Evaluation of Exoenzyme Activities, Biofilm Formation, and Co-hemolytic Effect in Clinical Isolates of Candida parapsilosis Species Complex.

Candida parapsilosis species complex is considered as important emerging pathogens and little is known about their pathogenicity factors and co-hemolytic activity with different bacteria species. The aim of this study was to determine in vitro exoenzyme activities, biofilm formation, and co-hemolytic effect of different bacteria species on clinical C. parapsilosis complex isolates. In total, 67 C. parapsilosis complex isolates consist of C. parapsilosis sensu stricto 63/67 and Candida orthopsilosis 4/67 were used in this study. To determine the hemolytic activity of these species, Sabouraud dextrose sheep blood agar was used. Evaluation of the CAMP-like phenomenon carried out in the presence of Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, and Streptococcus agalactiae. Tube test method with ethylenediaminetetraacetic acid-rabbit plasma was used to determine coagulase activity, and biofilm formation was assessed by the tube method in assist of Sabouraud glucose broth (8%) medium. Fisher's exact tests were used for data statistical analysis. Sixty-six of 67 (98.5%) and 3/67 (4.5%) of the species showed hemolysin and coagulase activity, respectively. Fifty-five of 67 (82.1%) of species had ability for biofilm formation, and none of the samples exhibited co-hemolytic effect in the presence of four mentioned bacteria. No significant difference was found between the level of enzyme production and biofilm formation among the isolates.

INTRODUCTION

Candida species have been recognized as normal flora of human skin. Although Candida albicans is the most frequently isolated fungal species from human infections, Candida parapsilosis has become the second or third most common cause of fungal infections in human.[1] Nowadays, these species have been reclassified as a C. parapsilosis species complex comprising three distinct species: C. parapsilosis, Candida orthopsilosis, and Candida metapsilosis, using different molecular methods.[2] Several factors, such as adherence, persistence, germ tube formation, phenotypic switching, interference with host defense systems, synergism with bacteria, and the production of hydrolases, have been proposed to be Candida spp. virulence factors.[3] The ability of C. albicans to form biofilms and adhere to host tissues is important pathogenesis factor. Biofilm formation can act as a reservoir of agents, allow coinfection with other pathogens, promote the persistence of infection, and increase mortality.[4] The role of exoenzymes as a virulence factor of C. albicans has been intensively investigated, but its contribution to the virulence of C. parapsilosis species complex remains uncertain.[56] The co-hemolytic effect which was named the “CAMP reaction,” first described by Christie, Atkins, and Munch-Peterson in 1944. The cooperative (CAMP-like) lytic processes are the result of the interaction of at least two membrane-active agents of bacteria, with biological membranes. Streptococcus agalactiae (B group streptococci) produces a thermostable, extracellular, diffusible protein that acts synergistically with the Staphylococcus aureus b-lysine to produce a zone of enhanced lysis in ovine and bovine erythrocyte cultures.[7]

According to our knowledge, there are no data about co-hemolytic effect of bacteria species which are commonly part of normal skin flora or any association in mucocutaneous area with infected lesions caused by Candida infections. The aim of this study was to evaluate hemolysin and coagulase activities, biofilm formation, and determination of co-hemolytic effect (CAMP-like factor) of C. parapsilosis species complex with standard strain of S. aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, and S. agalactiae.

MICROBIOLOGY REPORT

A subset of 67 clinical isolates including 63 C. parapsilosis sensu stricto and four C. orthopsilosis were included in this study. Hemolytic was evaluated with a sheep blood (7% v/v and 3% w/v glucose) plate assay[8] The presence of a distinctive translucent halo around the inoculum site indicated positive hemolytic activity. Coagulase activity was evaluated with standard tube test method using ethylenediaminetetraacetic acid-rabbit plasma.[9] The presence of a clot formation in tube tests expresses as positive coagulase test. Biofilm formation was evaluated by the tube method, visual detection (Sabouraud dextrose broth 8%).[10] The tubes were examined for the presence of an adherent visible film at the bottom of the tubes and the results were expressed as negative–, weak+, moderate++, and strong+++. Standard strains of bacteria including S. aureus (ATCC25923), S. saprophyticus (PTCC1440), S. epidermidis (PTCC1435), and S. agalactiae (PTCC1768) were used for the evaluation of co-hemolytic effects. After 2 days of incubation for assessing the hemolytic activity, a loop was used to streak each bacterium in straight lines across the plate at a distance of 10 mm from the edge of the border of yeast colony. A distinct arrowhead of hemolysis at the intersection of the tester strain and the Candida colony streaks considered as a positive result for CAMP-like reactions.[7] Chi-square and Fisher's exact tests were used for data statistical analysis. Out of 67clinical isolates, 66/67 (98.5%) presented hemolysin activity. Beta-hemolysin activity was detected in 52/67 (78.78%) isolates. Coagulase activity was detected in 3/4 (4.5%) isolates which all belongs to C. parapsilosis sensu stricto [Table 1]. There was no co-hemolytic effect between positive hemolytic isolates in association with bacteria species [Table 1 and Figure 1]. A total of 55 (82.1%) of the 67 Candida strains were positive for biofilm production [Figure 2].

Table 1

Distribution profile of enzymatic activity, biofilm production and CAMP-like effect on clinical species of C. parapsilosis complex

Figure 1

Hemolysin and co-hemolytic (CAMP-like) effect. (A), C. parapsilosis complex with Staphylococcus aureus (CAMP negative). (B -), C. albicans as control with Staphylococcus aureus (CAMP positive , a zone of half-moon shape hemolysis)

Figure 2

Biofilm formation: (a) Negative (control), (b) Positive (weak+), (c) Positive (mediate++), and (d) positive (strong+++)

CONCLUSION

In general, our study reveals that secretion of hemolysin and biofilm-forming ability are two major factors that play role in pathogenesis of C. parapsilosis species complex and none of the bacteria species did used in this study had any role in cooperative (CAMP-like) hemolytic activities with these strains.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.