Antifungal susceptibility profile and biofilm production of Candida spp. isolated from catheterised patients with urinary tract infection

Parthasarathy Kamini; Thayanidhi Premamalini; Kanagasabapathi Karthika; Sukumar Bavadharani

doi:10.4103/jacm.jacm_12_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 24 | Issue : 2 | Page : 76-83

Antifungal susceptibility profile and biofilm production of Candida spp. isolated from catheterised patients with urinary tract infection

Parthasarathy Kamini, Thayanidhi Premamalini, Kanagasabapathi Karthika, Sukumar Bavadharani
Department of Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Porur, Chennai, Tamil Nadu, India

Date of Submission	01-Aug-2022
Date of Acceptance	15-Sep-2022
Date of Web Publication	13-Dec-2022

Correspondence Address:
Thayanidhi Premamalini
Department of Microbiology, Sri Ramachandra Medical College and Research Institute, SRIHER, Porur, Chennai - 600 116
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jacm.jacm_12_22

Abstract

BACKGROUND: Candida species, which are present as normal flora in healthy individuals, are known to cause opportunistic infections with high rates of mortality, especially in immunocompromised individuals. Urinary tract infection (UTI) is the most common type of nosocomial infection, and about 10%-15% of them are due to Candida species. Catheter-associated infections due to Candida spp. are mainly due to biofilm formation.
OBJECTIVE: This study aims to speciate Candida isolates from catheterised patients with UTI infection and to compare the antifungal susceptibility pattern with their biofilm production.
MATERIALS AND METHODS: A total of 55 Candida species were isolated from the urine samples of catheterised patients with UTIs over a period of nine months. Patients' demographic details and risk factors were collected. All the isolates were identified and confirmed by routine phenotypic and genotypic techniques. The rare species that could not be identified by routine techniques were identified by sequencing the internal transcribed spacer region. Biofilm production was detected by tube method, and the antifungal susceptibility testing was done by broth microdilution method (CLSI M27-A3 guidelines).
RESULTS: In our study, 55 Candida species were identified, among which the most predominant species was found to be Candida tropicalis 23 (42%). Among 48 biofilm producers (87.2%), only 2 (4.1%) isolates of Candida albicans and 3 (6.2%) isolates of C. tropicalis were resistant to Amphotericin B and Fluconazole. The MIC values for Amphotericin B, Fluconazole and Itraconazole were high in both Candida auris (2) and Candida lusitaniae (1), although one isolate of each species was a potent biofilm producer. In Candida catenulata (4), three isolates had high MIC value for Amphotericin B and two isolates had high MIC value for Itraconazole and three isolates produced biofilm.

Keywords: Antifungal susceptibility pattern, biofilm production, Candida species, catheterised patient

How to cite this article:
Kamini P, Premamalini T, Karthika K, Bavadharani S. Antifungal susceptibility profile and biofilm production of Candida spp. isolated from catheterised patients with urinary tract infection. J Acad Clin Microbiol 2022;24:76-83

How to cite this URL:
Kamini P, Premamalini T, Karthika K, Bavadharani S. Antifungal susceptibility profile and biofilm production of Candida spp. isolated from catheterised patients with urinary tract infection. J Acad Clin Microbiol [serial online] 2022 [cited 2023 Mar 22];24:76-83. Available from: https://www.jacmjournal.org/text.asp?2022/24/2/76/363471

Introduction

Candida species form a part of normal flora in healthy persons and are known to produce opportunistic infections with increased mortality, particularly among immunocompromised people.^[1] Candiduria, which is characterised by the presence of Candida species in urine, is frequently observed in hospitalised patients.^[2],[3],[4] Predisposing factors, such as catheterisation, usage of higher class of antibiotics, lengthy stay in hospital and diabetes, all contribute to the development of candiduria in hospitalised patients.^[5] Most of the studies have observed Candida albicans as a chief aetiological agent, followed by Candida glabrata and Candida tropicalis.^[6] Recently, Candida non-albicans species have started emerging as a major causative factor for candiduria, especially C. tropicalis.^[7] Biofilms can be formed on mucosa and surfaces of indwelling catheters or devices and are inherently resilient to antifungal agents such as Fluconazole and Amphotericin B. Based on the Candida species, biofilm formation also differs.^[1],[8] The ability of the Candida species to form biofilm is a major virulence factor contributing to treatment failure.^[9] Urinary catheters offer an appropriate niche for the Candida species to form biofilm. Even though removal of the catheters plays a vital role in the treatment of biofilm-related infections, antifungal treatment may be mandatory after the removal in a few patients. Mortality with candiduria may increase in the existence of associated candidemia and several other associated comorbid conditions.^[7] Candiduria could also remain as a critical marker for disseminated Candida infection with increased mortality.^[10] Henceforth, knowing the Candida species and its antifungal susceptibility pattern is essential for appropriate timely management of the patient.^[11] Since there are less data on biofilm formation by Candida species and its antifungal susceptibility pattern among the catheterised patients, the current study was undertaken to explore the ability of biofilm formation among the catheterised candiduria patients and its antifungal susceptibility pattern.

Materials and Methods

Fifty-five Candida isolates which grew from urine samples of catheterised patients with UTI, collected over a period of nine months, were considered for the study. Based on the CDC guidelines, the presence of at least one signs or symptoms of UTI and ≥10³ colony-forming units (cfu)/ml in a single catheter urine specimen was considered for the diagnosis of CAUTI in this study.^[12] The demographic data and basic clinical details were collected from all patients.

Type of study

This was a hospital-based descriptive study.

Collection of isolates and characterisation of Candida species

The urine samples which showed the presence of budding yeast cells on direct microscopy and grew yeast-like colonies on culture were included in the study. The isolates were subcultured onto Sabouraud's dextrose agar slants and stored at 4°C. All the 55 Candida isolates were speciated by conventional phenotypic methods and later subjected to genotypic methods such as polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) for confirmation of identification. The rare species that could not be identified by PCR-RFLP were identified by sequencing the ITS region.

Control strains used for phenotypic and genotypic charecterisation

Candida albicans ATCC 90028, C. tropicalis ATCC 750, Candida krusei ATCC 6258, Candida glabrata ATCC 15126 and Candida parapsilosis ATCC 22019.

Phenotypic characterisation

Candida isolates were speciated phenotypically based on Gram staining, culture characteristics, germ tube formation, sugar fermentation, sugar assimilation, colour on tetrazolium reduction medium (TTZ) and CHROMagar with appropriate control strains.

Genotypic characterisation

DNA extraction

Genomic DNA was extracted from all the Candida test isolates and reference strains by phenol–chloroform method of Mirhendi et al., 2006, and Vijayakumar et al., 2012.^[13],[14]

Polymerase chain reaction

PCR amplification of ITS1 and ITS2 regions was carried out using universal fungal primers ITS 1 (5'– TCC GTA GGT GAA CCT GCG G– 3') and ITS4 (5'– TCC TCC GCT TAT TGA TAT GC– 3').^[13],[14] The PCR master mix was prepared containing 25 μl of PCR mix (Takara, Japan), 1 μl of forward (ITS1) and reverse primer (ITS4), 5 μl of template DNA and the volume made up to 50 μl with sterile nuclease-free water. The reaction mixtures were amplified in a thermal cycler (Veriti 96 well, Applied Biosystems, USA), with the following programme: 95°C for five minutes, followed by 35 cycles consisting of 95°C for 30 s, 56°C for 30 s and 72°C for 30 s, with a final extension period at 72°C for 10 min. After thermal cycling, 5 μl of the amplified product was run on a 1.5% (wt/vol) agarose gel, stained with ethidium bromide and visualised with ultraviolet light.

Restriction fragment length polymorphism

The PCR products were subjected to restriction analysis using MspI restriction enzyme (New England Biolabs), which cleaves the DNA at specific site producing fragments of varying length.^[13],[14] The reaction mixture was prepared containing 10 μl of amplified PCR product, 2 μl of 10X enzyme buffer, 5 units of MspI restriction enzyme and the volume made up to 20 μl with sterile nuclease-free water. The reaction mixtures were incubated at 37°C for one hour. After incubation, 5 μl of the amplified product was run on a 2% (wt/vol) agarose gel, stained with ethidium bromide and visualised with ultraviolet light. The species were identified based on the band size.

DNA sequencing and Basic Local Alignment Search Tool analysis

Candida species which could not be identified by PCR-RFLP were identified by sequencing the ITS region. DNA sequencing of PCR products was performed by AgriGenome Labs Pvt. Ltd., Cochin, Kerala. The sequence was then used for a nucleotide–nucleotide search using the Basic Local Alignment Search Tool (BLAST) algorithm at the NCBI website (http://www.ncbi.nlm.nih.gov/BLAST/), CBS database (http://www.westerdijkinstitute.nl/collections/) and ISHAM ITS (http://its.mycologylab.org/). Hits more than 99% were considered.

Biofilm formation

Candida isolates were tested for biofilm production by a modification of the standard method of Christensen et al.^[15] The scoring for tube method was done visually and compared to the results of the control strains. C. albicans ATCC 90028 was used as reference strain. Biofilm production was considered positive when a visible film lined the wall and the bottom of the tube. Ring formation at the liquid interface was not indicative of biofilm formation. The amount of biofilm formed was scored as 0 – negative, 1 – weak, 2 – moderate and 3 – high/strong. The procedure was performed in triplicates for better interpretation.

Antifungal susceptibility testing

The antifungal susceptibility was performed by the broth microdilution method for all the 55 Candida species isolated from catheterised patients with UTI, according to the CLSI M27-A3 guidelines (CLSI 2008) ('Reference method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard Third edition').^[16],[17]

Concentrations tested

Each antifungal agent was tested for the corresponding range of concentrations:

Amphotericin B (CMS462-5G, HiMedia Laboratories) – 0.06-16 μg/ml
Fluconazole (F8929-100MG, Sigma-Aldrich) – 0.25-64 μg/ml
Itraconazole (16657-100MG, Sigma-Aldrich) – 0.06-16 μg/ml
Posaconazole (32103-25MG, Sigma-Aldrich) – 0.06-16 μg/ml
Voriconazole (PZ0005-5MG, Sigma-Aldrich) – 0.06-16 μg/ml
Caspofungin (SML0425-5MG, Sigma-Aldrich) – 0.015-8 μg/ml.

Incubation and reading results

The results were interpreted according to the current breakpoints for the Candida species as per CLSI guidelines (M27-A3), after 24 h of incubation. As a quality check, the complete absence of turbidity in the sterility well was checked each time the test was performed.

Results

After speciation by phenotypic and genotypic methods, test for biofilm formation and antifungal susceptibility testing was performed for all the isolates. The results of biofilm production and antifungal susceptibility pattern were compared and interpreted.

Age distribution and gender distribution

The majority of patients in the study belonged to the age group of 61-70 years (19/55, 34.5%), followed by the age group of 71-80 years (12/55, 21.81%). Men (26/55, 47.2%) and women (29/55, 52.7%) were almost equally affected in our study. The male-to-female ratio was found to be 0.89:1.

Risk factors

Diabetes mellitus was found to be the most predominant risk factor (13/55 [24%]). The other risk factors associated with Candida infections in catheterised patients are shown in [Figure 1]. All the affected patients were on antimicrobial therapy. The common antimicrobials used in the treatment of these patients are shown in [Figure 2].

Figure 1: Risk factors of different Candida spp. causing UTI in catheterised patients. UTI: Urinary tract infection

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Figure 2: Antimicrobial therapy as a risk factor for Candida infections

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Identification and speciation of Candida isolates

All the 55 Candida isolates were initially identified as belonging to the genus Candida by phenotypic methods. Species identification of the Candida isolates by phenotypic methods such as sugar assimilation, fermentation and colour on chromogenic media could identify only four species, i.e. C. albicans 14/55 (25%), C. tropicalis 23/55 (42%), Candida glabrata 10/55 (18%) and Candida kefyr 1/55 (2%). PCR-RFLP was done for all the 55 isolates to rapidly speciate and confirm the results obtained by phenotypic method. The rare Candida isolates which were not identified by PCR-RFLP were further speciated by gene sequencing, which recognised them as Candida auris (2), Candida catenulata (4), Candida lusitaniae (1) and Candida kefyr (1).

Biofilm production

Among 55 Candida isolates tested for biofilm production using tube method, majority of the isolates produced biofilm (48/55 [87%]) and 7/55 (13%) did not produce biofilm. Among the Candida species, biofilm production was observed maximum in C. tropicalis (39.5%) followed by C. albicans (29.1%) [Table 1].

Table 1: Biofilm grading in positive Candida isolates

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[Table 2] shows the antifungal susceptibility pattern of all the tested Candida isolates.

Table 2: Interpretation of antifungal susceptibility testing by broth microdilution, the minimum inhibitory concentration₅₀ and minimum inhibitory concentration₉₀ values and the minimum inhibitory concentration range of various Candida isolates in the study

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For Amphotericin B, the resistance percentage of C. tropicalis (13%) was high followed by C. albicans (14.2%). All Candida glabrata isolates were susceptible to it. Rare isolates such as C. auris, C. lusitaniae and C. catenulata had high MIC values (4 μg/ml) for this drug whereas C. kefyr had low MIC value. For Fluconazole, the resistance percentage of C. tropicalis (13%) was high followed by C. albicans (14.2%). All Candida glabrata isolates were susceptible dose dependent to it. Rare isolates such as C. auris and C. lusitaniae had high MIC value (64 μg/ml) for this drug whereas C. kefyr and C. catenulata had low MIC values. For Itraconazole, the resistance percentage of C. albicans (7.1%) was high followed by C. tropicalis (4.3%). All Candida glabrata isolates were susceptible to it. Rare isolates such as C. auris, C. lusitaniae and C. catenulata also had high MIC values. All Candida isolates were susceptible to Voriconazole, Posaconazole and C Caspofungin. However, breakpoints have not been set for testing Candida glabrata against Voriconazole. MIC₅₀ and MIC₉₀ values of Posaconazole, Voriconazole and Caspofungin were low for C. albicans, C. tropicalis and C. glabrata. Since the rare isolates were low in number, MIC₅₀ and MIC₉₀ was not calculated for any of them.

The breakpoints have not been set by M27-A3 document for C. auris, Candida catenulata, C. lusitaniae and Candida kefyr. Hence, these rare isolates could not be ascertained as susceptible, intermediate or resistant. For C. auris, the mean MIC value of Caspofungin and Posaconazole was low whereas it was high in Voriconazole.^[18]

According to the tabular column, all the rare isolates have high MICs for Amphotericin B, Fluconazole and Itraconazole. The MIC values of these rare isolates are given in [Table 3].

Table 3: Antifungal susceptibility patterns of rare isolates

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Comparison of biofilm production and antifungal susceptibility patterns

Among 48 (87.2%) biofilm producers, only 2 (4. 1%) isolates of C. albicans, 3 (6. 2%) isolates of C. tropicalis and 1 isolate of C. lusitaniae were resistant to Amphotericin B and Fluconazole. Three isolates of C. catenulata were resistant to Amphotericin B alone. They were sensitive to all other azoles and Caspofungin [Table 4].

Table 4: Biofilm production and azole resistance patterns associated with the study isolates

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Discussion

Urinary tract infection (UTI) in catheterised patients is the most common healthcare-associated infection in hospitalised patients.^[19] Candida species is one of the most common causative agents causing nosocomial UTI.^[20] Studies have shown that many of the Candida species are able to attach to catheters and produce biofilms. These isolates are resistant to different antimicrobial agents.^[21],[22]

Diabetes mellitus was the predominant risk factor (24%) in our study. Diabetes mellitus is a metabolic disorder that predisposes individuals to fungal infections, including those related to Candida species, due to an immunosuppressive effect on the patient.^[23] Previous use of antibiotics was a universal risk factor in our patients. The risk was highest after treatment with Imipenem and Meropenem group of drugs (52.7%) followed by beta-lactamase inhibitor combinations (38.1%), which was similar to a previous study by Jain et al., 2007, where the risk factor for Imipenem and Meropenem group of drugs was reported as 75.7%.^[24]

About 14.2% of C. albicans and 13% of C. tropicalis isolates were resistant to Amphotericin B in our study. Most of the other studies have reported 100% susceptibility to Amphotericin B for C. albicans and C. tropicalis.^[25],[26] However, there are some reports that describe the development of resistance in some strains.^{[1],[27],[28]} The MIC₉₀ values of Amphotericin B for C. albicans and C. tropicalis were found to be high in this study. Hence, this drug may not be the drug of choice to treat infections caused by C. albicans and C. tropicalis. All the five isolates which were resistant to Amphotericin B also showed resistance to Fluconazole also. Previous studies by Pfaller et al., 2006, had identified a mutation in the gene ERG-3 which can lead to the production of a sterol compound other than ergosterol, thereby contributing to Amphotericin B resistance along with Fluconazole resistance.^[29] All the Candida glabrata isolates were susceptible to Amphotericin B. A study by Mirchevska et al., 2016, had reported all the Candida glabrata isolates as being susceptible, which was consistent with the findings of our study,^[26] though their MIC₉₀ was found to be 1 μg/ml. All the rare isolates had high MIC values for Amphotericin B except for one isolate each of Candida catenulata and Candida kefyr. Another study on rare isolates by Young et al., 2003, showed high MIC values for Amphotericin B, which was corresponding to the observations of our study.^[30]

Pfaller et al., 2010, showed moderate resistance to Fluconazole and in 2018 Munmun B. Marak et al., had reported increased resistance for both C. albicans (40.90%) and C. tropicalis (36.36%) to Fluconazole.^[1],[31] Similarly, in our study, 14.2% of C. albicans and 13% of C. tropicalis isolates showed Fluconazole resistance. All the Candida glabrata isolates were in the susceptible dose-dependent range in our study, which was similar to another study by Hatice Turg Dagi et al., 2016, where 97% of the isolates were in that range.^[32] The MIC₉₀ values of Fluconazole for C. albicans, C. tropicalis and Candida glabrata were also found to be high. Hence, this drug can be used for treatment only after performing antifungal susceptibility testing. All the rare Candida species had very high MIC values for Fluconazole, except for two isolates, one each of Candida catenulata and Candida kefyr. This finding was similar to the finding in other studies by Chowdhary et al., 2013, and Le et al., 2011, which also showed high MIC values for C. auris.^[33],[34] Frequent use of Fluconazole contributes to the emergence of resistance for the different Candida species. Hence, therapeutic use of Fluconazole should be restricted to selected high-risk patients, to minimise the risk of emergence of azole-resistant strains of Candida.

The resistance percentage of C. albicans and C. tropicalis to Itraconazole was found to be very low in our study. None of the Candida glabrata isolates was resistant to Itraconazole. Furthermore, MIC₉₀ values for all these three isolates were found to be low. This finding was analogous to the study done by Manik et al., 2015, where all the C. albicans, C. tropicalis and Candida glabrata isolates were susceptible to Itraconazole.^[35] In contrast, the MIC values of the rare Candida isolates to Itraconazole were found to be high, except for Candida kefyr. Yeast infections with common Candida species such as C. albicans, C. tropicalis and Candida glabrata that are not responsive to the conventional drugs can be treated with second-line broad-spectrum antifungal such as Itraconazole, if treatment with Fluconazole fails. However, this cannot be applied for the rare Candida isolates, since their MIC values for Itraconazole were high.

Majority of the C. albicans and C. tropicalis isolates were susceptible to Voriconazole except for one isolate of C. albicans and two isolates of C. tropicalis, which was in the susceptible dose-dependent range. We did not observe resistance to Voriconazole and Posaconazole, and also, their MIC₉₀ values were found to be low. This observation was similar to the findings in other studies by Pfaller et al., 2011, and Lockhart et al., 2012.^[36],[37] The MIC values of Voriconazole for rare Candida species were also found to be low in our study. Hence, Voriconazole can be used as a better drug for treatment of Fluconazole-resistant Candida species. Since urine levels of Voriconazole and Posaconazole are low, with only around 5% and 10% of the active drug excreted, its efficacy in treating Candida UTIs is quite limited.^{[38],[39],[40]} However, the information about their susceptibility pattern can be useful in treating patients with urosepsis.

Although few studies have reported resistance to Caspofungin in C. albicans, C. tropicalis and Candida glabrata,^{[41],[42],[43]} all tested isolates were susceptible to Caspofungin in our study and also their MIC values were low. Rare Candida species also had low MIC values for Caspofungin. This finding was comparable to the observation in another study by Hatice Turg Dagi et al., 2016.^[32] However, all the echinocandins are extensively metabolised, and very little active drug can be recovered in the urine.^[44] Therefore, eradication of Candida in the vascularised cortex and interstitium of the kidney by echinocandins is more likely than in the collecting system, and clinical experience is minimal.^[45] In a retrospective review of data from the Caspofungin database, this agent was found to be efficacious in three patients who had Candida pyelonephritis of ascending origin and in whom other antifungal therapy had failed.^[46]

In a study by Kumar et al., 2016, out of 54 biofilm producers, 19 were resistant to Fluconazole,^[47] whereas in our study, only 5 were resistant to Fluconazole and Amphotericin B, out of 48 biofilm producers. The less correlation between biofilm formation and antifungal resistance in our study may be due to less antifungal resistance percentage reported in our study. Although many authors have also stated that there is no statistical correlation between biofilm formation and antifungal susceptibility testing,^[1] the correlation between biofilm production from sessile cells and antifungal resistance has not been considered in these studies. Hence, it is mandatory to perform antifungal testing for sessile biofilm-producing cells for proper validation of the results. Among the rare isolates, the MIC values for Amphotericin B, Fluconazole and Itraconazole for most of the biofilm-producing isolates were found to be high. Hence, Voriconazole, Posaconazole or Caspofungin can be used to treat infections caused by such isolates.

Conclusion

The production of virulence factors (biofilm production) by Candida species signifies its pathogenicity. Infections with biofilm-producing Candida isolates resistant to antifungal agents may result in persistent infection. Hence, reducing the incidence of biofilm-related candidiasis in hospitals is a requirement in the search for optimised patient care. Furthermore, the information on the capacity of a Candida isolate to produce biofilm would help the clinician to evaluate its virulence and devise an appropriate antifungal treatment plan for the patient.

Acknowledgement

My heartfelt sincere thanks to the management for providing facilities to conduct research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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