2. Materials and Methods
This study was carried out in the Intensive Care Unit of the Imam Khomeini and Golestan Hospitals, Ahvaz, during January–September 2015. One hundred forty ICU patients with tracheal tubes who were intubated and mechanically ventilated were surveyed for endotracheal tube biofilms. The length of hospitalization was at least two weeks prior to sampling. Collected endotracheal tubes of patients who had clinical manifestation of pneumonia including cough, purulent respiratory secretion, fever, and new or progressive infiltration of lung in CXR were placed in sealed sterile bottles and referred immediately to the laboratory for processing. From the central region of each endotracheal tube 1 cm section was cut and processed for quantitative microbial culture.
2.1. Morphological and Molecular Identification
The specimens were inoculated onto Sabouraud Dextrose Agar (SDA, Difco) supplemented with chloramphenicol and incubated at 37°C for two days. Primarily, yeast colonies were identified by conventional tools such as colony color on CHROMagar Candida medium (CHROMagar Company, Paris, France), germ tube tests in serum at 37°C for 2-3 h, and microscopic morphology on cornmeal agar (Difco Laboratories, Detroit, Mich., USA) with 1% Tween 80. Confirmation molecular approaches were adjusted. Genomic DNA was extracted, using the method of glass bead disruption, and the PCR-RFLP method was performed as described previously [14].
2.2. Antifungal Susceptibility Testing
MICs (minimum inhibitory concentrations) of identified Candida isolates were determined according to recommendations stated in the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document [15]. Amphotericin B (Sigma, St. Louis, MO, USA), fluconazole (Pfizer, Groton, CT, USA), itraconazole (Janssen Research Foundation, Beerse, Belgium), voriconazole (Pfizer, Groton, CT, USA), and caspofungin (Merck, Whitehouse Station, NJ, USA) were obtained as reagent-grade powders from the respective manufacturers for preparation of the CLSI microdilution trays. Inoculum was prepared by gently scraping the surface of the fungal colonies with a sterile cotton swab moistened with sterile physiological saline. Conidial suspensions were adjusted to transmission of 75% to 77% at 530 nm (approximate 1 × 106–5 × 106 CFU/mL). The inoculum suspensions, including mostly nongerminated conidia, were diluted 1 : 1000 in RPMI 1640 medium and the final inoculum in assay wells was between 0.5 × 103 and 5 × 103 CFU/mL. The microdilution trays were incubated at 35°C for 24–48 h. MICs were determined visually by comparison of the growth in the wells containing the drug with the drug-free control. Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258) reference strains were for quality control.
4. Discussion
Candida is opportunistic pathogen which causes a life-threatening infection with high rates of mortality especially in immunocompromised individuals [16]. The pathogenicity of Candida species is attributed to certain virulence factors, mostly production of biofilm [17, 18]. Candida species are now recognized as major agents of hospital-acquired infection. Almost invariably, an implanted device such as an urinary catheter or endotracheal tube is associated with these infections [18]. Candida species can cause significant problems of medical settings as persistent and recurrent device related infections [19, 20]. This properties also differed among different species of Candida [17, 21]. In this study C. albicans (35.7%) was the most common species obtained from endotracheal tube, compatible with other studies that mentioned that C. albicans is considered as major etiologic agent in candidiasis [16, 17, 20]. Other studies reported that the ability of biofilm production in C. parapsilosis and C. glabrata was significantly less than C. albicans [17, 21]. Biofilm phenotype in non-C. albicans species is the cause of the survival and well adapted to colonization of tissues and indwelling devices [20]. This difference in our results probably is due to variety of biofilms formation among Candida species from different sources. Mahmoudabadi et al. showed a higher percentage C. albicans (41.7%) which have recovered from blood samples in comparison with other sources [21]. In our investigation, other species of Candida included C. glabrata (25.2%), C. parapsilosis (16.8%), C. tropicalis (12.6%), and C. krusei (9.4%) obtained from endotracheal tubes. These data are in agreement with the findings of a previous study [22–25]. Also Shokohi et al., Richter et al., and Papon et al. mentioned C. glabrata as the most common non-C. albicans species in their investigation [24–27]. Deorukhkar et al.’s study indicated C. tropicalis (29.4%) as the major non-C. albicans species isolate followed by C. glabrata (20.7%) that is incompatible with these studies [28, 29]. The obtained antifungal susceptibility patterns indicated that C. albicans isolates were highly susceptible to caspofungin (100%) (MIC ≤ 2 μg/mL). These findings are in agreements with other studies that reported [30, 31]. In this investigation 26.4% of C. albicans strains were resistant to fluconazole (MIC ≥ 64 μg/mL), whereas other studies reported the rates of this resistance as 45.83%, 11.9%, 74.2%, 2.7%, and 38.7%, respectively [27, 32–35]. Studies by Shokohi et al., Al-Mamari et al., Aher et al., Awari, and Roy et al. indicated the resistance of C. albicans to itraconazole as 5.4%, 10.3%, 36.9%, 35%, and 19.3%, respectively [26, 32, 36, 37]. However, in our finding 35.2% of C. albicans strains were shown to be resistant to itraconazole MIC ≥ 1 μg/mL. In our study 14.7% of C. albicans isolates were resistant to voriconazole (MIC ≥ 1 μg/mL) that was different from results of Zang et al. and Badiee and Alborzi’s studies [23, 38]. MIC range (0.016–≥16 μg/mL) and MIC90 (8 μg/mL) for voriconazole in present study were different from results by Zhang et al. and Badiee and Alborzi which reported MIC range and MIC90 as 0.0313–4 μg/mL and 0.25 μg/mL and 0.003–16 μg/mL and 4 μg/mL, respectively [23, 38]. In addition, 17.6% of C. albicans isolates in our study were indicated to be resistant to amphotericin B MIC ≥ 2 μg/mL. The result was to some extent similar to the result reported by of Aher et al. (13.8%) and differs from results by Njunda et al. (54.4%), Awari et al. (7.5%), and Zhang et al. (1.1%) [26, 33, 35, 37–39]. MIC range (0.062–2 μg/mL) and MIC90 (2 μg/mL) for amphotericin B in present study differ from the data reported by Bosco-Borgeat et al. which reported MIC Range and MIC90 as 0.13–1 μg/mL and 0.5 μg/mL, respectively [39]. Our data indicated that 79.1% of C. glabrata were resistance to itraconazole. These data are in disagreement with the rate of itraconazole resistance C. glabrata in studies by Shokohi et al. (12.5%), Haddadi et al. (21%), and Deorukhkar et al. (46.2%) [27, 29, 40]. Also Aher reported 46.4% and 40% resistance to fluconazole and itraconazole, respectively; these mentioned rates differ from the results of our investigation [26]. Our study has shown that fluconazole MIC90 values (8, 64, and 2 μg/mL), itraconazole (16, 16, and 0.5 μg/mL), and voriconazole (8, 16, and 0.25) against C. albicans, C. glabrata, and C. parapsilosis, respectively, according to study by Badiee and Alborzi in regard to fluconazole rates are lower (16, 128, and 4) and itraconazole and voriconazole have higher amount (2, 16, and 0.25 and 4, 3, and 0.033), respectively [23]. In fact, MIC90 in C. glabrata as the main non-Candida albicans to fluconazole, itraconazole, and voriconazole was higher than C. albicans and C. parapsilosis. MIC90 fluconazole, itraconazole, and voriconazole for C. glabrata in our investigation were 64, 16, and 16. Long term fluconazole and itraconazole prophylaxis were accompanied with reduction in sensitivity to these agents and recently C. glabrata known as naturally less susceptible to azoles compared to other Candida species [35, 41, 42]. Our study has shown that amphotericin B and caspofungin MIC90 values were 2, 2, and 0.5 μg/mL and 0.5, 0.75, and 0.5 μg/mL against C. albicans, C. glabrata, and C. parapsilosis, respectively. However in other studies MIC90 values of amphotericin B were lower (0.25, 0.5, and 0.25 μg/mL) [23]. In present study 87.5%, 37.5%, and 75% of C. parapsilosis strains were susceptible to fluconazole, itraconazole, and voriconazole, respectively. This result is similar to that by Shokohi et al. who found no resistance species among them. Badiee et al. obtained 6.9% and 3.5% resistance to fluconazole and itraconazole, respectively. In addition they find no voriconazole resistance C. parapsilosis among them. In addition Zhang et al.’s findings show 15.4% resistance to fluconazole; however there was no resistance to itraconazol and voriconazole [23, 27, 38]. In our study we find that 41.6%, 66.6%, and 41.6% of C. tropicalis were resistant to fluconazole, itraconazole, and voriconazole, respectively. In contrast, Shokohi et al. did not find resistance species in their study. Also Zhang et al. and Aher et al. obtained 10.7% and 4.8% and 52% and 56% of isolates which were resistant to fluconazole and itraconazole, respectively [26, 27, 38]. However C. tropicalis isolates were highly susceptible to caspofungin and amphotericin B (100%, 83.3%). Therefore these antifungals seem to be the most active drug for candidiasis treatment. In our study MIC90 of fluconazole, itraconazole, and voriconazole for C. parapsilosis were 2, 0.5, and 0.25 μg/mL whereas Zhang et al., Tay et al., and Bonfietti et al. obtained 2, 0.062, and 0.25 μg/mL, 4, 0.19, and 0.047 μg/mL, 2, 0.06, and 0.03 μg/mL, respectively, as MIC90 to mention antifungal drugs [38, 43, 44]. Our results indicated that C. parapsilosis isolates from endotracheal tube were highly susceptible to caspofungin and amphotericin B; this data also shows that the concentration of 0.5 μg/mL of this medicine is able to inhibit 90% of mentioned isolates.