| Abstract|| |
Background and Objectives: Biofilms are colonies of microbial cells encased in a self-produced organic polymeric matrix. The biofilm production is more important for nonalbicans Candida (NAC); as C. albicans possess many other mechanisms to establish infections. Correct identification of Candida species has gained importance due to persistent rise in infections caused by NAC. We sought to isolate, identify Candida species in clinical isolates and study biofilm formation. Materials and Methods: Modified microtiter plate method was performed to study biofilm formation by isolates in Sabouraud's dextrose broth. It was then quantitatively assessed using a spectrophotometer. Biofilm formation was graded as negative, +1, +2, +3 and + 4 on the basis of percentage absorbance. Results: Biofilm formation was observed in 16 of 40 (40.0%) isolates of C. albicans as compared to 39 of 78 (50.0%) of isolates of NAC. Strong (+4) biofilm production was seen in maximum biofilm producers in C. tropicalis (12 of 27) followed by C. albicans (8 of 16). Total biofilm producers were significantly more among high vaginal swab isolates 63.2% (12 of 19) and urine isolates 59.2% (29 of 49), when compared to blood isolates 34.2% (13 of 38) as well as other isolates 27.5% (11 of 40). Interpretation and Conclusions: NAC species are qualitatively and quantitatively superior biofilm producers than C. albicans. Biofilm production is the most important virulence factor of NAC species and compared to other lesions, it is more significantly associated with luminal infections.
Keywords: Biofilm, Candida albicans, nonalbicans Candida, virulence factor, yeast
|How to cite this article:|
Agwan V, Butola R, Madan M. Comparison of biofilm formation in clinical isolates of Candida species in a tertiary care center, North India. Indian J Pathol Microbiol 2015;58:475-8
|How to cite this URL:|
Agwan V, Butola R, Madan M. Comparison of biofilm formation in clinical isolates of Candida species in a tertiary care center, North India. Indian J Pathol Microbiol [serial online] 2015 [cited 2019 Aug 20];58:475-8. Available from: http://www.ijpmonline.org/text.asp?2015/58/4/475/168873
| Introduction|| |
Candida species are normal inhabitants of the skin and mucosa. The importance of epidemiological monitoring of yeasts involved in pathogenic processes is unquestionable due to the increase of these infections over the last decade; so are the changes observed in species causing candidiasis. The most external layers of Candida cells are essential for the adherence to host surface, thereby playing a pivotal role in the pathophysiology of candidiasis. Biofilms are colonies of microbial cells encased in a self-produced organic polymeric matrix and represent a common mode of microbial growth. Recently, microbial biofilms have gained prominence because of the increase in infections related to indwelling medical devices. The advantages of forming a biofilm for the organism include protection from the environment, nutrient availability, metabolic cooperation, and acquisition of new genetic traits.
Biofilms may help maintain the role of fungi as commensal and pathogen, by evading host immune mechanisms, resisting antifungal treatment, and withstanding the competitive pressure from other organisms. Consequently, biofilm related infections are difficult to treat. The biofilm production is also associated with high level of antimicrobial resistance of the associated organisms.
The proportion of infections due to nonalbicans Candida (NAC) species is persistently increasing the need for a correct identification of Candida isolates at the species level. Although C. albicans remains the most common fungal isolate recovered from blood, recent reports indicate a trend toward an increasing prevalence of infections caused by NAC.,,,
The proportion of biofilm production is much higher among isolates of NAC species recovered from blood than other sites. In contrast to C. albicans candidemia, biofilm positivity of NAC species is more significantly associated with clinical characteristics of candidemia, including number of positive blood cultures, presence of central venous catheter-related candidemia, total parenteral nutrition and clinical significance candidemia.
The biofilm production is more important for NAC strains and C. albicans possess mechanisms other than biofilm production to establish infections.Candida species are now recognized as major agents of hospital acquired infection worldwide.
This study was therefore undertaken, to study distribution of various Candida species isolated from various clinical samples and their capacity to form biofilm.
| Materials and Methods|| |
The present study was conducted from January 2013 to December 2013, on 118 nonrepeat samples received in clinical Microbiology laboratory of tertiary care center, in western Uttar Pradesh. Candida species were isolated from clinical specimens received from tertiary care hospital, identified and studied for biofilm formation.
The study was approved by the research and ethics committee.
Candida species were isolated from blood, urine, pus, high vaginal swab (HVS) and other clinical samples. The clinical isolates of Candida were identified up to species level by standard laboratory techniques.C. albicans ATCC 90028 strain was used as control. The modified microtiter plate method was used for biofilm formation by isolates in Sabouraud's dextrose broth. Its formation was observed and confirmed by inverted microscope, at the end of 24 h incubation. It was then quantitatively assessed using a spectrophotometer. The percentage absorbance (%A) value for each test sample was calculated by subtracting percentage absorbance value of reagent blank from the percentage absorbance value for the sample, difference giving a measure of the amount of light blocked by biofilm when passing through the bottom of wells. Biofilm formation was graded as negative (%A: <5), +1 (%A: 5–20), +2 (%A: 20–35), +3 (%A: 35–50) and +4 (%A: More than 50).
| Result|| |
A total of 118 nonrepeat clinical isolates of Candida species were included in this study and screened for biofilm production. The clinical specimens from which these species were isolated included urine 49 (41.5%), blood 38 (32.2%), HVS 19 (16.1%), pus 7 (5.9%), vault swab 3 (2.5%) and 1 (0.9%) each of bone with tissue and central line [Table 1].
|Table 1: Comparison of biofilm producers in isolates from different clinical samples|
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The isolated Candida species included 40 (33.9%) C. albicans and 78 (66.1%) NAC.
ATCC 90028 strain of C. albicans did not show biofilm formation. Biofilm formation was observed in 16 of 40 (40.0%) isolates of C. albicans as compared to 39 of 78 (50.0%) of isolates of NAC. The Chi-square test was applied which interpreted a nonsignificant variation in the number of biofilm producers and nonproducers (χ2 = 1.06 and P = 0.30) [Table 2].
The NAC species were further identified up to individual species level. Various NAC species isolated were C. tropicalis 40 (33.9%), C. parapsilosis 22 (18.7%), C. krusei 6 (5.1%), C. glabrata 4 (3.4%), C. kefyr 3 (2.5%) and C. guilliermondii 3 (2.5%).
Biofilm formation was seen in all the isolates (3 of 3) of C. kefyr and C. guilliermondii. It was also more prominently observed in isolates of C. tropicalis (67.5%), followed by C. albicans ( 40%), C. glabrata ( 25%) and C. parapsilosis (22.7%). None of the isolates of C. rusei (00%) produced any biofilm.
Among the biofilm producers, strong (+4) biofilm production was seen in maximum number in C. tropicalis (12 of 27), followed by C. albicans (8 of 16), C. parapsilosis (3 of 5), C. guilliermondii (2 of 3), C. kefyr (1 of 3) and C. glabrata (1 of 1). None of the Candia krusei isolates showed biofilm production [Table 3].
|Table 3: Distribution of different grade of biofilm in isolates of Candida species|
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Twenty-nine of 49 urine, 13 of 38 blood and 12 of 19 HVS isolates were biofilm producers. Biofilm producers among the 12 other samples consisted 1 of 7 pus, 1of 3 vault swab, 1 of 1 central venous line and 1 of 1 bone + tissue [Table 1].
| Discussion and Conclusions|| |
The biofilm growth protects the microorganism from the host defense and antimicrobial agents. In this line, biofilm formation is a risk factor that increases the mortality rate in candidiasis in critically ill patients or immunocompromised individuals.
Recent studies have documented a shift toward NAC species from C. albicans. Some studies have reported increasing trend of incidences of infections caused by NACs, gradually surpassing C. albicans as cause of candidemia in some regions. Factors such as increased use of antifungal drugs and broad spectrum antibiotics, long-term use of catheters and increase in the number of immunocompromised patients have contributed to the emergence of NAC species in increasing numbers.,,
In the present study, the clinical isolates of Candida species (118) consisted of NAC (78) which exceeded C. albicans (40), in concordance with various other studies worldwide.,,,,,,
Isolates of NAC had more biofilm producers (50%) compared to C. albicans (40.0%), in concordance with other studies. More number of biofilm producers of NAC show strong (+4) biofilm production compared to biofilm producer C. albicans [Table 3]. Other studies have reported similar findings.,, Although in our study the percentage of strong biofilm producers of C. guilliermondiii and C. kefyr is very high (100%), this finding could be incidental and cannot be generalized as their sample size is very small and needs to be studied in higher number of isolates.
Biofilms represent the most prevalent type of microbial growth in nature and are crucial to the development of clinical infections. The ability to form extensive biofilms on the surface of catheters, and other prosthetic devices, also contributes to the high prevalence of the organism as etiologic agent of intravascular nosocomial infections. Biofilm formation should be considered as an important virulence determinant during candidiasis.
The virulence factors vary in relation to type, site and stage of infection. We found that biofilm producers were significantly higher in numbers amongHVS isolates 63.2% (12 of 19) and urine isolates 59.2% (29 of 49), compared to blood isolates 34.2% (13 of 38) as well as other isolates 27.5% (11 of 40). Biofilm as a virulence factor thus appears to contribute most in pathogenesis of urinary tract infection and other luminal infections, compared to other clinical conditions.
Due to the increasing incidence of Candida infections, there is great interest in Candida virulence factors, which are in turn important in the establishment of the strategies for control and prevention of candidiasis.
Nonalbicans Candida species cannot be overlooked as mere contaminant or nonpathogenic commensals. Research on prevalent Candida species along with their virulence factors in a given set up would be an important tool to prove the relation between the infective species of Candida and infection. The changing patterns of the Candida isolation from various clinical samples have made identification of Candida species producing virulence factors compulsory for diagnostic Microbiology service.
This changing trend of causative role of Candida in different studies from different parts of the world and from India and the emergence of NAC species and their association with virulence factors cannot be overlooked. Increased isolation and complete identification of Candida species in more Microbiology laboratories might be instrumental in reports of rising NAC emergence. More multilocational studies on larger sample size will definitely go a long way in revealing epidemiology, emergence and spread of NAC.
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Conflicts of interest
There are no conflicts of interest.
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Dr. Vivek Agwan
Department of Microbiology, Subharti Medical College, Meerut - 250 005, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2], [Table 3]