Biofilm profile of candidaemia isolates

Sir,

Invasive candidiasis is the most common deep-seated fungal infection. A study done at a trauma centre, New Delhi, observed that 96 per cent of all the opportunistic mycoses were caused by Candida spp1. It is the fourth most common bloodstream isolate of nosocomial infections in the United States of America2. In India, its occurrence is 6-18 per cent, among which Candida tropicalis features as the predominant causative agent of candidaemia. In recent times, Candida auris has been in the centre of discussion as an important pathogen due to its multidrug-resistant nature and capability of causing localized outbreak in a hospital setup3.

Candida species can evade host’s defence mechanism by expressing several virulence factors including biofilm production, adhesins, hydrolytic enzymes, toxins and pseudohyphae formation4. Among these, the ability of Candida species to produce biofilm contributes a major role in its pathogenicity. Tube method and microtitre plate method are the common methods used for testing biofilm production5,6. Biofilm production by different species is variable and depends on the site of infection, geographical localization and time. Biofilm production was studied from vaginal isolates in the same institute in 2015, where Candida glabrata was the most common isolate but with only 30 per cent (7/23) biofilm positivity5. There is an increased detection of C. auris globally and also at our institution. There are limited studies on biofilm production among candidaemia isolates in India and especially biofilm production by C. auris strains; therefore, the study was undertaken to know the biofilm profile of candidaemia isolates obtained from patients admitted in a tertiary care hospital.

The present study was a cross-sectional descriptive study, which was conducted between March 2020 and July 2020 at the department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India. The Institute Ethics Committee granted waiver of consent from the study participants (JIP/IEC/2020/016). There were 70 candidaemia isolates obtained and revived during the study period. All 70 Candida isolates were included in the study, of which, 35 (50%) were C. tropicalis, 11 (15.7%) Candida albicans, nine (12.9%) Candida parapsilosis, nine (12.9%) C. auris, five (7.1%) C. glabrata and one (1.4%) Candida krusei.

Candida isolates were revived on Sabouraud dextrose agar (SDA) and biofilm production was tested by semiquantitative test tube method as described by Branchini et al6 with minor modifications. Briefly, a loopful of colony was inoculated into 10 ml of SDA broth with an additional supplement of eight per cent glucose and was incubated at 37°C for 24 h. A known biofilm producer clinical isolate of C. krusei was used as a positive control and un-inoculated SDA broth tube was used as a negative control. After incubation, the broth was decanted and the walls of the tubes were stained with 0.5 per cent safranin for seven minutes. Thereafter, the tubes were washed with distilled water. Biofilm production was visualized and interpreted based on the colour intensity, and it was graded as negative, weak positive (1+), moderate positive (2+) and strong positive (3+)6. The results were expressed in proportions or percentages.

Seventy candidaemia isolates were tested for biofilm production; 54 (77.1%) isolates of Candida spp. were biofilm producers, which is in concordance with the other similar studies7-9. All isolates of C. auris, C. parapsilosis and C. glabrata, 22 strains of C. tropicalis, 8 strains of C. albicans and a single isolate of C. krusei were biofilm producers (Table). Among the biofilm-producing isolates, 22 (31.4%) isolates were identified as weak positive (1+), 16 (22.9%) were moderate positive (2+) and 16 (22.9%) were strong positive (3+).

Majority of the strains of C. tropicalis (25/35) and C. albicans (6/11) were observed as either non-biofilm producers or weak biofilm producers in this study (Table); findings similar to the ones generated through other studies7,8. Detailed research on other pathogenic mechanisms of C. tropicalis is required for better understanding of pathogenesis as it was identified as the prevalent clinical isolate.

Even though C. auris and C. parapsilosis were isolated low in numbers than C. tropicalis and C. albicans, the maximum number of strains of C. parapsilosis (n=5), were observed as strong biofilm producers followed by C. auris (n=4). We are of the opinion that biofilm production is an important virulent factor, especially among non-albicans Candida species. While Candida strains may cause mucosal colonizations/infections irrespective of their capability of producing biofilms, we believe that candidaemia is caused by biofilm producing non-albicans and non-tropicalis Candida strains. Maximum number of strains showing strong biofilm production were among C. parapsilosis, followed by C. auris in this study. A biofilm production study was conducted on vaginal isolates earlier by the department of Microbiology, JIPMER; however, biofilm production were not observed in two (2/2) vaginal isolates of C. parapsilosis5. It again emphasizes our hypothesis that the non-biofilm producing strains of C. parapsilosis had colonized the vagina, but invasion was caused by biofilm producing strains of C. parapsilosis, although we did not study candidaemia among vaginal candidiasis cases in our previous study5.

All nine candidaemia isolates of C. auris in the present study produced biofilm. Four out of nine strains of C. auris were strong biofilm producers. This is one of the few studies, which characterizes biofilm production in C. auris strains. However, Singh et al10 reported that colonizing isolate of C. auris was more biofilm producers when compared with other candidaemia isolates, which is in contrast with our hypothesis. Recently, another study conducted in the USA identified C. auris as a biofilm producer11. Only one C. krusei was identified in our study which was a strong biofilm producer. Mohandas et al7 also identified C. krusei as a strong biofilm producer. We observed strains of C. auris, C. parapsilosis and C. glabrata as the strong biofilm producers. All these three species readily colonize skin and mucosal surfaces. They are also difficult to treat and one of the reasons is attributed to their biofilm production ability.

A few of the limitations of the present study were that it was conducted only in one tertiary care hospital, sample size was low and duration of the study was short. However, strong biofilm production was observed in C. parapsilosis and C. auris strains of candidaemia isolates, whereas C. tropicalis was the most common isolate, which was a weak biofilm producer. It indicates that other virulence factors also play a major role in the pathogenesis in this context.