|Year : 2016 | Volume
| Issue : 3 | Page : 308-314
Identification, antifungal resistance profile, in vitro biofilm formation and ultrastructural characteristics of Candida species isolated from diabetic foot patients in Northern India
D Kumar1, T Banerjee1, J Chakravarty2, SK Singh3, A Dwivedi3, R Tilak1
1 Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of General Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Department of Endocrinology and Metabolism, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Submission||08-Oct-2014|
|Date of Acceptance||09-Jan-2016|
|Date of Web Publication||12-Aug-2016|
Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Purpose: Diabetic foot ulcers are a serious cause of diagnostic and therapeutic concern. The following study was undertaken to determine the fungal causes of diabetic foot ulcers, with their phenotypic and genotypic characterisation. Materials and Methods: A total of 155 diabetic foot ulcers were studied for 1 year. Deep tissue specimen was collected from the wounds, and crushed samples were plated on Sabouraud dextrose agar with chloramphenicol (0.05 g). Identification was done by growth on cornmeal agar, germ tube formation and urease test. For molecular identification, conserved portion of the 18S rDNA region, the adjacent internal transcribed spacer 1 (ITS1) and a portion of the 28S rDNA region were amplified, using the ITS1 and ITS2 primers. Antifungal susceptibility against voriconazole, fluconazole and amphotericin B was determined by standard broth microdilution method. Biofilm formation was studied in three steps. First, on the surface of wells of microtiter plates followed by quantification of growth by fungal metabolism measurement. Finally, biofilms were analysed by scanning electron microscopy (SEM). Results: Fungal aetiology was found in 75 patients (48.38%). All were identified as Candida species (100%). The prevalence of different species was Candida tropicalis (34.6%), Candida albicans (29.3%), Candida krusei (16.0%), Candida parapsilosis (10.6%), Candida glabrata (9.33%). All were susceptible to amphotericin B (100%). On microtiter plate, all the isolates were viable within 48 h showing biofilms. The metabolic activity of cells in the biofilm increased with cellular mass, especially in the first 24 h. On SEM, majority showed budding yeast form. Conclusion: Non-albicans Candida spp. with potential biofilm forming ability are emerging as a predominant cause of diabetic foot ulcers.
Keywords: Biofilm, Candida, foot, molecular, non-albicans, resistance
|How to cite this article:|
Kumar D, Banerjee T, Chakravarty J, Singh S K, Dwivedi A, Tilak R. Identification, antifungal resistance profile, in vitro biofilm formation and ultrastructural characteristics of Candida species isolated from diabetic foot patients in Northern India. Indian J Med Microbiol 2016;34:308-14
|How to cite this URL:|
Kumar D, Banerjee T, Chakravarty J, Singh S K, Dwivedi A, Tilak R. Identification, antifungal resistance profile, in vitro biofilm formation and ultrastructural characteristics of Candida species isolated from diabetic foot patients in Northern India. Indian J Med Microbiol [serial online] 2016 [cited 2020 Jan 28];34:308-14. Available from: http://www.ijmm.org/text.asp?2016/34/3/308/188320
| ~ Introduction|| |
The alarming rise in the incidence of diabetes mellitus, taking on pandemic proportions, has become a cause of global concern. With the predicted estimates of 380 million to be affected with this disease by the year 2025, the highest prevalence of diabetic patients amongst the 191 WHO member states were reported in India.  Being the most common cause of non-traumatic amputation of a lower extremity, 15% of diabetics develop a lower-extremity ulcer during their lifetime.  The pathogenesis of diabetic foot is highly complex including polyneuropathy, peripheral vascular disease and compromised immunity, slower wound healing, trauma and infection. In this context, mycosis is a serious diagnostic and therapeutic problem and cause of mortality in diabetics.
Majority of the studies on aetiology of diabetic foot ulcer focus on bacterial causes and very few studies report on varying prevalence of fungal pathogens. , However, the emergence of fungal pathogens warrants a detailed study of them. In India, a handful of studies have suggested the predominance of non-albicans Candida spp. in diabetic foot ulcers. , Nevertheless, there is a lack of studies from the subcontinent on the virulence traits of these isolates that is often responsible for their isolation from such type of ulcers.
The following study was undertaken to determine the role of fungal aetiology in the causation of diabetic foot ulcers along with phenotypic and genotypic characterisation of the isolates and to assess the associated risk factors.
| ~ Materials and Methods|| |
Site of study and study population
The study was conducted in the Department of Microbiology and the Associated University Hospital in Varanasi, North India. A total of 155 patients with type 2 diabetes mellitus and foot ulcer who were hospitalised for surgical management of lower-extremity wounds from July 2012 to August 2013 were included in the study. Relevant clinical data were collected regarding demographic details, duration of lower-limb lesion, duration of type 2 diabetes mellitus and wound assessment. The investigational setting was expanded to include the quality of blood glucose control glycated haemoglobin and neurological parameters, such as neuropathy symptoms and deficit scores as well as assessment of sudomotoric capacity.
Isolation and identification
A deep tissue specimen from the junction of viable and nonviable tissue, often invading the stratum corneum was collected from the wounds following thorough normal saline wash during surgery in sterile containers and sent for fungal cultures. A concomitant histological smear, however, could not be done which could have predicted the invasiveness of the isolates better. Further, the specimen was processed in a type IIB biological safety cabinet. The tissue was sliced and put into tissue grinder and crushed at 2000 rpm for 1 min. These crushed tissues were placed directly into Sabouraud dextrose agar with chloramphenicol (0.05 g) and submerged slightly beneath the surface by an inoculating loop. The slants were incubated at 37°C and observed for 2 weeks. KOH (10%) and Gram's stain were performed. Further identification was done by morphological study on cornmeal agar, germ tube formation and urease test followed by sugar assimilation and fermentation tests based on reference standard. 
Molecular identification of the isolates
DNA extraction and purification were performed using Genomic DNA Extraction Kit (AccuPrep Genomic DNA extraction kit, Bioneer Corporation, Korea) according to manufacturer guidelines. A conserved portion of the 18S rDNA region, the adjacent internal transcribed spacer 1 (ITS1) and a portion of the 28S rDNA region were amplified, using the ITS1 and ITS2 primers described elsewhere  to amplify Candida albicans ATCC 14053 (218 or 219, and 110 bp), Candida glabrata CBS2175 (482 or 483 bp), Candida parapsilosis CBS2195 (229 bp), Candida tropicalis CBS94 (218 bp) and Candida krusei CBS573 (182 bp), respectively. A positive control of DNA of C. albicans ATCC 90028 and a negative control without any DNA were run for each of the experiment.
Antifungal susceptibility testing
Antifungal susceptibility was determined by broth microdilution method according to CLSI guidelines.  Standard antifungal powders (potency 99.99% as per manufacturer) of voriconazole, fluconazole and amphotericin B (Lifecare Ltd., India) were obtained from the manufacturer. These antifungals were tested based on their frequent use in our hospital setup. Stock solutions were prepared in dimethyl sulphoxide (voriconazole and Amphotericin B), and water (fluconazole). Serial twofold dilutions were prepared exactly as outlined in CLSI document M27-A3.  Final dilutions were made in RPMI 1640 medium (Sigma, USA) buffered to pH 7.0 with 0.165M morpholinepropanesulfonic acid buffer (Sigma, USA). The final concentration of solvent did not exceed 1% in any well. Aliquots (100 μl) of each antifungal agent at a two-fold final concentration were dispensed into the wells of plastic microdilution plate. The trays were sealed and frozen at −70°C until they were used.
Fungal suspension having turbidity matching with standard McFarland concentration of 0.5 OD was made which yielded yeast stock suspension of 1−5 × 10 6 cells/ml. Working suspension was made by 1:100 dilution followed by 1:20 dilution of stock suspension in RPMI 1640 broth medium which resulted in a final concentration of approximately 5 × 10 2 -2.5 × 10 3 cells/ml. The final concentrations of the antifungal agents were 0.007-8 μg/ml for voriconazole and, 0.06-4 μg/ml amphotericin B, and 0.06-128 μg/ml for fluconazole. The trays were incubated at 35°C, and minimal inhibitory concentration (MIC) endpoints were read after 24 h of incubation. Drug-free and yeast-free controls were included. MIC results for all agents were read visually following 24 h of incubation as the lowest concentration of drug that caused a significant diminution (≥50% inhibition) of growth compared with control.  The recently revised CLSI clinical breakpoints were used to identify strains of C. glabrata and for fluconazole the MIC values ≥64 μg/ml was considered as resistance. A breakpoint of 16-32 μg/ml was considered to be indicative of susceptible-dose dependent (SDD) and <8 μg/ml susceptible for voriconazole. Quality control was performed by testing the CLSI recommended strains C. krusei ATCC 6258 and C. parapsilosis ATCC 22019.
Study of biofilm formation
The study of biofilm formation was done by three different procedures. In the first step, biofilm formation on the surface of wells of microtiter plates was studied. For this, biofilms were prepared in commercially sterilised, polystyrene and flat-bottomed 96-well microtiter plate (Tarsons, India). Twenty microliter of standardised yeast cell suspension (3 × 10 6 CFU/ml were deposited into each well of a microtiter plate containing 180 μl of SDB supplemented with 8% glucose. The plates were covered and incubated for 24 h at 35°C in an orbital shaker ( Remi Laboratory Instruments, USA). Biofilm formation was measured by ELISA plate reader at 405 nm wavelength. The results were expressed in absorbance and then converted to transmittance. Therefore, adherent biofilm layer was scored as either negative; *(%T <5); **(%T = 20 and 35); ***(%T = 35 and 50); ****(%T ≥50), as previously proposed study.  On the basis of these scores, biofilm-producing samples were further classified into weakly (*), moderately (**, ***) and strongly (****) positive.
In the next step, quantification of growth by fungal metabolism measurement was done. Biofilm was quantified biochemically according to the method described by Tumbarello et al.  with some modifications. Briefly, 180 μl of SDB (Hi-media, Mumbai India) supplemented with 8% glucose, and 20 μl of standardised yeast cell suspension (3 × 10 6 CFU/ml) were placed into each well of 96-well microtiter plate. The plates were incubated for different time periods as 2 h, 4 h, 24 h and 48 h. After incubation, a 100 μl aliquot of a saline solution containing 2H-tetrazolium-5-carboxanilide (XTT) (100 μg/ml) and phenazine methosulphate (10 μg/ml) (Sigma, USA) was inoculated into each prewashed biofilm, and also into control wells (wells without yeast suspension). The plates were further incubated in the dark at 35°C for 3 h to allow the conversion of XTT into its formazan derivative. For each sample, experiments were designed in quadruplicates, on three different days. The amount of XTT formazan was measured by spectrophotometric reader at 492 nm of the microtiter plate.
Finally, biofilms were analysed by scanning electron microscopy (SEM).  For this, bottom of 96-well microtiter plates were equally cut off and placed onto 12-well plates. Then, 2 ml of yeast suspension (3 × 10 6 CFU/ml) was transferred in to each well and the plate was incubated at 37°C during for 48 hrs with shaking at 70-100 rpm. After that, the plates were removed from the shaker and wells were gently washed three times with sterile saline solution in order to remove the loosely adherent biofilm yeast cells. For SEM analysis, a biofilm suspension was fixed in 2.5% glutaraldehyde overnight. The fixed specimen was then washed again by 0.1 M sodium cacodylate buffer pH 7.4 at 4°C and left for 3 h. Above suspension was dehydrated by sequential dipping into alcohol from 30% to 90% followed by a dip into absolute alcohol and air-dried for 20 min. The dehydrated samples were sputter-coated with gold under sputtering device and observed under a Zeiss EVO-LS-10 SEM. The images obtained were processed using imaging software SmartSEM® VS10- Central facility, Department of Zoology, Banaras Hindu University, Varanasi.
Demographic details and associated risk factors were compared between those with fungal foot ulcers and without fungal ulcers. Biofilm formation and MIC of antifungal drugs was compared. The data were analysed using the SPSS software (Statistical Package for the Social Sciences, version 16.0, SPSS Inc., Chicago, IL, USA). Percentage for proportions and odds ratios for categorical variables has been reported, where appropriate.
| ~ Results|| |
Isolation and identification of fungal isolates
Of the total 155 cases studied, fungal aetiology was found in 75 patients (48.38%). Of these, all were identified as Candida species (100%). The prevalence of different species of Candida was C. tropicalis (34.6%), C. albicans (29.3%), C. krusei (16.0%), C. parapsilosis (10.6%) and C. glabrata (9.33%). The morphological and biochemical features varied according to the species. While on Gram's staining, 4-6 μm oval yeast cells were seen for C. albicans and C. tropicalis, the former produced thick walled, terminal chlamydospores on corn meal agar as against single blastospores in the latter. Oval to cylindrical (4-8 μm) yeast cells and round (2-4 μm) yeast cells were seen for C. parapsilosis and C. glabrata, respectively.
The species distribution and Wagner's grading of ulcer have been presented in [Table 1]. Initially, the number of isolates increased with Wagner's grading up to level 3. There was perfect concordance with the conventional and molecular methods for detection of different species [Figure 1]. All the species assigned by morphological and biochemical tests were confirmed by polymerase chain reaction. The mean age (± standard deviation) of the 155 participants was 43.4 (±8.2) years. There was a significant association between the age of participants and detection rates of C. albicans (P < 0.05). The results of the analysis indicated that older participants were less likely to be infected with C. albicans. Elevated blood sugar and increased total leucocyte count were significantly associated with fungal infection in the patients with foot ulcer [Table 2].
|Figure 1: Multiplex polymerase chain reaction using primers internal transcribed spacer 1, internal transcribed spacer 2 for identification of Candida spp.|
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|Table 1: Prevalence of Candida spp. and Wagner's grading of the foot ulcers |
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Antifungal susceptibility testing
In vitro susceptibility results of the Candida isolates are summarised in [Table 3]. The MICs for the quality control strains were within the acceptable limits. The determined MIC ranges were 0.12-1, 0.12-64 and 0.03-0.25 μg/ml for amphotericin B, fluconazole and voriconazole respectively. All the isolates were susceptible to amphotericin B. The isolates demonstrated very low MIC values against amphotericin B, with 98.6% (74/75) showing values of 0.03 μg/ml. For voriconazole, 94.6% (71/75) presented MIC values of 0.03 μg/ml, whereas 89.3% (67/75) were susceptible for fluconazole, 2 isolates were resistant. Six isolates showed SDD against fluconazole and 4 isolates for voriconazole.
|Table 3: In vitro susceptibility testing of isolates of Candida spp. against three antifungal agents |
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Biofilm formation and metabolic activity
Biofilm formation was seen in varying extent among the Candida isolates. On microtiter plate assay, all the isolates were viable within 48 h showing biofilms. The metabolic activity of cells in the biofilm increased over time along with the increase in the cellular mass, especially in the first 24 h. As the biofilm matured, the metabolic activity gradually reached a plateau but remained high. Among the Candida spp., C. tropicalis and C. parapsilosis simultaneously showed a high biofilm formation and metabolic activity as against C. glabrata. A difference in the metabolic viability was observed between different yeasts, but no significant difference was detected (P = 0.347). C. albicans, C. parapsilosis and C. krusei showed their metabolic peak within 24 h [Figure 2]a and b.
|Figure 2: (a) Results of 2H-tetrazolium-5-carboxanilide reduction assay, (b) biofilm production in microtiter plates and metabolic activity of the Candida spp. Mean of each experiment executed in quadruplicate|
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Antifungal susceptibility profile and biofilm formation
Of the 75 isolates, 54 (72%) isolates showed moderate to strong biofilm formation (7, 87.5% C. parapsilosis, 19, 86.36% C. albicans, 13, 50% C. tropicalis). Of these, 2 (28.57%) isolates of C. parapsilosis (P = 0.71), 10 (52.63%) isolates of C. albicans (P = 0.54) 7 (53.84%) isolates of C. tropicalis (P = 0.11) were also associated with higher MIC values especially against fluconazole.
Ultrastructural characteristics on scanning electron microscopy
On microscopy by SEM, various cellular morphologies within the biofilm were seen. Most of the Candida spp. showed budding yeast form, closely attached to the surface. C. parapsilosis was the only isolate that demonstrated adhesion in budding yeast and hyphae-like forms. Most of the yeasts adhered to each other only when in blastoconidia form after 48 h of contact. In the case of C. glabrata biofilm structure, [Figure 3] dense biofilms were not seen.
|Figure 3: Scanning electron photomicrographs showing morphology of yeasts adhered to the plate surface after 48 h of contact|
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| ~ Discussion|| |
Foot ulceration is one of the most serious and costly complications of diabetes worldwide. Situations are even worse in developing countries where one in every six people with diabetes develop foot ulcer.  Interestingly, as against most of the studies, , majority of the patients were females. Factors such as age, body mass index and duration of diabetes were comparable in both groups and were not found to significantly affect the occurrence of foot ulcers. Like other studies, poor glycaemic control was significantly associated with these ulcers. The real problem is the requirement of amputation in such cases that has been often observed.
Prevalence of nearly 50% for fungal causes of diabetic foot ulcer was seen in this study. Studies in the past from India have reported a much lower rate of isolation (9%) from superficial swabs from diabetic wound patients.  However, a recent study has shown a higher prevalence of 27.9% of fungal agents, with majority being Candida spp. (76.6%),  from deep tissue wound. Worldwide varying prevalence of 18-55% of Candida spp. from diabetic patients has been isolated from several sites including foot ulcers.  The collection of deep tissue from ulcers might have accounted for larger prevalence rate detected in this study. However, it should be emphasised that just as suspicion of fungal agents along with the more commonly assumed bacterial infections in diabetic foot ulcers is important, processing of deep tissue specimen is equally essential for determining the complete spectrum of pathogens.
The emergence of non-albicans Candida spp. over C. albicans has been observed in several reports from various sites of infection.  C. tropicalis, which was the major species isolated in this study has become an important cause of candidemia.  Similarly, C. glabrata which was previously considered as a non-pathogenic saprophyte is now increasingly being recognised as a pathogen.  Some of the above-mentioned studies have also reported C. tropicalis as the major spp. ,, However, no filamentous fungi were isolated in this study. Molecular detection and confirmation of species has been a well-established method that has contributed immensely to widen the spectrum of non-albicans Candida spp. In this study too, routine biochemical tests helped in accurate identification of Candida spp. that was reaffirmed by molecular detection.
Antifungal susceptibility testing revealed that in vitro susceptibility was highest for amphotericin B and voriconazole and 89.3% for fluconazole. However, 10 isolates that showed SDD against fluconazole and/or voriconazole also warrants their emergence as resistant isolates in the future. In this study, high MIC 90 especially against fluconazole was noted. This could be due to the following facts. Fluconazole resistance among non-albicans Candida against a background of widespread use of azoles have been proposed though there is lack of any established data on this regard.  On the other hand, reports of fluconazole being the most frequently prescribed antifungal is very much evident.  Unfortunately, in our set up too, use of polyenes and azoles was high. Empirical use of antifungals owing to the long time period required for fungal culture positivity in some cases was one of the major causes of antifungal use in our hospital. In addition, even though an antimicrobial policy including administration of antifungals exist in the hospital, most of the times it could not be implemented due to the fact that ours being a tertiary care centre, majority of the patients were referred from other hospitals with on-going antifungal treatment. Consequently, our hospital was left with no choice other than continuing the treatment. For all such situations, a prudent approach would be to use antifungals only following a susceptibility test. One of the limitations in antifungal susceptibility testing in this study was that determination of geometric mean titres along with MIC 50 and 90 values could have given a better concept on the resistance patterns of these isolates.
Biofilm formation is one of the most important virulence factors exhibited by Candida spp. Biofilms are responsible for persistence of fungal infections and thus cause significant clinical and economic problem. C. albicans has the second highest rate of colonisation to infection.  In contrast, in this study, Candida albicans was not the only species that showed greater biofilm formation, but C. parapsilosis showed the greatest biofilm formation. The problem with biofilms is that yeast cells can detach from the adherent biofilms leading to chances of fungaemia and serious systemic infections. In addition, the problem of steadily increasing biofilm formation ability by the non-albicans Candida isolate is a cause of concern. Studies performed elsewhere , have shown that fungi tested from the biofilms were much more resistant to antifungals than their planktonic forms as biofilm formation acts like an additional barrier to penetration of the drugs. In this study, though MIC determination from both planktonic and biofilm colonies was not done, yet majority of the strong/moderate biofilm producing isolates were associated with higher MIC values especially against fluconazole (P < 0.05), although in the susceptible range. Many studies have focused on biofilms because they are more resistant to antimicrobials and host defences. Most of them have reported a near-total resistance to antifungal agents exhibited by biofilm-associated Candida. C. albicans biofilms in vivo, and in vitro demonstrated higher susceptibility when treated with a combination of amphotericin B and posaconazole than when treated with single drugs. , Combining the use of echinocandins with other drugs that have antifungal activity is becoming an important alternative form of therapy in mycoses caused by fungi that are resistant to standard antifungal monotherapy or in certain biofilm-associated diseases.
In this study, SEM confirmed the varying biofilm forming capacity of different Candida spp. and therefore helped in predicting the consequent complications that can arise when affected by a specific Candida spp. Biofilms are resistant to broad range of antifungals used clinically. On the other hand, under planktonic conditions, one of the mechanisms of azole resistance is active efflux of the drugs. Interestingly, these efflux pumps are also upregulated during different phases of biofilm formation and it has been seen that in early adherence phase of biofilm formation, isolates are more susceptible than a latter phase.  Therefore, knowledge of biofilm formation in these isolates can provide insight into the importance of initiation of early and robust treatment especially in cases of diabetic foot ulcers as a life saving measure. A vivid understanding of the mechanisms and variations of biofilm formation will make the advent of methods to curb their formation possible.
This study depicts the increasing prevalence of fungal aetiology in the causation of diabetic foot ulcers in North India. Further studies in this area will also contribute towards the identification of new targets for future therapeutics against these recently emerged pathogens.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]