Fluconazole-resistant Candida parapsilosis clonally related genotypes: first report proving the presence of endemic isolates harbouring the Y132F ERG11 gene substitution in Spain.

OBJECTIVES: We report here for the first time the presence of Candida parapsilosis isolates harbouring the Y132F ERG11 gene substitution in patients admitted to a Spanish hospital. METHODS: We studied the available (n = 104) C parapsilosis isolates from patients admitted to the Son Espases reference hospital in the Balearic Islands from 1 April 2019 to 30 November 2020. Isolates were sourced from 70 patients: catheter (n = 41), blood cultures (n = 37), lower respiratory tract (n = 15), intra-abdominal (n = 8), and other samples (n = 3). Isolates were genotyped and tested for antifungal susceptibilities to amphotericin B, triazoles, anidulafungin, and micafungin using EUCAST E.Def 7.3.2. The ERG11 gene was sequenced in fluconazole-resistant isolates. RESULTS: Among the 104 isolates, fluconazole and voriconazole resistance was found in 87 (84%) and 30 (29%) isolates, respectively; all isolates were fully susceptible to echinocandins and amphotericin B. All fluconazole-resistant isolates harboured the Y132F substitution in the ERG11 gene and were grouped into 11 clonally related genotypes. A genotype accounted for 70% (61/87) of fluconazole-resistant isolates. Genotypes involving the fluconazole-resistant isolates were different from those found in the remaining fluconazole-susceptible genotypes. Fifty-six patients harboured fluconazole-resistant genotypes, and 35 of the 56 had candidaemia (48%), abdominal candidiasis (17%), or other forms of candidiasis (35%). Only 20% of the study patients infected by fluconazole-resistant genotypes had a history of azole use. DISCUSSION: Fluconazole resistance in C parapsilosis isolates from patients admitted to this reference hospital is not attributable to prior azole use, but rather to the presence of a group of fluconazole-resistant C parapsilosis genotypes that have become endemic.

Whole-genome sequence-guided PCR for the rapid identification of the Pseudomonas aeruginosa ST175 high-risk clone directly from clinical samples.

OBJECTIVES: Pseudomonas aeruginosa frequently show MDR/XDR profiles, which are associated with worldwide-disseminated high-risk clones (HRCs). We developed a PCR assay for the detection in clinical samples of ST175, an HRC that is widespread in European countries. METHODS: The whole-genome sequence was obtained for one ST175 isolate using a PacBio RSII sequencer. Reads from multiple isolates belonging to ST175 and the PAO1 reference strain were mapped against the ST175 genome to identify potentially specific regions. Once curated, using the BLAST database to search for the presence of those regions in any other organism, we designed a specific PCR for the detection of ST175. RESULTS: Assembly of the ST175 PacBio-sequenced genome resulted in three contigs with a total length of 7 087 985 bases, encoding 6566 coding sequences. Specific regions for ST175 genomes were detected and a PCR targeting a 318 bp fragment located within a 3177 bp ORF coding for a putative reverse transcriptase was designed. The PCR test was first evaluated in silico against 229 XDR P. aeruginosa genomes (73 ST175) from two multicentre studies, yielding 100% sensitivity and specificity. Then, the PCR was evaluated in vitro in 25 isolates (12 ST175) and in 120 clinical samples (30 urine samples, 30 blood cultures, 30 sputum samples and 30 rectal swabs) of which 10% contained ST175, yielding again 100% sensitivity and specificity. CONCLUSIONS: The PCR assay developed, showing high sensitivity and specificity for the detection of the ST175 HRC directly from clinical samples, could become a useful tool for guiding infection control and treatment strategies in areas with a high prevalence of this clone.