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The Identification of Meyerozyma Guilliermondii from Blood Cultures

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The Identification of Meyerozyma Guilliermondii from Blood Cultures + Models MYCMED-716; No. of Pages 8 Journal de Mycologie Médicale (2017) xxx, xxx—xxx Available online at ScienceDirect www.sciencedirect.com ORIGINAL ARTICLE/ARTICLE ORIGINAL The identification of Meyerozyma guilliermondii from blood cultures and surveillance samples in a university hospital in Northeast Turkey: A ten-year survey a, b b N. Cebeci Güler *, ˙I. Tosun , F. Aydin a Department of Medical Microbiology, Faculty of Medicine, Giresun University, Giresun, Turkey b Department of Medical Microbiology, Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey Received 8 March 2017; accepted 17 July 2017 KEYWORDS Summary Meyerozyma (Pichia) guilliermondii exists in human skin and mucosal surface Meyerozyma microflora. It can cause severe fungal infections like candidemia, which is an opportunistic guilliermondii; pathogen. One hundred and forty-one M. Guilliermondii isolates, consisting of 122 blood culture Candida isolates, belonging to 126 patients; 13 total parenteral nutrition solution isolates; and two rectal membranifaciens; swab isolates were identified according to carbohydrate assimilation reactions in a university Blood culture; hospital in Turkey between January 2006 and December 2015. Following Candida albicans ITS; (34.0%) and C. Parapsilosis (21.2%), the third yeast species most commonly isolated from blood IGSAF; cultures in the Farabi Hospital was M. guilliermondii (20.6%). The patients were hospitalised in Antifungal MIC 27 different departments. A total of 50% of the patients were in pediatric departments, 49.2% were in intensive care units, and 17.2% were in haematology-oncology departments. Molecular identification of the isolates was performed using DNA sequence analysis of ribosomal ITS gene regions and IGS amplification-AluI fingerprinting (IGSAF). With molecular identification, 140 isolates were identified as M. guilliermondii and one isolate was identified as Candida mem- branifaciens. It was observed that the ITS1 region specifically helps in identifying these species. It was demonstrated that biochemical and molecular methods were 99.3% consistent in identifying M. guilliermondii. The Wild-Type (WT) Minimum Inhibitory Concentrations (MICs) distribution of fluconazole, voriconazole, itraconazole, and flucytosine were determined using * Corresponding author. E-mail address: [email protected] (N. Cebeci Güler). http://dx.doi.org/10.1016/j.mycmed.2017.07.007 1156-5233/# 2017 Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Cebeci N, et al. The identification of meyerozyma guilliermondii from blood cultures and surveillance samples in a university hospital in Northeast Turkey: A ten-year survey. Journal De Mycologie Médicale (2017), http://dx.doi.org/10.1016/ j.mycmed.2017.07.007 + Models MYCMED-716; No. of Pages 8 2 N. Cebeci Güler et al. the Sensititre YeastOne YO2V system after 24 h of incubation. One M. guilliermondii strain was determined to be non-WT for fluconazole, voriconazole, itraconazole and flucytosine. In total, three M. guilliermondii strains, for fluconazole, were determined to be non-WT in this study. # 2017 Elsevier Masson SAS. All rights reserved. Introduction Nevertheless, antimycotic sensitivities of this fungus have not been clarified. Isolates identified as Candida guilliermondii (teleomorph In the Farabi Hospital, the M. guilliermondii has been the Pichia guilliermondii) were included in the new Meyerozyma third yeast species most frequently isolated from blood genus by Kurtzman and Suzuki in 2010 [1]. The cultures in the past 10 years. The objective of the present M. guilliermondii complex is a genetically heterogeneous study was to assess whether the biochemical identification complex comprising several phenotypically indistinguishable was correct using nucleotide sequencing and Restriction taxa, including M. guilliermondii, Candida fermentati, Can- Fragment Length Polymorphism Analysis of PCR-Amplified dida carpophila, and Candida xestobii [2—5]. However, the Fragments (PCR-RFLP) and to determine in vitro sensitivity of species Candida famata (teleomorph Debaryomyces hanse- M. guilliermondii to fluconazole, voriconazole, itraconazole nii) and M. guilliermondii are extremely difficult to diffe- and flucytosine. rentiate phenotypically. They have a phylogenetically close relationship [6,7]. Materials and Methods M. guilliermondii is widely distributed in nature and is a common constituent of the normal human microflora [8] and is reported to be responsible for severe fungal infections Yeast strains such as candidemia, a human opportunistic pathogen. M. guilliermondii is defined as a newly emerging, rare This retrospective study evaluates a ten-year-period bet- pathogen that is responsible for a small percentage of all ween January 2006 and December 2015 at Farabi Hospital, candidemia. Moreover, in the past 20 years, it was observed which is a university hospital in Trabzon, Turkey. The that this pathogen was responsible for 1—11.7% of all candi- M. guilliermondii isolate, which was first isolated from the demia, with increasing incidence [9—11]. This species is a blood cultures of each patient, was included in the study. more common cause of candidemia in cancer patients than it M. guilliermondii strains isolated at intervals of 4 weeks, is in general hospital populations [11—14]. M. guilliermondii from successive specimens of the same patient, were consi- fungemia may occur in children with underlying conditions dered as different candidemia episodes. One isolate repre- other than cancer [15,16]. Besides sporadic candidemia senting each episode was included in the study. One hundred cases, candidemia outbreaks (real or pseudo) caused by and forty-one M. guilliermondii isolates were identified from the species were seen [17—19]. Candidemias are infections blood cultures (126 isolates) of 122 patients. Surveillance with high mortality [20]. Furthermore, this species is espe- specimens consisted of total parenteral nutrition (TPN) solu- cially notable for its greater propensity to express multidrug tions (13 isolates) and rectal swabs (two isolates). Most of the resistance than other organisms of the genus Candida [21]. isolates (87.23%) belong to times of outbreak (Fig. 1). Figure 1 Distribution of M. guilliermondii isolates isolated from blood cultures and surveillance specimens in the university hospital in Northeast Turkey. Please cite this article in press as: Cebeci N, et al. The identification of meyerozyma guilliermondii from blood cultures and surveillance samples in a university hospital in Northeast Turkey: A ten-year survey. Journal De Mycologie Médicale (2017), http://dx.doi.org/10.1016/ j.mycmed.2017.07.007 + Models MYCMED-716; No. of Pages 8 Meyerozyma guilliermondii in the blood cultures and surveillance samples 3 Biochemical identification final extension step at 72 8C for 7 min. A negative control was performed with each run by replacing the template DNA with Blood cultures were processed in the Clinical Microbiology sterile water in the PCR mixture. All amplicons were purified 1 laboratory by an automated blood culture system (Bactec, using the NucleoSpin Extract II (Macherey-Nagel, Ger- BD, Paramus, NJ, USA). Yeasts were initially observed on many). The DNA fragments were sequenced using a Genetic Gram-stained preparations, subcultured on Sabouraud Dex- Analyzer 3130 automated DNA sequencer (Applied Biosys- 1 trose Agar (SDA; Oxoid, England). Surveillance samples were tems, USA) with a BigDye Terminator v3.1 Cycle Sequencing directly inoculated to SDA. Germ tube-positive strains were Kit (Applied Biosystems). All amplicons were sequenced identified as C. albicans and C. dubliniensis. The other using reverse primer ITS4 for the whole ITS region. ITS1, strains were identified using carbohydrate assimilation reac- ITS2, ITS3 and ITS4 primers were placed on the whole ITS DNA tions (API 20C AUX, bioMérieux, Marcy l’Etoile, France). sequence of the study isolates in silico. The sequence bet- ween ITS1 and ITS4 primers was evaluated as the whole ITS region sequence, the sequence between ITS1 and ITS2 pri- DNA preparation mers was evaluated as the ITS1 region sequence, and the sequence between ITS3 and ITS4 primers was evaluated as Meyerozyma guilliermondii isolates were subcultured on SDA the ITS2 region sequence. Species were identified by search- and incubated at 28 8C for 24 to 48 h. Colonies were suspen- ing databases using the Basic Local Alignment Search Tool ded in saline to obtain a turbidity of a 0.5 McFarland standard (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/). A at a 530 nm wavelength. One millilitre of the cell suspension sequence identity of > 99% was used for species identifica- was centrifuged at 5,000 g for 3 min in a microcentrifuge. The tion. Pairwise comparisons and multiple sequence align- genomic DNA was extracted by following isolation of nucleic ments were also performed with CLUSTAL W2 (http:// acids from bacteria or yeast protocol, using a High Pure PCR www.ebi.ac.uk/Tools/msa/clustalo/). Template Preparation Kit (Roche, Germany) in accordance with the manufacturer’s instructions. The extracted DNA was stored at À20 8C until later use. Nucleotide sequence accession numbers GenBank accession numbers of whole ITS sequences of study Amplification and sequencing of the ITS region isolates are given in Table 1. Amplification of the whole internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was performed with fungus- Amplification and AluI fingerprinting of the IGS specific universal ITS1 (5"-TCCGTAGGTGAACCTGCGG-3")
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