LGCmain
Indian Journal of Pathology and Microbiology
Home About us Instructions Submission Subscribe Advertise Contact e-Alerts Ahead Of Print Login 
Users Online: 6927
Print this page  Email this page Bookmark this page Small font sizeDefault font sizeIncrease font size
IJPM is coming out with a Special issue on "Genitourinary & Gynecological pathology including Breast". Please submit your articles for these issues


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2015  |  Volume : 58  |  Issue : 4  |  Page : 475-478
Comparison of biofilm formation in clinical isolates of Candida species in a tertiary care center, North India


Department of Microbiology, Subharti Medical College, Meerut, Uttar Pradesh, India

Click here for correspondence address and email

Date of Web Publication4-Nov-2015
 

   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 Top


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.[1] 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.[2] 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.[3] The advantages of forming a biofilm for the organism include protection from the environment, nutrient availability, metabolic cooperation, and acquisition of new genetic traits.[4]

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.[5] The biofilm production is also associated with high level of antimicrobial resistance of the associated organisms.[6]

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.[7],[8],[9],[10]

The proportion of biofilm production is much higher among isolates of NAC species recovered from blood than other sites.[11] 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.[12]

The biofilm production is more important for NAC strains and C. albicans possess mechanisms other than biofilm production to establish infections.[13]Candida species are now recognized as major agents of hospital acquired infection worldwide.[14]

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 Top


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.[15]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.[16] 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).[17]


   Result Top


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

Click here to view


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].
Table 2: Distribution of isolates according to biofilm production

Click here to view


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

Click here to view


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 Top


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.[18]

Recent studies have documented a shift toward NAC species from C. albicans.[19] Some studies have reported increasing trend of incidences of infections caused by NACs, gradually surpassing C. albicans as cause of candidemia in some regions.[20] 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.[21],[22],[23]

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.[8],[24],[25],[26],[27],[28],[29]

Isolates of NAC had more biofilm producers (50%) compared to C. albicans (40.0%), in concordance with other studies.[6] 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.[6],[17],[30] 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.[15] 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.[31] Biofilm formation should be considered as an important virulence determinant during candidiasis.[5]

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.[32]

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.[33]

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.[22] 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.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
   References Top

1.
Mujica MT, Finquelievich JL, Jewtuchowicz V, Iovannitti CA. Prevalence of Candida albicans and Candida non-albicans in clinical samples during 1999-2001. Rev Argent Microbiol 2004;36:107-12.  Back to cited text no. 1
    
2.
Senet JM. Candida adherence phenomena, from commensalism to pathogenicity. Int Microbiol 1998;1:117-22.  Back to cited text no. 2
    
3.
Mukherjee PK, Zhou G, Munyon R, Ghannoum MA. Candida biofilm: a well-designed protected environment. Med Mycol 2005;43:191-208.  Back to cited text no. 3
    
4.
De Bernardis F, Agatensi L, Ross IK, Emerson GW, Lorenzini R, Sullivan PA, et al. Evidence for a role for secreted aspartate proteinase of Candida albicans in vulvovaginal candidiasis. J Infect Dis 1990;161:1276-83.  Back to cited text no. 4
    
5.
Baillie GS, Douglas LJ. Candida biofilms and their susceptibility to antifungal agents. Methods Enzymol 1999;310:644-56.  Back to cited text no. 5
    
6.
Ozkan S, Kaynak F, Kalkanci A, Abbasoglu U, Kustimur S. Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents. Mem Inst Oswaldo Cruz 2005;100:319-23.  Back to cited text no. 6
    
7.
Eggimann P, Garbino J, Pittet D. Epidemiology of Candida species infections in critically ill non-immunosuppressed patients. Lancet Infect Dis 2003;3:685-702.  Back to cited text no. 7
    
8.
Pfaller MA, Jones RN, Messer SA, Edmond MB, Wenzel RP. National surveillance of nosocomial blood stream infection due to species of Candida other than Candida albicans: frequency of occurrence and antifungal susceptibility in the SCOPE Program. SCOPE Participant Group. Surveillance and Control of Pathogens of Epidemiologic. Diagn Microbiol Infect Dis 1998;30:121-9.  Back to cited text no. 8
    
9.
Blumberg HM, Jarvis WR, Soucie JM, Edwards JE, Patterson JE, Pfaller MA, et al. Risk factors for candidal bloodstream infections in surgical intensive care unit patients: the NEMIS prospective multicenter study. The National Epidemiology of Mycosis Survey. Clin Infect Dis 2001;33:177-86.  Back to cited text no. 9
    
10.
Miller LG, Hajjeh RA, Edwards JE Jr. Estimating the cost of nosocomial candidemia in the United States. Clin Infect Dis 2001;32:1110.  Back to cited text no. 10
[PUBMED]    
11.
Hawser SP, Douglas LJ. Biofilm formation by Candida species on the surface of catheter materials in vitro. Infect Immun 1994;62:915-21.  Back to cited text no. 11
    
12.
Ibàñez-Nolla J, Nolla-Salas M, León MA, García F, Marrugat J, Soria G, et al. Early diagnosis of candidiasis in non-neutropenic critically ill patients. J Infect 2004;48:181-92.  Back to cited text no. 12
    
13.
Ramesh N, Priyadharsini M, Sumathi CS, Balasubramanian V, Hemapriya J, Kannan R. Virulence factors and anti fungal sensitivity pattern of Candida Sp. isolated from HIV and TB patients. Indian J Microbiol 2011;51:273-8.  Back to cited text no. 13
    
14.
Calderone RA. Candida and Candidiasis. Washington Dc: ASM Press; 2002. p. 1-5.  Back to cited text no. 14
    
15.
Chander J. Textbook of Medical Mycology. 3rd ed. New Delhi: Mehta Publishers; 2008. p. 278-83.  Back to cited text no. 15
    
16.
Djordjevic D, Wiedmann M, McLandsborough LA. Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl Environ Microbiol 2002;68:2950-8.  Back to cited text no. 16
    
17.
Shin JH, Kee SJ, Shin MG, Kim SH, Shin DH, Lee SK, et al. Biofilm production by isolates of Candida species recovered from nonneutropenic patients: comparison of bloodstream isolates with isolates from other sources. J Clin Microbiol 2002;40:1244-8.  Back to cited text no. 17
    
18.
Tumbarello M, Posteraro B, Trecarichi EM, Fiori B, Rossi M, Porta R, et al. Biofilm production by Candida species and inadequate antifungal therapy as predictors of mortality for patients with candidemia. J Clin Microbiol 2007;45:1843-50.  Back to cited text no. 18
    
19.
Dan M, Poch F, Levin D. High rate of vaginal infections caused by non-C. albicans Candida species among asymptomatic women. Med Mycol 2002;40:383-6.  Back to cited text no. 19
    
20.
Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 2007;20:133-63.  Back to cited text no. 20
    
21.
Kothavade RJ, Kura MM, Valand AG, Panthaki MH. Candida tropicalis: its prevalence, pathogenicity and increasing resistance to fluconazole. J Med Microbiol 2010;59:873-80.  Back to cited text no. 21
    
22.
Kumari V, Banerjee T, Kumar P, Pandey S, Tilak R. Emergence of non-albicans Candida among candidal vulvovaginitis cases and study of their potential virulence factors, from a tertiary care center, North India. Indian J Pathol Microbiol 2013;56:144-7.  Back to cited text no. 22
[PUBMED]  Medknow Journal  
23.
Shivaprakasha S, Radhakrishnan K, Karim PM. Candida spp. other than Candida albicans: a major cause of fungaemia in a tertiary care centre. Indian J Med Microbiol 2007;25:405-7.  Back to cited text no. 23
[PUBMED]  Medknow Journal  
24.
Abi-Said D, Anaissie E, Uzun O, Raad I, Pinzcowski H, Vartivarian S. The epidemiology of hematogenous candidiasis caused by different Candida species. Clin Infect Dis 1997;24:1122-8.  Back to cited text no. 24
    
25.
Chow JK, Golan Y, Ruthazer R, Karchmer AW, Carmeli Y, Lichtenberg D, et al. Factors associated with candidemia caused by non-albicans Candida species versus Candida albicans in the intensive care unit. Clin Infect Dis 2008;46:1206-13.  Back to cited text no. 25
    
26.
Nguyen MH, Peacock JE Jr, Morris AJ, Tanner DC, Nguyen ML, Snydman DR, et al. The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. Am J Med 1996;100:617-23.  Back to cited text no. 26
    
27.
Baran J Jr, Muckatira B, Khatib R. Candidemia before and during the fluconazole era: prevalence, type of species and approach to treatment in a tertiary care community hospital. Scand J Infect Dis 2001;33:137-9.  Back to cited text no. 27
    
28.
Price MF, LaRocco MT, Gentry LO. Fluconazole susceptibilities of Candida species and distribution of species recovered from blood cultures over a 5-year period. Antimicrob Agents Chemother 1994;38:1422-4.  Back to cited text no. 28
    
29.
Pappas PG, Rex JH, Lee J, Hamill RJ, Larsen RA, Powderly W, et al. A prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients. Clin Infect Dis 2003;37:634-43.  Back to cited text no. 29
    
30.
Mohandas V, Ballal M. Distribution of Candida species in different clinical samples and their virulence: biofilm formation, proteinase and phospholipase production: a study on hospitalized patients in southern India. J Glob Infect Dis 2011;3:4-8.  Back to cited text no. 30
    
31.
Matsumoto FE, Gandra RF, Ruiz LS, Auler ME, Marques SA, Pires MF, et al. Yeasts isolated from blood and catheter in children from a public hospital of São Paulo, Brazil. Mycopathologia 2002;154:63-9.  Back to cited text no. 31
    
32.
Salyers A, Witt D, editors. Virulence factors that promote colonization. In: Bacterial Pathogenesis: a Molecular Approach. Washington, D.C.: ASM Press; 1994. p. 30-46.  Back to cited text no. 32
    
33.
Deorukhkar SC, Saini S. Non albicans Candida species: its isolation pattern, species distribution, virulence factors and antifungal susceptibility profile. Int J Med Sci Public Health 2013;2:533-8.  Back to cited text no. 33
    

Top
Correspondence Address:
Dr. Vivek Agwan
Department of Microbiology, Subharti Medical College, Meerut - 250 005, Uttar Pradesh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.168873

Rights and Permissions



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Result
    Discussion and C...
    References
    Article Tables

 Article Access Statistics
    Viewed2457    
    Printed44    
    Emailed0    
    PDF Downloaded224    
    Comments [Add]    

Recommend this journal