We compared six colistin susceptibility testing (ST) methods on 61 carbapenem-nonsusceptible Klebsiella pneumoniae ( n = 41) and Acinetobacter baumannii ( n = 20) clinical isolates with provisionally elevated colistin MICs by routine ST. Colistin MICs were determined by broth microdilution (BMD), BMD with 0.002% polysorbate 80 (P80) (BMD-P80), agar dilution (AD), Etest, Vitek2, and MIC test strip (MTS). BMD was used as the reference method for comparison. The EUCAST-recommended susceptible and resistant breakpoints of ≤2 and >2 μg/ml, respectively, were applied for both K. pneumoniae and A. baumannii . The proportions of colistin-resistant strains were 95.1, 77, 96.7, 57.4, 65.6, and 98.4% by BMD, BMD-P80, AD, Etest, MTS, and Vitek2, respectively. The Etest and MTS methods produced excessive rates of very major errors (VMEs) (39.3 and 31.1%, respectively), while BMD-P80 produced 18% VMEs, AD produced 3.3% VMEs, and Vitek2 produced no VMEs. Major errors (MEs) were rather limited by all tested methods. These data show that gradient diffusion methods may lead to inappropriate colistin therapy. Clinical laboratories should consider the use of automated systems, such as Vitek2, or dilution methods for colistin ST.

Trichophyton isolates with reduced susceptibility to antifungals are now increasingly reported worldwide. We therefore studied the molecular epidemiology and the in vitro antifungal susceptibility patterns of Greek Trichophyton isolates over the last 10 years with the newly released EUCAST reference method for dermatophytes. Literature was reviewed to assess the global burden of antifungal resistance in Trichophyton spp. The in vitro susceptibility of 112 Trichophyton spp. molecularly identified clinical isolates (70 T. rubrum, 24 T. mentagrophytes, 12 T. interdigitale and 6 T. tonsurans) was tested against terbinafine, itraconazole, voriconazole and amorolfine (EUCAST E.DEF 11.0). Isolates were genotyped based on the internal transcribed spacer (ITS) sequences and the target gene squalene epoxidase (SQLE) was sequenced for isolates with reduced susceptibility to terbinafine. All T. rubrum, T. interdigitale and T. tonsurans isolates were classified as wild-type (WT) to all antifungals, whereas 9/24 (37.5%) T. mentagrophytes strains displayed elevated terbinafine MICs (0.25–8 mg/L) but not to azoles and amorolfine. All T. interdigitale isolates belonged to ITS Type II, while T. mentagrophytes isolates belonged to ITS Type III* (n = 11), VIII (n = 9) and VII (n = 4). All non-WT T. mentagrophytes isolates belonged to Indian Genotype VIII and harbored Leu393Ser (n = 5) and Phe397Leu (n = 4) SQLE mutations. Terbinafine resistance rates ranged globally from 0–44% for T. rubrum and 0–76% for T. interdigitale/T. mentagrophytes with strong endemicity. High incidence (37.5%) of terbinafine non-WT T. mentagrophytes isolates (all belonging to ITS Type VIII) without cross-resistance to other antifungals was found for the first time in Greece. This finding must alarm for susceptibility testing of dermatophytes at a local scale particularly in non-responding dermatophytoses.

Two colistin-susceptible/colistin-resistant (Col ^s /Col ^r ) pairs of Acinetobacter baumannii strains assigned to international clone 2, which is prevalent worldwide, were sequentially recovered from two patients after prolonged colistin administration. Compared with the respective Col ^s isolates (Ab248 and Ab299, both having a colistin MIC of 0.5 μg/ml), both Col ^r isolates (Ab249 and Ab347, with colistin MICs of 128 and 32 μg/ml, respectively) significantly overexpressed pmrCAB genes, had single-amino-acid shifts in the PmrB protein, and exhibited significantly slower growth. The Col ^r isolate Ab347, tested by proteomic analysis in comparison with its Col ^s counterpart Ab299, underexpressed the proteins CsuA/B and C from the csu operon (which is necessary for biofilm formation). This isolate also underexpressed aconitase B and different enzymes involved in the oxidative stress response (KatE catalase, superoxide dismutase, and alkyl hydroperoxide reductase), suggesting a reduced response to reactive oxygen species (ROS) and, consequently, impaired colistin-mediated cell death through hydroxyl radical production. Col ^s isolates that were indistinguishable by macrorestriction analysis from Ab299 caused six sequential bloodstream infections, and isolates indistinguishable from Ab248 caused severe soft tissue infection, while Col ^r isolates indistinguishable from Ab347 and Ab249 were mainly colonizers. In particular, a Col ^s isolate identical to Ab299 was still invading the bloodstream 90 days after the colonization of this patient by Col ^r isolates. These observations indicate considerably lower invasiveness of A. baumannii clinical isolates following the development of colistin resistance.

Single-locus bla _OXA-51-like sequence-based typing (SBT) was evaluated for its ability to determine correctly sequence types (STs) in Acinetobacter baumannii clinical isolates, in comparison with the Pasteur's multilocus sequence typing (MLST) reference method and 3-locus sequence typing (3-LST). The comparative study was performed in 585 multidrug-resistant (MDR) A. baumannii clinical isolates recovered from 21 hospitals located throughout Greece, Italy, Lebanon, and Turkey. The isolates belonged to nine clonal complexes (CCs) that correspond to 12 distinct sequence types (STs) and to one singleton ST. These clonal lineages predominate worldwide among nosocomial MDR A. baumannii strains. The most common clone was CC2 (ST2 and ST45; n = 278 isolates) followed by CC1 (ST1 and ST20; n = 155), CC25 ( n = 65), ST78 ( n = 62), CC15 (ST15 and ST84; n = 9), CC10 ( n = 4), CC3 ( n = 4), CC6 ( n = 3), CC54 ( n = 3), and CC83 ( n = 2). Using the bla _OXA-51-like SBT method, all 585 isolates of the study were typed and assigned correctly to the nine CCs and the singleton ST78. The 3-LST method was not able to classify isolates belonging to CC6, CC10, CC54, and CC83, which are not yet characterized in its database. The low-cost and convenient bla _OXA-51-like SBT method, compared with 3-LST and MLST, discriminated all epidemic and sporadic lineages of our collection and could be effectively applied to type rapidly A. baumannii strains.

In two pairs of clinical colistin-susceptible/colistin-resistant (Cst ^s /Cst ^r ) Acinetobacter baumannii strains, the Cst ^r strains showed significantly decreased biofilm formation in static and dynamic assays ( P < 0.001) and lower relative fitness ( P < 0.05) compared with those of the Cst ^s counterparts. The whole-genome sequencing comparison of strain pairs identified a mutation converting a stop codon to lysine (*241K) in LpsB (involved in lipopolysaccharide [LPS] synthesis) in one Cst ^r strain and a frameshift mutation in CarO and the loss of a 47,969-bp element containing multiple genes associated with biofilm production in the other.