiasis may involve the stomach, intestine or hepatobiliary system (Singhi et al.,
2009).
RESPIRATORY CANDIDIASIS
Respiratory candidiasis may involve airways from pharynx and epiglottis to bronchi.
Candida albicans is a frequent colonizer of the upper respiratory tract, especially in
hospitalized patients. The symptoms can be hoarseness of the voice, low fever,
tachypnea and sometimes there are no specific findings on physical examination
(Singhi et al., 2009).
VULVO-VAGINAL CANDIDIASIS
Vulvo-vaginal candidiasis (VVC) is one of the most common infections of the vulva and
the vagina. Approximately 3 out of 4 sexually active women will experience VVC at least
once in their lifetime (Ferrer, 2000; Irie et al., 2006; Li et al., 2008).
SIGNS AND SYMPTOMS

Symptoms of candidiasis vary depending on the area affected(Dolin et al.,2010). Most
candidial infections result in minimal complications such as redness, itching and
discomfort, though complications may be severe or fatal if left untreated in certain
populations. In immunocompetent persons, candidiasis is usually a very localized
infection of the skin or mucosal membranes, including the oral cavity (thrush), the
pharynx or esophagus, the gastrointestinal tract, the urinary bladder, or the genitalia (
Walsh TJ et al.,1996).
Candidiasis is a very common cause of vaginal irritation, or vaginitis, and

can also occur on the male genitals. In immunocompromised patients, Candida
infections can affect the esophagus with the potential of becoming systemic, causing a
much more serious condition, a fungemia called candidemia (Fidel PL 2002). Thrush is
commonly seen in infants. It is not considered abnormal in infants unless it lasts longer
than a few weeks.
Infection of the vagina or vulva may cause severe itching, burning,

soreness, irritation, and a whitish or whitish-gray cottage cheese-like discharge, often
with a curd-like appearance. These symptoms are also present in the more common
bacterial vaginosis (Terri Warren, RN 2010). In a 2002 study published in the Journal of
Obstetrics and Gynecology, only 33% of women who were self-treating for a yeast
infection actually had a yeast infection, while most had either bacterial vaginosis or a
mixed-type infection (Ferris DG et al., 2002).Symptoms of infection of the male genitalia
include red, patchy sores near the head of the penis or on the foreskin, severe itching,
or a burning sensation. Candidiasis of the penis can also have a white discharge,
although uncommon.
DIMORPHISM
Although often referred to as dimorphic, C. albicans is in fact polyphenic. When
cultured in standard yeast laboratory medium C. albicans grows as ovoid yeast cells.
However, mild environmental changes in temperature and pH can result in a
morphological shift to pseudohyphal growth. Pseudohyphae share many similarities with
yeast cells but their role during candidiasis remains unknown (Berman J et al., 2002).
When C. albicans cells are grown in medium which mimic the physiological environment
of a human host, they grow as true hyphae. The ability of C. albicans to form hyphae
has been proposed as a virulence factor as these structures are often observed
invading tissue and C. albicans strains which are unable to form hyphae are defective in
causing infection. In a process that superficially resembles dimorphism, C. albicans
undergoes a process called phenotypic switching, in which different cellular
morphologies are generated spontaneously. One of the classically studied strains that
undergo phenotypic switching is WO-1, which consists of two phases: one that grows as
round cells in smooth white colonies and one that is rod-like and grows as flat gray
colonies (Rikkerrink E et al., 1988). Another potential regulatory molecule is Efg1p, a
transcription factor found in the WO-1 strain that regulates dimorphism, and more
recently has been suggested to help regulate phenotypic switching. Efg1p is expressed
only in the white and not in the gray cell-type, and over expression of Efg1p in the gray
form causes a rapid conversion to the white form (Srikantha T et al., 2000).
CANDIDA GENOME
One of the most interesting features of the C. albicans genome is the occurrence of
numeric and structural chromosomal rearrangements as means of generating genetic
diversity, named chromosome length polymorphisms (contraction/expansion of repeats),
reciprocal translocations, chromosome deletions and trisomy of individual
chromosomes. These karyotypic alterations lead to changes in the phenotype, which is
an adaptation strategy of this fungus. These mechanisms will be better understood with
the complete analysis of the C. albicans genome.
The C. albicans genome for strain SC5314 was sequenced at the Stanford DNA
sequencing and technology centre (Braun B et al., 2005). The genome of the WO1
strain was sequenced by the Broud institute of MIT and Harvard. The sequencing of the
C. albicans genome and subsequently of the genomes of several other medically
relevant Candida species has profoundly and irreversibly changed the way Candida
species are now investigated and understood (Denfert C et al.,2007). The C. albicans
genome sequencing effort was launched in October 1996. Successive releases of the
sequencing data and genome assemblies have occurred in the last 10 years,
culminating in the release of the diploid assembly 19, which provided a haploid version
of the genome along with data on allelic regions in the genome (Denfert C et al., 2007).
A refined assembly 20 with the eight assembled C. albicans chromosomes was
released in the summer of 2006. Importantly, the availability of sequencing data prior to
the completion of the genome sequence has made it possible to start C. albicans post-
genomics early on. In this regard, genome databases have been made available to the
research community providing different forms of genome annotation. These have been
merged in a community-based annotation hosted by the Candida Genome Database.
The availability of the genome sequence has paved the way for the implementation of
post-genomic approaches to the study of C. albicans: macroarrays and then
microarrays have been developed and used to study the C. albicans transcriptome;
proteomics has also been developed and complements transcriptional analyses;
furthermore, systematic approaches are becoming available to study the contribution of
each C. albicans gene in different contexts. Other Candida genome sequences have
been, or are being, determined: C. glabrata, C. dubliniensis, C. parapsilosis, C.
guilliermondii, C. lusitaniae, and C. tropicalis. These species will soon enter the post-
genomic era as well and provide interesting comparative data. An interesting feature of
the Candida clade is that the CUG codon, which normally specifies leucine, specifies
serine in these species, an unusual example of a departure from the universal genetic
code. The genome sequences obtained for the different Candida species along with
those of non-pathogenic hemiascomycetes provide a wealth of knowledge on the
evolutionary processes that shaped the hemiascomycete group, as well as those that
may have contributed to the success of different Candida species as pathogens
(Denfert C et al., 2007).The genome of C. albicans is highly dynamic, and this
variability has been used advantageously for molecular epidemiological studies of C.
albicans and population studies in this species. A remarkable discovery arose from the
genome sequence in identifying the presence of a parasexual cycle (no meiotic division)
in C. albicans (Butler G et al., 2009). This parasexual cycle is under the control of
mating-type loci and switching between white and opaque phenotypes. Investigating the
role the mating process plays in the dynamics of the C. albicans population or in other
aspects of C. albicans biology and pathogenicity will undoubtedly represent an
important focus for future research.
BIOFILM PRODUCTION
A biofilm is a microbially derived sessile community characterized by cells that are

irreversibly attached to a substratum or interface or to each other, embedded in a matrix
of extracellular polymeric substances that they have produced, and exhibiting an altered
phenotype with respect to growth rate and gene transcription. This is the definition
formulated by Donlan and Costerston in 2002.
The biofilm is attached to a surface which can be biotic (the human body)
or abiotic (implanted, medical devices such as urinary or intravascular catheters,
prosthetic heart valves, shunts, endotracheal tubes, voice prostheses, cardiac
pacemakers and joint replacements). The consequences for health include biofilm
resistance to antimicrobial agents, device failure, resistance to host defences and arise
of persistent infections (Donlan, 2001; Kojic et al., 2004; Ramage et al., 2006; Nett et
al., 2006).
Candida albicans lives on several simple carbon and energy sources; it can grow on
glucose, fructose, lactose and galactose (Seneviratne et al., 2008). C. albicans can
grow well in the presence of oxygen, grows much better on simple substrates and is
resistant to acidic environment. Candida biofilms consist of nearly 40% carbohydrate,
with the major carbohydrate component varying between Candida species.
BIOFILM FORMATION AND DEVELOPMENT

Biofilm formation by Candida species is a complex process. There are three important
steps in the development of biofilms namely adherence and colonization on the
substrate, growth and proliferation of the yeast cells and maturation of the biofilm. The
early stage of biofilm formation is characterized by adherence and development of the
yeast blastospores into microcolonies.
After 1824 h, the bilayered structure of the Candida albicans

biofilm consists of budding yeasts, germ tubes, and young hyphae. In this phase,
extracellular matrix production occurs. The extracellular matrix is mainly composed of
DNA, proteins and polysaccharides. During maturation, the biofilm becomes a thick
extracellular matrix layer with a dense network of yeasts, pseudohyphae, and hyphae
( Blankenship et al., 2006; Nobile et al., 2006;). Mature Candida albicans biofilms have
a complex 3D structure with typical water channel architecture and a large
heterogeneity (Hawser et al., 1994;Baillie et al., 1999;). This complex structure is an
optimal environment for influx of nutrients, efflux of waste products and ramification of
water channels (Ramage et al., 2006).
Several in vitro models (Nobile et al., 2006) have been used to
clarify the stages and processes required for C. albicans biofilm formation. The
difference between yeast cells that form biofilms and yeast cells that live as free-living
organisms has also been studied. For example, Candida biofilms are 30 to 2000 times
more resistant than planktonic cells to various antifungal agents depending on the
strain.
DIAGNOSIS
Diagnosis of a yeast infection is done either via microscopic examination or culturing.

For identification by light microscopy, a scraping or swab of the affected area is placed
on a microscope slide. A single drop of 10% potassium hydroxide (KOH) solution is then
added to the specimen. The KOH dissolves the skin cells, but leaves the Candida cells
intact, permitting visualization of pseudohyphae and budding yeast cells typical of many
Candida species. For the culturing method, a sterile swab is rubbed on the infected skin
surface. The swab is then streaked on a culture medium. The culture is incubated at
37C for several days, to allow development of yeast or bacterial colonies. The
characteristics (such as morphology and colour) of the colonies may allow initial
diagnosis of the organism causing disease symptoms (Srikumar C 2010)
TREATMENT OF CANDIDIASIS
In clinical settings, candidiasis is commonly treated with the antifungal drugs.
Commonly used antifungal drugs to treat candidiasis are topical clotrimazole, nystatin,
fluconazole, and topical ketoconazole. For example, a one-time dose of fluconazole
(150-mg tablet taken orally) has been reported as being 90% effective in treating a
vaginal yeast infection (Moosa MY et al., 2004) .This dose is only effective for vaginal
yeast infections, and other types of yeast infections may require different dosing. In
severe infections, amphotericin B, caspofungin, or voriconazole may be used. Local
treatment may include vaginal suppositories or medicated douches. Gentian violet can
be used for thrush in breastfeeding babies, but when used in large quantities, it can
cause mouth and throat ulcerations, and has been linked to mouth cancer in humans
and to cancer in the digestive tract of other animals (Craigmill A 1991). Chlorhexidine
gluconate oral rinse is not recommended to treat candidiasis, but is effective as
prophylaxis agent (Ferretti GA et al., 1988); chlorine dioxide was found to have similar
in vitro effectiveness against Candida (Uludamar A et al.,2010).
ANTIFUNGAL DRUG RESISTANCE

Most of the yeasts now become resistant to antimicrobial drugs. Drug resistance
appears to be dependent on different mechanisms (Donlan et al., 2002). C. albicans
can develop resistance to antifungal drugs (Cowen LE et al .,2002). Because of the
biofilm and the formation of an extracellular matrix, the cells are shielded from the
environment and it becomes more difficult for the antibiotics to reach the yeast cell. Also
the efflux pumps and the presence of a subpopulation of persisted cells can play a role
in antifungal drug resistance ( Pierce et al., 2008). There is only a limited series of
antifungal drugs available for the treatment of Candida albicans but resistance is
increasing. (Liu et al., 2008).
ERG11 AND OTHER ERG GENES

In all fungal species, ERG11 (also described as ERG16, CYP 51A1) is the gene
encoding ERG11p or lanosterol 14- demethylase, an essential enzyme for ergosterol
synthesis. Resistance to azole antifungal drugs has been associated with ERG11 gene
overexpression and/or point mutations and also alterations in the ergosterol biosynthetic
pathway. Over expression of ERG11 causes an increased copy number of the enzyme
lanosterol 14-demethylase and results in increased ergosterol synthesis which
overwhelms the capacity of the antifungal drug. The effect of ERG11 gene
overexpression on antifungal susceptibility has been described by several studies in C.
albicans(Lamb DC et al .,1997,Perera S et al.,2001,Lupetti A et al.,2002) and also in C.
glabrata and C. dubliniensis clinical isolates (Perera S et al.,2002, Marichal P et
al.,1997, Casalinuova I.A et al.,1999). Enhanced expression of the ERG11 gene in C.
albicans as a consequence of azoles exposure was observed in matched sets of clinical
isolates from the same strain (Lopez-ribot JL et al.,1998, White TC et al.,1997). In vitro
azole-dependent ERG11 up regulation was demonstrated in additional Candida species
such as C. tropicalis, C. glabrata and C. krusei (Henry KW et al.,2000). Recently, in an
analysis of unmatched sets of clinical isolates it was found that resistance did not
correlate with overexpression of ERG11 (White TC et al.,2002). Moreover, it has been
reported that depletion of the ERG11 gene in C. glabrata review of mechanisms 72
glabrata results in the accumulation of (Perera S et al., 2002 , Snydman DR et al.,
2003 ) demethylzimosterol, which did not cause defective growth of fungal cells in vitro
and in vivo( Nakayama H et al.,2001).
Fluconazole and other azoles resistance has also been associated with point
mutations of the ERG11 gene (Franz R et al .,1998, White TC et al.,1998 ); these
mutations result in conformational changes that reduce effective binding between
azoles and their target. Several investigators found sequence differences of the ERG11
gene in fluconazoleresistant C. albicans and in S. cerevisiae transformants(Sanglard D
et al.,1998). A list of different aminoacid exchanges has been provided by different
studies that could simply reflect allelic variations(Morchhauser J 2002). In fluconazole-
resistant C. albicans isolates frequently observed nucleotide changes were concerned
with two aminoacids located near the heme binding site (R467K [arginine 467 replaced
by lysine] and G464S [glycine 464 replaced by serine]); this probably resulted in
structural or functional alterations reducing fluconazole affinity in Erg11p.
The correlation between decreased susceptibility to azole drugs and nucleotide

changes in the ERG11 sequence was not always observed( Marichal P et al .,1999).
Recently, other nucleotide substitutions in ERG11 gene were identified (K143R [lysine
143 replaced by arginine], E266D [glutamic acid 266 replaced by aspartic acid], V404L
[valine 404 replaced by leucine], V488I [valine 488 replaced by isoleucine]) in three C.
albicans isolates( Maebashi K et al., 2003); these mutations were associated with the
fluconazole resistance phenotype.
In C. albicans, increased expression of ERG1 gene encoding squalene
epoxidase and of ERG2 gene encoding C8-sterol isomerase was associated with
fluconazole resistance (Smith WL et al., 2002). In contrast, other studies found that the
ERG1 gene was repressed in resistant isolates (Cowen LE et al.,2002). The ERG3
gene encoding C5,6-desaturase was observed first in S. cerevisiae(Watson PF et
al.,1989). Defective sterol C5,6-desaturase was attributed as the cause of fluconazole
resistance in C. albicans clinical isolates from AIDS patients(Kelly SL et al.,1997). Such
isolates accumulated ergosterol precursors including ergosta-7-enol and ergosta-7,22-
dienol. The molecular mechanisms associated with ERG3 defects are still
unclear(Jackson CJ et al., 2003).
EXPRESSION OF TWO MAJOR EFFLUX PUMPS
Efflux pumps belong to two different classes: the ATP-Binding Cassette (ABC)
transporters and the Major Facilitators Superfamily (MFS). The ABC transporters are
energy-dependent by ATP hydrolysis; the MFS transporters operate through a proton
gradient. Two ABC transporters genes, CDR1 and CDR2 (Candida Drug Resistance),
as well as that encoding a major facilitator, CaMDR1 (Candida albicans Multidrug
Resistance), have been shown to be overexpressed in C. albicans azole-resistant
isolates (Wirsching S et al., 2000). CaMDR1 is specific for fluconazole resistance but
not for other azoles(Sanglard D et al., 1995). Upregulation of these efflux pumps
reduces the effective concentrations of fluconazole in the fungal cell and is correlated to
azole resistance in C. albicans. Genetic deletion of the CDR1 gene resulted in
hypersusceptibility to azole drugs(Sanglard D et al., 1996), whereas CDR2 gene
disruption did not cause hypersusceptibility to these agents. The latter gene is closely
related to CDR1 and disruption of CDR1 and CDR2 resulted in increased
hypersusceptibility to azole antifungals (Sanglard D et al., 1996). Microarrays
technology was used to examine differences in gene expression and identification of
new genes associated or not associated with drug resistance. These data suggest that
the efflux pumps may be regulated by combined expression of several genes. Analysis
of these differentially regulated genes requires further investigation and opens up the
possibility of finding new targets for antifungal therapeutics.
OTHER CHANGES IN FLUCONAZOLE RESISTANCE

Recently, antifungal resistance results in biofilm-associated infections (Kuhn DM et al.,
2002). Efflux pumps do not appear to contribute to fluconazole resistance in C. albicans
at late (intermediate and mature) stages in biofilm formation (Mukherjee PK et
al .,2003), but solely in the early-phase. On the contrary, changes in sterol profile were
expressed by resistant phenotypes at intermediate and mature phases. Therefore,
phasespecific mechanisms are suggested to be operative in antifungal resistance of
biofilm cells (Mukherjee PK et al ., 2003).Some of the C. albicans cell wall
glycoproteins have been found to be highly immunogenic and differently modulated
according to fungal growth (Spagnoli GC et al., 1985).
In vitro studies on the cell wall of fluconazole- susceptible and -resistant
C. albicans strains detected altered distribution of cell wall glucan-associated proteins
(Angiolella L et al., 2002). These results suggests that fluconazole treatment could have
an effect on fungal cell wall metabolism and structure( Hazen KC et al.,2000), and these
effects may be stably incorporated into the cell wall upon acquisition of resistance
(Angiolella L et al.,2002). The asexual and diploid nature of C. albicans complicates the
characterization of gene expression in antifungal drug resistance (Lockhart SR et al.,
2003). Several studies investigating changes in chromosome copy number, loss (or not)
of heterozygosity, gene disruption at definite loci and other genetic strategies have been
also linked to fluconazole resistance among Candida species (Debacker MD et
al .,2003).
MATERIALS
AND
METHODS
MATERIAL AND MAETHOD
MEDIA USED AND COMPOSITION

CORN MEAL AGAR
Corn Meal 50gm
Agar 15gm
Distilled water 1000ml
Ph 6.2
Mix corn meal and agar in 1000 ml of distilled water. Sterilized by autoclaving at 121 0C
for 10 min.
SABOURAUD DEXTROSE AGAR (SDA)
Glucose 40gm
Peptone 10gm
Agar 20gm
Ph 5.5
Add glucose, peptone and agar in 1000ml of distilled water.sterlized by autoclaving at

1210C for 10 min.
SABOURAUD DEXTROSE BROTH
Glucose 40gm
Peptone 10gm
Ph 5.5
Add glucose and peptone in 1000ml of distiiled water and sterilized by autoclaving at
1210 C for 15 min.
YEAST PHOSPHATE DEXTROSE (YPD)
Bacto peptone 10gm
Glucose 20gm
Yeast extract 10gm
Add bacto peptone, glucose and yeast extract in 1000ml of distilled water and sterilized
at 1210 C for 15 min.
YEAST NITROGEN BASE (YNB)
Potassium dihydrogen orthophosphate 1.0gm
Magnesium sulfate 0.5gm
Ammonium sulfate 5.0gm
Noble agar 25.0gm
Add all the above ingredients in 1000ml of distilled water and autoclaved at 115 0C for
15 min.
MUELLER-HINTON AGAR
Beef infusion 300ml
Casein hydrolysate 17.5g
Starch 1.5g
Agar 23-30g

Add all the above ingredients in 1000ml of distilled water and autoclave at 121 0C for 15
min.
CHEMICALS AND REAGENTS
1.Lysis buffer
Lysis buffer was perpared by adding 1% sodium dodecyl sulfate,2.5 Mm EDTA,25Mm

sodium acetate and 267ug/ml proteinase K in 100 ml of dustilled water.
2.TE buffer
TE buffer was prepared by adding Tris Hcl 100Mm and EDTA 10Mm in 100 ml of
distilled water.
3.phenol chloroform isoamyl alcohol mix
Phenol chloroform isoamyl alcohol mix was prepared by adding 25.0ml phenol,24.0ml
chloroform and 1.0ml isoamyl alcohol.
4.3M Sodium acetate3M Sodium acetate was prepared by adding 24.6gm sodium
acetate in 100 ml of distilled water.
5.70% Ethanol
70% Ethanol was prepared by adding 70.0ml absolute ethanol in 30 ml of distilled water.
6.TBE Buffer 5X
TBE Buffer 5x was prepared by adding 27.0 gm Tris base,13.7gm Boric acid, and
10.0ml of of 0.5 M EDTA in 500ml of distilled water.
7.RNase solution
8.Ethidium bromide solution
9.0.8% Agarose
Agarose was prepared by adding 0.8 gm agarose in 100 ml of water.
10.Normal saline
Normal saline was prepared by adding 0.85 gm of Nacl in 100 ml of water.
11. Enzyme ECORI
12. 10X assay buffer
13. molecular grade water.
14. dNTP mix
15.15Mm Mgcl2
16. Taq DNA polymerase(1unit)
17.Reverse and Forward primer.
18. 10X PCR buffer.
GLASSWARE AND APPARATUS
Petri plates
Test tubes
Conical flasks
Flasks
Measuring cylinders
Beakers
Micropipette
Inoculating loops
Non absorbent cotton.
Filter paper
Aluminium foil
Paper disk
Incubator
Laminar air flow
Autoclave
Microtips
Weiging balance
METHODS
COLLECTION OF SAMPLES
A total of 30 samples (Urine and Throat) were collected from zonal hospital and SILB
college students at Solan (H.P). Out of 30 samples, 22 were urine samples obtained
from zonal hospital and 8 were throat samples collected from girls living in SILB hostel
Solan. All the samples were collected in sterile air tight container.
TRANSPORT OF SAMPLES
Samples were transported in sterile, humidified, leak proof and air tight container from
zonal hospital Solan and girls hostel to SILB Solan for further processing following
standard protocols of isolation.
ISOLATION OF CANDIDA SPECIES

SDA was prepared for the isolation of Candida species from the various samples
collected from the different places. SDA was prepared according to the manufacturer
instruction. Media was autoclaved at 121 0 C for 15 min. After autoclaving different
samples were streaked on SDA slants under sterile condition and the slants were
incubated at 250 C for 2-3 days. Various colonies of Candida were observed after
incubation.
PRESERVATION OF SAMPLES
10% Glycerol were prepared and then transferred in small screw capped bottles. These
screw capped bottles which containing 10% Glycerol were autoclaved at 121 c for 10
minutes. Transfer the loopful of culture from already prepared slants into these small
screw capped bottles containing glycerol and then stored at -20 0C as per described by
the standardized protocol of Arunaloke C et al ,.2002.
CHARACTERIZATION OF CANDIDA ISOLATES.
Isolated Candida species were identified up to species level through examination of

morphological characteristics and biochemical tests. The phenotypic traits like germ
tube production, pseudohyphae and chlamydospore formation were observed as per
methodology described by Arunaloke C et al.,2002.
GERM TUBE TEST
Germ tube test was done for the presumptive identification of candida albicans. It is a
rapid screening test where the germ tube was produced within two hours in contact with
the serum. Test started with a fresh growth from a pure culture. A very light suspension
of the test organism was made in 0.5ml of sterile serum incubated at 37 0C for exactly
2hrs.Placed one drop from the incubated serum on a slide with a cover slip. Observed
the slide under microscope for production of germ tube. Germ tube represents initiation
of hyphal growth arising from the yeast cell.
PSEUDOHYPHAE PRODUCTION
The ability to produce pseudohyphae was determined by observing their morphology on

corn meal agar supplemented with tween 80..With the help of sterile needle yeast
colonies were streaked on CMA plate, sterile cover slip was placed over streaked
colonies. The plates were incubated for 3-5 days at 25 0C. The plates were observed
after 3-5 days under microscope to see the presence of pseudohyphae.
CHLAMYDOSPORE PRODUCTION
Chlamydospore production ability of Candida species were checked by using corn meal
agar supplemented tween 80. The samples which are previously grown in SDA were
seeded as 4 parallel streaks on petri plates containing corn meal agar and the plates
were incubated at 300C for 3-5 days for the production of chlamydospore (Fisher F et
al., 1998). The plates were visualized under optical microscope (Gatica JLM et al.,
2002). The double walled rounded spore were observed as chlamydospore.
MORPHOLOGY ON CHROM AGAR
The isolates of Candida species were cultured on SDA at 30 0 C for 48 hour. After this
they were seeded on chromgenic agar and incubated at 30 0 C for 48 hour. CHROM agar
allows the selective yeast isolation and identifying colonies of C.albicans,
C.dubliniensis, C.tropicalis and C.krusei by colour identification (Hospenthol DR et
al.,2006).The strain were identified according to the colour of the colonies, C.albicans or
C.dubliniensis as green colonies, C.tropicalis as steel blue colonies.
SUGAR FERMENTATION TEST

Sugar fermentation test for various Candida species was done as described by
Arunaloke C.et.al 2002. Pepared liquid fermentation medium containing peptone (1%),
sodium chloride (5%), andradess indicator (0.005%). Sterilized by autoclaving at 110 0
C for 10min. Added filtered sterilized sugar at the concentration of 2% to the medium.
Poured into the sterile test tubes (approx. 5ml) and placed sterile Durhams tubes into
each tube. Plugged the tubes with colour coded cotton plugs. Inoculum preparation
was done by suspending heavy inoculums of yeast grown on sugar free medium.
Inoculated each carbohydrates broth with approx. 0.1ml of inoculum. Incubated the
tubes at 250 C up to 1 weak. Examined the tubes every 48-72hrs interval for the
production of acid and gas (in Durhhams). Production of gas in the tube was taken as
fermentation positive while only acid production may indicated that carbohydrate is
assimilated.
SUGAR ASSIMILATION TEST
The sugar assimilation test was performed for the assimilation of various sugars by
Candida species described as per protocol described by Arunaloke C.et.al., 2002.
Prepared a yeast suspension from a 24-48hrs old culture in 2ml of YNB by adding
heavy inoculum. Added this suspension to the 18ml of molten agar and mixed well.
Poured the entire volume in to a 90mm petri plate. Allowed the Petri plates at room
temp until the agar surface hardens. Placed the various carbohydrates-impregnated
discs on the surface of agar plate. Sugar discs can be obtained commercially or can be
prepared by punching 6mm-diameter disc from Whatman no. 1 filter paper. Sterilized
the disc by placing them in hot air oven for 1hr. Add a drop of 10% filter sterilize
solution to each disc. Dry the disc at 37 0 C and store at 40 C in airtight container.
Incubated the plates at 37 0C for 3-4 days. The presence of growth around the disc is
considered as positive for that particular carbohydrate.
ANTIFUNGAL SUSCEPTIBILITY TESTING
The antifungal susceptibility of Candida strains was performed by disk diffusion method
as per CLSI M44-A protocols.
PREPARATION OF INOCULUM
Four-five colonies from pure growth of each organism were transferred to 5 ml of
Mueller- Hinton broth. The broth was incubated at 37C for 18-24 hours. The turbidity of
the culture was compared with 0.5 McFarland Nephelometer standard to get 150 106
CFU/ml. The standardized inoculum suspension was inoculated within 15-20 minutes .
DISK DIFFUSION METHOD
The antifungal susceptibility of Candida strains was performed by disk diffusion method
as per CLSI M44-A protocol. In this method Muller-Hinton agar supplemented with 2%
glucose and 0.5g/ml methylene blue dye (GMB) medium were used. The disks of
antifungal was used and stored at 8 0 C or below, or freeze at -14 C or below, in a non
frost-free freezer until needed. Inoculum was prepared by picking five distinct colonies
of approximately 1 mm in diameter from a 24-hour-old culture of Candida species.
Colonies was suspended in 5 ml of sterile 0.145 mol/L saline (8.5 g/100mL NaCl; 0.85%
saline).The resulting suspension was vortexed for 15 seconds and its turbidity were
adjusted either visually or with a spectrophotometer. Within 15 minutes after adjusting
the turbidity of the inoculum suspension, a sterile cotton swab was dipped into the
suspension. The dried surface of a sterile Mueller-Hinton + GMB agar plate was
inoculated by evenly streaking the swab over the entire agar surface. This procedure
was repeated by streaking two more times, rotating the plate approximately 60 C each
time to ensure an even distribution of inoculum. As a final step, the rim of the agar was
swabbed. Antimicrobial disks was dispensed onto the surface of the inoculated agar
plate. The plates were incubated inverted position at 25 0 C ( 2 C) within 15 minutes
after the disks were applied. Each plate was examined after 20 to 24 hours of
incubation for zone of inhibition. Zone diameter was measured to the nearest whole
millimeter at the point at which there was a prominent reduction in growth.
EXTRACTION OF GENOMIC DNA
Genomic DNA of the Candida species was extracted as per methodlogy described by
Arunaloke C et al., 2002. A loopful of culture grown on SDA was inoculated in 5ml of
yeast phosphate dextrose broth in 200 ml flask. Incubated overnight at 30 0 C. Pellet
down the growth in 1.5 ml of microfuge tube at 7500rpm for 2 min. Washed the pellet
twice with sterile distilled water and centrifuged again for 4 min. Suspended the pellet in
200ul of lysis buffer and 0.3 gm of sterile glass beads. Mixed well by vortexing and then
added 200ul of equal amount of phenol chloroform to the cell suspension. Vortexed this
mixture vigourously for one min followed by cooling on ice for 30 sec. Repeated the
process of vortexing and cooling for five more times. Added 200ul of TE buffer and
again vortex briefly. Centrifuged the mixture at 13000 rpm at room temperature for 5
min. Transferred the aqueous phase in a clear sterile microfuge tube and equal volume
of chloroform isoamyl alcohol added. Centrifuged at 10000 rpm for 7 min. Collected
aqueous phase and added 1/10 th volume of 3M Sodium acetate and equal volume of
chilled isopropanol.mix and incubate at -20 0 C for 2 hr for DNA precipitation. Collected
the DNA precipitate at 10000 rpm for 5 min and decant the supernatant. Added 0.5 ml
70% ethanol, vortex briefly to wash the pellet. Centrifuged at 10000rpm for 5 min,
aspirate out the supernatant carefully and allow the pellet to dry at 37 0 C for one hour.
Resuspended the pellet in appropriate volume of TE buffer. The DNA was allowed to
dissolve for atleast 6 hr at room temperature with intermittent mixing. One ul of RNase
was added and incubated at 370C for 30 min. The DNA was electrophoresed on a 0.8%
agarose gel to check the quality and quantity.
POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION OF ERG 11

GENE
Amplification of ERG11 gene was done by using polymerase chain reaction in five drug
resistant Candida species. Cells were inoculated in fresh YPED medium with constant
shaking at 30C for overnight. Genomic DNA was isolated by using DNA extraction
method and was used as template for amplification of coding region of ERG11 genes
with the following primers: 55-GTTTCTACTGGATCCCATGG-35 and 55-
TACATCTGTGTGTCTACCACC-35. PCR was carried out in 50l volume containing 10 x
PCR buffer 5l, genomic DNA 2 l, 2.5mmol/L of each dNTP 5l, 25 mmol/L MgCl 2 5l,
10pmol/L each primer 2.5l and 3U/l Taq polymerase 2.5l. Amplification was
performed in thermal cycler for 1 cycle of 4 min. at 94 C and then for 35 cycles, each of
which consisted of 30s at 94C, 1min. at 55C and 1min. at 72 C; this was followed by 1
final cycle of 10 min. at 72C. The PCR products were then analysed by electrophoresis
on 0.8% agarose gel and were visualized under transilluminator after staining the gel
with ethidium bromide (ZHOU Yong et al., 2011).
RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)

ANALYSIS
Genetic variability among different Candida isolates was checked by employing RFLP
analysis. Total amplified product of the of the five drug resistant Candida isolates were
digested by using restriction enzymes EcoRI (Smith et al.,1989) .The 5 ul of of amplified
product of the 5 Candida strains ,i.e. two strains of Candida albicans (H2 and H15),two
strains of Candida tropicalis (H1 and H7) and one strain of Candida glabrata(H6) were
transferred into the 1.5ml of fresh microfuge tubes. To each tube 5 ul of EcoRI assay
buffer, 16 ul of molecular grade water and one 1ul of enzyme 40 U EcoRI was added.
All the 5 tube was incubated overnight at 37 0C. After overnight incubation the digests
was electrophoresed in 0.8% agarose gel contaning ethidium bromide in 0.5xTE buffer
at 50 v for 4 hour and visualized under UV transilluminator to observe the band pattern
(Bostock et al.,1993).
RESULTS
RESULTS
A total of 30 samples were collected from zonal hospital Solan and college students of
SILB Solan. All the samples obtained from hospital were urine samples collected from
patients suspected for UTI while, throat samples were taken from college students
suspected with Candida infection. Out of 30 samples, Different Candida species were
isolated from 24 samples while 6 samples were found negative (Table-1).
TABLE 1 : DETAILS OF SAMPLE COLLECTION
Sample .No. Sample source Sample type Candida infection
1 Hospital Urine +
2 Hospital Urine +
3 Hospital Urine +
4 Hospital Urine +
5 Hospital Urine +
6 Hospital Urine +
7 Hospital Urine +
8 Hospital Urine +
9 Hospital Urine +
10 Hospital Urine +
11 Hospital Urine +
12 Hospital Urine +
13 Hospital Urine _
14 Hospital Urine +
15 Hospital Urine +
16 Hospital Urine +
17 Hospital Urine +
18 Hospital Urine +
19 Hospital Urine +
20 Hospital Urine +
21 Hospital Urine +
22 Hospital Urine _
23 Student Throat _
24 Student Throat _
25 Student Throat _
26 Student Throat +
27 Student Throat _
28 Student Throat +
29 Student Throat +
30 Student Throat +
H:- hospital sample; S:- student sample

TABLE 2 : PHENOTYPIC CHARACTERIZATION OF ISOLATED
CANDIDA SPP.
Strain Isolated CHROMaga CMA with SDA Germ

No. organism r tween 80 tube
1 C.tropicalis Dark blue abundant Cream _
pseudohyphae,pin coloured
colony
(H1) e forest with slightly
arrangement. mycelia
border
2 C.albicans Green colony Terminal White to +

chlamydoconidia coloured,
(H2) smooth,
soft

(H3) smooth,
soft
4 C.krusei Whitish pink Elongated yeast, White to _

abundant cream
colony
(H4) pseudohyphae coloured,
(match stick like butyrous
appearance)
5 C.parapsilosis Pink/violet Giant hyphae, Cream _

blastospores at coloured to
colony
(H5) nodes yellowish,
glistening
and soft,
mostly
smooth or
winkled
6 C.glabrata Light pink Yeast only White to _

cream
colony
(H6) coloured,
soft, glossy
and
smooth
7 C.parapsilosis Pink/violet Giant hyphae, Cream _

blastospores at coloured to
colony
(H7) nodes yellowish,
glistening
and soft,
mostly
smooth or
winkled

(H8) smooth,
soft

(H9) smooth,
soft

colony
border
11 C.guilliermondi Whitish blue Scant White to _

pseudohyphae cream
i colony
with chains of coloured,
(H11) blastoconidia butyrous

colony
border
14 C.guilliermondi Violet colony Scant White to _

pseudohyphae cream
i
with chains of coloured,
(H13) blastoconidia butyrous
15 C.tropicalis Blue colony abundant Cream _

(H14)
e forest with slightly
border

(H15) smooth,
soft

colony
border

colony
border

(H18) smooth,
soft

(H19) smooth,
soft

(H20) smooth,
soft

(S21) smooth,
soft

(S22)
smooth,
soft

(S23)
smooth,
soft

(S24)
smooth,
soft
MORPHOLOGY ON CORN MEAL AGAR

C. albicans showed the terminal chlamydoconidia ,C. guiliermondii showed the chain of
blastoconidia with pseudohyphae, C.glabrata form the yeast only, C.tropicalis form the
abundant pseudohyphae having the pine forest arrangement terminal chlamydospore in
cluster,C.parapsilosis form the giant hyphae, and blastospore at nodes on corn meal
agar.
MORPHOLOGY ON CHROM AGAR

C.tropicalis form the dark blue colonies, C.albicans form the green colonies, C
.guilliermondii forms the whitish blue colony, C.glabrata forms light pink colonies,
C.parapsilosis form pink or violet colonies and C.krusei forms the whitish pink colonies
on chrom agar.
GERM TUBE TEST

Germ tube production test was positive only for the C. albicans.
MORPHOLOGY ON SDA
C.albicans showed White to coloured, smooth, soft. C.guilliermondii White to cream
coloured, butyrous. C.glabrata showed White to cream coloured, soft, glossy and
smooth. C.parapsilosis showed Cream coloured to yellowish, glistening and soft, mostly
smooth or winkled. C.krusei showed White to cream coloured, butyrous. C.tropicalis
Cream coloured with slightly mycelia border.
TABLE-3. RESULTS OF SUGAR FERMENTATION TEST.

Strai Species Sugar fermentation
n no. glucos Maltos sucros lactos
e e e e
H1 C.tropicalis A A A -
H2 C.albicans A A - -
C. tropicalis showed the
acid production in
H4 C.krusei A - - -
glucose, sucrose and
H5 C.parapsilosi A - - -
maltose where as the
s
Candida albicans
H6 C.glabrata A - - -
showed the acid
H7 C.parapsilosi A - - -
production in glucose and
s
maltose, H8 C.albicans Candida
A A - -
guilliermondii showed the
acis production in
glucose and sucrose and
the rest of the H11 C.guilliermon A - A - other
Candida spp. dii showed the
acid production in
H13 C.guilliermon A - A -
glucose only.
dii
S21 C.albicans A A - -
TABLE-4. RESULTS OF SUGAR ASSIMILATION TEST.
Strai Species Glu Mal Suc Lac Gal Mel Cel Ino Xyl Raf Tre Dul
n no.
H1 C.tropicalis + + + - + - + - + - + -
H2 C.albicans + + + - + - - - + - + -
H3 C.albicans + + + - + - - - + - + -
H4 C.krusei + - - - - - - - - - - -
H5 C.parapsilosis + + + - + - - - + - + -
H6 C.glabrata + + - - - - - - - - + -
H7 C.parapsilosis + + + - + - - - + - + -
H8 C.albicans + + + - + - - - + - + -
H9 C.albicans + + + - + - - - + - + -
H10 C.tropicalis + + + - + - + - + - + -
H11 C.guilliermondi + + + - + + + - + + + +
i
H12 C.tropicalis + + + - + - + - + - + -
H13 C.guilliermondi + + + - + + + - + + + +
i
H14 C.tropicalis + + + - + - + - + - + -
H15 C.albicans + + + - + - - - + - + -
H16 C.tropicalis + + + - + - + - + - + -
H17 C.tropicalis + + + - + - + - + - + -
H18 C.albicans + + + - + - - - + - + -
H19 C.albicans + + + - + - - - + - + -
H20 C.albicans + + + - + - - - + - + -
S21 C.albicans + + + - + - - - + - + -
S22 C.albicans + + + - + - - - + - + -
S23 C.albicans + + + - + - - - + - + -
S24 C.albicans + + + - + - - - + - + -
Glu glucose, mal- maltose, suc- sucrose, lac- lactose
Gal- galactose, mel- mellibiose, cel- cellobiose, ino- inositol
Xyl- xylose, raf- raffinose, tre- trehalose, dul- dulcitol
C.albicans showed the zones of precipitation around glucose, maltose, sucrose,

galctose xylose and trehalose disc, C.tropicalis showed the zones of precipitation
around the glucose, maltose, sucrose, galctose xylose, trehalose and cellobiose disc,
C.guiliermondii showed the zones of precipitation around the glucose, maltose, sucrose,
galctose xylose, trehalose, melbiose ,raffinose, cellobiose and raffinose disc, C.krusei
showed the zones of precipitation around the glucose disc only, C.parapsilosis showed
the zones of precipitation around the glucose, maltose, sucrose, galctose xylose and
trehalose disc, and C. glabrata showed the zones of precipitation around the glucose,
maltose, and trehalose disc .
TABLE-5 RESULTS OF ANTIFUNGAL SUSCEPTIBILITY TESTING.
S.NO. Strain Species Voriconazole Amphotericin Results of Results of

no. B Voriconazole amphoteric
in B
1. H1 C.tropicalis 0mm 6mm Resistant Resistant
2. H2 C.albicans 0mm 4mm Resistant Resistant
3. H3 C.albicans 0mm 16mm Resistant Sensitive
4. H4 C.krusei 40mm 9mm Sensitive Resistant
5. H5 C.parapsilosis 0mm 7mm Resistant Resistant
6. H6 C.glabrata 0mm 8mm Resistant Resistant
7. H7 C.parapsilosis 42mm 9mm Sensitive Resistant
9. H9 C.albicans 37mm 0mm Sensitive Resistant
10. H10 C.tropicalis 0mm 17mm Resistant Sensitive
11. H11 C.guilliermondii 42mm 15mm Sensitive Sensitive
12. H12 C.tropicalis 0mm 10mm Resistant S-DD
13. H13 C.guilliermondii 47mm 0mm Sensitive Resistant
16. H16 C.tropicalis 45mm 11mm Sensitive S-DD
18. H18 C.albicans 37mm 16mm Sensitive Sensitive
19. H19 C.albicans 38mm 8mm Sensitive Resistant
20. H20 C.albicans 0mm 10mm Resistant S-DD
21. S21 C.albicans 43mm 0mm Sensitive Resistant
22. S22 C.albicans 36mm 15mm Sensitive Sensitive
Significant number of isolates were found resistant to two antifungal used. 50% isolates
were resistant to the voriconazole and 60% isolates were resistant to the amphotercin B
where as 13% isolates found semi dose dependent against amphotercin B.
TABLE-6 RESULTS OF RESTRICTION FRAGMENT LENGTH
POLYMORPHISM (RFLP) ANALYSIS OF AMPLIFIED ERG 11 GENE.
Strain no. Species Restriction Restriction Size of

Enzyme site bands
H1 Candida tropicalis EcoRI GAATTC 410bp
300bp
200bp
H2 Candida albicans EcoRI GAATTC 300bp
200bp
H6 Candida glabrata EcoRI GAATTC 340bp
280bp
200bp
170bp
H8 Candida albicans EcoRI GAATTC 410bp
300bp
200bp
H15 Candida tropicalis EcoRI GAATTC 410bp
300bp
200bp
The drug resistant Candida isolates, which were resistant to both the antifungal agent
were selected for RFLP analysis. A total of 4 polymorphic RFLP bands of different sizes
were detected. The results shows that the RFLP profile of 3 bands (410,300 and 200bp)
were the most common and the profile of 4 bands were only found in one isolate.
Generally, for most strains, the specific enzyme digestion product corresponded to its
genotype. Several strains of similar species showed different RFLP profiles. For
example, Candida albicans (H2 and H8) had different RFLP profiles with 2 and 3 DNA
bands. While Candida tropicalis (H1 and H15) had similar band pattren respectively,
Strain Candida glabrata (H6) showed 4 bands. Comparison of the RFLP profiles
showed that the restriction profiles of the most of the strains were identical, whereas
only two species Candida glabrata (H6) and Candida albicans (H2 and H8) showed
different band pattern. Moderate degree of variation was observed among drug resistant
Candida isolates.
FIG 1: - CHROM agar plate showing green coloured colonies of Candida albicans and
blue coloured colonies of Candida tropicalis.
Germ tube
FIG 2: - Germ tube formation by Candida albicans.
FIG 3: - Candida krusei showed budding cell broadly ellipsoidal to cylindrical.

Pseudomycelium often present, robust.
FIG 4: - Results of sugar fermentation test for C krusei.
FIG 5:- Results of sugar assimilation test showing the zones of precipitation around
different sugar discs for C.glabrata.
FIG 6- Zone of inhibition around the antifungal discs (voriconazole and amphotercin B)
for Candida albicans.
FIG 7 -Plate showing fluconazole ane amphotercin B resistant Candida albicans.

FIG 8- RFLP band pattern of amplified product while digesting with EcoR1 restriction
endonuclease enzyme. M=1Kb marker, lane-1 =Candida tropicalis (H1), lane-2 Candida
tropicalis (H15), lane 3= Candida albicans (H2), lane-4= Candida albicans (H8) and
lane-5 = Candida glabrata (H6).
DISCUSSION
DISCUSSION
Candida is yeast and the part of the normal microflora of human body. Candida is
known as opportunistic fungal pathogens as it causes infection when person become
immunocompromised, immunonosuppressed or diabetic. In immune compromized
individuals Candida causes diseases like oral thrush, intestinal candidiasis, vaginal
thrush and onchomycosis (Chamaine 2005, Bennett JE et al., 1992,). In the present
study Candida species were isolated from the UTI patients of zonal hospital Solan and
college students of SILB. Farina et al .,1999, studied the prevalence of Candida
infection during the period of 1988 to 1997 at the regional hospital of Bergamo In Italy
168 cases of fungaemia were studied, out of which 70% cases were due to the Candida
species and this shows the dominance of Candida species in immunocompromised
host. Candida is a leading pathogen causing the different types of candidiasis such as
vaginal, respiratory,urinary and invasive candidiasis etc. since 1990, it has become
clear that Candida species continue to become an important etiological agent of
nosocomial infection. The Candida species have been typed and classified using a
wide variety of techniques and proportion of such infection is increased due to the
involvement of non albicans species (Fridkin et al ., 1996).To prevent and control the
nosocomial infection, identification of Candida species upto the species level is most
important (Pfaller et al .,1997). In the present study phenotypic traits such as
chlamydospore production, pseudohyphae formation, germ tube production has been
studied. C.albicans showed the terminal chlamydoconidia ,C.guiliermondii showed the
chain of blastoconidia with pseudohyphae, C.glabrata formed the yeast only,
C.tropicalis formed the abundant pseudohyphae having the pine forest arrangement
terminal chlamydospore in cluster, C.parapsilosis formed the giant hyphae and
blastospore at nodes on corn meal agar. Germ tube production test was positive only for
the Candida albicans. Fluconazole, voriconazole and amphotericin B are the choice of
drugs used for the treatment of fungal infection. However, due to continues use of these
drugs, most of the Candida sp. developed resistance against these drugs. Casalinuova
IA et al.,1999 studied that fluconazole, voriconazole and amphotericin B were the drugs
of choice used for fungal infection. Most of the Candida sp. are now becoming resistant
due to their continous use. In the current study it was found that maximum number of
Candida isolates were resistant to voriconazole and amphotericin B. 50% strain were
resistant to the voriconazole and 60% strains were resistant to the amphotercin B where
as 13% strains were interpreted as semi dose dependent against amphotercin B.
Results of Antifungal susceptibility testing shows that various species of Candida
becomes resistant to a number of antifungal agents such as voriconazole and
fluconazole. Xiao-dong S et al.,2008 studied that genotypes of strains isolated from
cutaneous and vaginal infections were significantly different within these two body site.
They studied the mechanisms that influence genotype of colonizing of C. albicans in
cutaneous and vagina of the patients.
A number of different methods of detecting variations in genetic
sequences in Candida strains have been developed, including RFLP analysis(Smith et
al.,1989).In the present study the DNA of the 10 drug resistant Candida sp. were
isolated and ERG 11 of these Candida sp was amplified by PCR. RFLP of the amplified
product was done with restriction enzyme EcoRI. The RFLP technique with the
restriction enzyme EcoRI used in this study proved to be a reliable, sensitive method for
assessing the genetic relatedness of different Candida species. A total of 4 polymorphic
RFLP bands of different sizes were detected.The results shows that the RFLP profile of
3 bands (410,300 and 200bp) were the most common and the profile of 4 bands were
only found in one isolate. Several strains of similar species showed different RFLP
profiles Genotypic studies have the potential to provide more reproducible
classifications of organisms. These studies will help us in understanding identification of
Candida species, pathogenecity pattern and genetic relatedness between different
species.
SUMMARY
SUMMARY
Candida is the leading agent of various types of diseases in immunocompromised

individuals. It is the part of normal microfloa of humans and acts as opportunistic
pathogen.candida infection is increasing in recent years. In the present study 22 urine
samples collected from zonal hospital Solan and 8 throat samples collected from girls
hostel SILB Solan. Samples were transported from these places to SILB Solan through
air tight container and streaked on slants of SDA. The slants of SDA were incubated at
250 C for 3-5 days. After incubation a loopfull of culture was inoculated in 10% glycerol
for the preservation and the glycerol bottels were stored at -20 0 C. Out of 30 samples 24
samples shows the presence of Candida. These 24 strains of Candida were isolated
from the positive samples and phenotypic characteristics of all the isolates were
examined such as pseudohyphae, chlamydospore formation and germ tube production.
Germ tube production test was done by incubating strains in human serum. Germ tube
production test was positive only for Candida albicans. Out of 24 strains 12 strains
shows the germ tube production belonging to Candida albicans C. albicans shows the
terminal chlamydoconidia ,C.guiliermondii shows the chain of blastoconidia with
pseudohyphae, C.glabrata form the yeast only, C.tropicalis form the abundant
pseudohyphae having the pine forest arrangement terminal chlamydospore in cluster,
C.parapsilosis forms the giant hyphae, and blastospore at nodes on corn meal agar.
C.tropicalis form the dark blue colonies, C.albicans form the green colonies,
C.guilliermondii forms the whitish blue colony, C.glabrata forms light pink colonies,
C.parapsilosis form pink or violet colonies and C.krusei forms the whitish pink colonies
on chrom agar. Sugar fermentation test was also done for the acid gas production or
acid production. C. tropicalis shows the acid production in glucose, sucrose and maltose
where as the Candida albicans shows the acid production in glucose and maltose,
Candida guilliermondii showed the acid production in glucose and sucrose and the rest
of the other Candida spp. shows the acid production in glucose only. Sugar assimilation
test was also done for the assimilation Candida sp. around the carbohydrate disc .
C.albicans shows the sugar assimilation around glucose,maltose, sucrose, galctose
xylose and trehalose disc, C.tropicalis shows the sugar assimilation around the
glucose,maltose, sucrose, galctose xylose, trehalose and cellobiose disc,
C.guiliermondii shows the sugar assimilation around the glucose,maltose, sucrose,
galctose xylose, trehalose,melbiose ,raffinose,cellobiose and raffinose disc, C.krusei
shows the sugar assimilation around the glucose disc only, C.parapsilosis shows the
sugar assimilation around the glucose,maltose, sucrose, galctose xylose and trehalose
disc, and C.glabrata shows the sugar assimilation around the glucose,maltose, and
trehalose disc . Antifungal susceptibility testing was also done by placing amphotercin B
and voriconazole disc on Muller Hinton agar. Different diameter of zones was formed
around the respective antifungal disc. The results were interpreted as resistant and
sensitive against these drugs after measuring diameter of zones of inhibition. ERG 11
gene of 5 drug resistant Candida species was amplified and RFLP patterns were
generated using EcoR1 restriction endonuclese anzyme in order to study the genetic
variability in Candida resistant species. A total of 4 polymorphic RFLP bands of different
sizes were detected. The results shows that the RFLP profile of 3 bands (410,300 and
200bp) were the most common and the profile of 4 bands were only found in one
isolate. Several strains of similar species showed different RFLP profiles. The study will
help in identification and resistance pattern against various antifungal agents and also
to observe genetic variability among these species.
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Species identification, antifungal susceptibility testing

and genetic variability among Candida species

A Research Project Report

(Affiliated to H.P. University, Shimla)

The assistant and help received during the course of this

Prof. M.P. Vohra Dr. Ajay Kumar Mr. Amit Kumar

And all ambition are backed by strength given by God.

I am also heartiest thankful to my friends who proved to be supportive at any good or

SDB- SABOURAUD DEXTROSE BROTH

YNB- YEAST NITROGEN BASE

YPD- YEAST PHOSPHATE DEXTROSE

CMA-CORN MEAL AGAR

KOH- POTASSIUM HYDROXIDE

PCR-POLYMERSE CHAIN REACTION

TE BUFFER- TRIS ETHYLENE DIAMINE TETRACETIC ACID

dNTP DEOXYNUCLEOTIDE TRIPHOSPHATE

RFLP- RESTRICTION FRAGMENT LENGTH POLYMORPHISM

2. AIM AND OBJECTIVES 3

3. REVIEW OF LITERATURE 4-17

4. MATERIAL AND METHOD 18-29

AIM AND OBJECTIVES

Candida species isolated from clinical samples.

1. Isolation of Candida spp from various clinical samples.

2. Characterization and antifungal susceptibility testing of Candida species.

3. To check genetic variability among drug resistant Candida species.

Candida albicans is an opportunistic pathogen that usually lives as commensal in the

According to Ha et al., 2010, candidemia has become an increasingly important

OTHER CANDIDA INFECTIONS

SIGNS AND SYMPTOMS

Candidiasis is a very common cause of vaginal irritation, or vaginitis, and

Infection of the vagina or vulva may cause severe itching, burning,

A biofilm is a microbially derived sessile community characterized by cells that are

BIOFILM FORMATION AND DEVELOPMENT

After 1824 h, the bilayered structure of the Candida albicans

Diagnosis of a yeast infection is done either via microscopic examination or culturing.

ANTIFUNGAL DRUG RESISTANCE

ERG11 AND OTHER ERG GENES

The correlation between decreased susceptibility to azole drugs and nucleotide

OTHER CHANGES IN FLUCONAZOLE RESISTANCE

MATERIAL AND MAETHOD

MEDIA USED AND COMPOSITION

Corn Meal 50gm

Distilled water 1000ml

SABOURAUD DEXTROSE AGAR (SDA)

Distilled water 1000ml

Add glucose, peptone and agar in 1000ml of distilled water.sterlized by autoclaving at

SABOURAUD DEXTROSE BROTH

Distilled water 1000ml

Bacto peptone 10gm

Yeast extract 10gm

Distilled water 1000ml

YEAST NITROGEN BASE (YNB)

Potassium dihydrogen orthophosphate 1.0gm

Magnesium sulfate 0.5gm

Ammonium sulfate 5.0gm

Noble agar 25.0gm

Distilled water 1000ml

Beef infusion 300ml

Casein hydrolysate 17.5g

Distilled water 1000ml

CHEMICALS AND REAGENTS

Lysis buffer was perpared by adding 1% sodium dodecyl sulfate,2.5 Mm EDTA,25Mm

3.phenol chloroform isoamyl alcohol mix