Susceptibility evaluation and study of the resistance gene of Candida albicans isolates obtained from clinical specimens of UNICAMP Hospital and Clinics

Author(s)

Isabela Haddad Peron

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2016-03-30

Examining board members

Angélica Zaninelli Schreiber; Wanda Pereira Almeida; Celia Regina Garlipp

• Advisor: Angélica Zaninelli Schreiber

Abstract

Candida species are present in the human body microbiota and, when there is an imbalance in the relationship between microbiota and host, these yeasts can become opportunistic pathogens, being Candida albicans the most frequently found species. The systemic candidiasis presents localized infectious symptoms and is able to spread to other organs through the haematogenous route. The fluconazole antifungal inhibits the synthesis of ergosterol ¿ an essential component of the fungal cell membrane ¿ and is the most common prophylactic choice when treating candidiasis. While some species are intrinsically resistant to fluconazole (C.krusei) or are more prone to develop resistance (C.glabrata), reports on the resistance of C.albicans to fluconazole are rare. However, this species is in fact able to develop such resistance mechanisms, including the mutations of the ERG11 gene, which codifies the production of the enzyme that acts in the biosynthesis of the ergosterol. This report aimed to perform an antifungal susceptibility surveillance study on C.albicans bloodstream samples, as well as investigating mutations in the ERG11 gene on two clinical C.albicans isolates (LIF-E10 and LIF 12560) that had previously presented in vitro resistance to fluconazole. This experiment was provided with 147 samples of C.albicans isolates from hemocultures that had been conducted in the Hospital and Clinics of the State University of Campinas from 2006 to 2010, as well as the LIF-E10 and LIF 12560 isolates, obtained, respectively, from oral cavity and esophageal cavity. The bloodstream isolates had been previously identified by conventional methods and Vitek¿ 2 Systems (bioMérieux), and were reactivated through Sabouraud dextrose agar cultures, being subsequently placed in identification panels in the BD Phoenix¿ equipment. The in vitro tests to susceptibility to antifungals micafungin, amphothericin B, 5-flucytosine, fluconazole, voriconazole, itraconazole and caspofungin were performed by the broth microdilution methodology in accordance with the Clinical and Laboratory Standards Institute (CLSI) documents M27-A3 and M27-S4. The screening of the ERG11 gene mutations of the LIF-E10 and LIF 12560 isolates was obtained by amplifying three genic regions through specific PCR primers ErgSec1A/1B, ErgSec2A/2B and ErgSec3A/3B. The resulting product was submitted to both sequencing and analysis of the DNA fragments, amplified by comparing them to the standard fluconazole-sensible sample C.albicans ATCC 90028. From the 147 clinical isolates that went through reactivation, 143 were recovered, whereas four did not present any growth. From the 143 submitted to reidentification in the BD Phoenix¿ equipment, 128 (89.5%) were identified as C.albicans, and 15 (10.5%) were identified as Candida dubliniensis. Since such result presented by the BD Phoenix¿ differed from the previous identification by the Vitek¿ 2, the sequencing was necessary (primers ITS4/ITS5 and NL1/NL4), resulting in the confirmation of the 15 isolates as belonging to the C.albicans species. Regarding the in vitro susceptibility tests, all hemoculture isolates were characterized within the susceptible range (S) when facing all of the tested antifungals, corroborating with the epidemiological data that shows the dominance of the susceptible profile of the C.albicans. The nucleotide sequence analysis of the ERG11 gene of both C.albicans isolates LIF-E10 and LIF 12560 revealed 6 nucleotide mutations (E116D, T128K, E266D, A298V, G448V, and G464S to LIF 12560), all of them previously reported on the data as associated to the phenomenom of the C.albicans fluconazole resistance (AU)

• FAPESP's process: 14/08693-8 - Susceptibility and resistance gene evaluation of Candida albicans clinical isolates obtained from clinical specimen in the Hospital de Clinicas UNICAMP
• Grantee: Isabela Haddad Peron
• Support Opportunities: Scholarships in Brazil - Master