Antifungal susceptibility of yeast bloodstream isolates collected during a 10-year period in Austria.

BACKGROUND: Candida-associated infections put a significant burden on western healthcare systems. Development of (multi-)resistant fungi can become untreatable and threaten especially vulnerable target groups, such as the immunocompromised. OBJECTIVES: We assessed antifungal susceptibility and explored possible influence factors of clinical Candida isolates collected from Austrian hospitals between 2007 and 2016. METHODS: Thousand three hundred and sixty clinical Candida spp. isolated from blood cultures were subjected to antifungal susceptibility testing (AFST) in a liquid-handling aided continuous microdilution assay. We tested against fluconazole, voriconazole, posaconazole, itraconazole, isavuconazole, anidulafungin, caspofungin and micafungin according to EUCAST with additional recording of growth curves. We performed rigid quality control on each assay via growth curve assessment and included two standard reference strains. Minimal inhibitory concentrations (MIC) were quantified according to EUCAST guideline E.DEF 7.3.1, and susceptibility was evaluated using EUCAST clinical breakpoints. RESULTS: The isolate collection consisted of Candida albicans (59%), C. glabrata (19%), C. parapsilosis (9%), C. tropicalis (5%) and C. krusei (3%) and few other Candida species and fungi (5%). During the observed time period, species abundance and antifungal resistance rates remained constant. Multi-resistance was rare and we found no single isolate which was resistant to both azoles and echinocandins. Within the antifungal resistance profile of our strain collection, we observed clusters along species boundaries. CONCLUSIONS: Over the last decade, the distribution of Candida species and its level of antifungal resistance remained constant in Austria. Our data compare well with other European countries. Principal component analysis of the susceptibility profile of this collection revealed species-specific clusters and substantial intra-species variation, especially for C. glabrata.

LMB-1, a novel family of class B3 MBLs from an isolate of Enterobacter cloacae.

Objectives: To identify and characterize a novel MBL gene conferring carbapenem resistance to an isolate of Enterobacter cloacae from Austria. Methods: The novel MBL gene was heterologously expressed in Escherichia coli TOP10 to conduct comparative MIC studies and biochemical assays. Furthermore, WGS was performed using Illumina MiSeq and Oxford Nanopore MinION instruments to analyse the genetic environment of the novel MBL gene. Results: The novel MBL showed highest sequence homology to a predicted MBL precursor from the marine bacterium Rheinheimera pacifica and hence belongs to Ambler subgroup B3. The comparative MIC studies and biochemical assays showed activity of the novel enzyme against penicillins, cephalosporins and carbapenems, but not against aztreonam. It was named Linz MBL (LMB-1). The blaLMB-1 gene was shown to be located on a 108 kb plasmid of Inc type IncFIB(K). Of note, a gene adjacent to blaLMB-1 coded for a glycerophosphoryl diester phosphodiesterase that was also previously detected in R. pacifica. Conclusions: Homologies of the MBL gene itself and another gene located on the same plasmid to genes detected in marine bacterial species strongly suggest that this novel MBL was transferred to E. cloacae from a marine bacterium. This underlines the importance of natural reservoirs supplying hitherto unknown resistance genes to clinically relevant bacterial species and the importance of ongoing surveillance and research.