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 g/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.T he increasing occurrence of infections due to multidrug-resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacteriaceae led to the revival of old and neglected antibiotics that may remain active, such as polymyxins (polymyxin B and colistin) (1, 2). Colistin is increasingly being used as a last-resort treatment option for infections caused by MDR organisms (2, 3), particularly carbapenem-resistant (CR) Gramnegative bacteria (4). However, during the last years, increasing colistin resistance emerged worldwide, especially among Klebsiella pneumoniae and A. baumannii isolates, further limiting treatment options (5-7). In Europe, the evolving colistin resistance is more pronounced in southern countries (notably Greece, Romania, and Italy) (8-10).Rapid and reliable colistin susceptibility testing (ST) is needed in routine clinical laboratories to allow appropriate therapeutic decision-making. Thus far, few studies have assessed the performance of colistin ST methods, displaying controversial results, and thus, the most accurate one is still challenging (11).Disk diffusion, commonly used in many clinical laboratories, yielded high error rates compared to MIC-based methods and is considered unreliable for the detection of colistin resistance (12)(13)(14). Among commercial methods, gradient diffusion strips are convenient tests for determining colistin MICs, but their performance is not well established. Some studies demonstrated very good correlations between the results of Etest (bioMérieux, Marcy l'Etoile, France) and broth microdilution (BMD) or agar dilution (AD) methods for colistin , while other reports questioned the reliability of Etest (18,19). Another gradient diffusion test, MIC test strip (MTS) (Liofilchem SRL, Italy), has not been evaluated for colistin ST to the best of our k...

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 g/ml), both Col r isolates (Ab249 and Ab347, with colistin MICs of 128 and 32 g/ml, respectively) significantly overexpressed pmrCAB genes, had single-aminoacid 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.

c 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.

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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. Colistin-resistant (Cst r ) Acinetobacter baumannii clinical isolates are increasingly recovered worldwide (1), which is causing major clinical concerns. Thus far, two primary colistin resistance mechanisms have been described in A. baumannii, (i) modification of the lipid A moiety of lipopolysaccharide (LPS) mediated by mutations and/or overexpression in the two-component regulatory system pmrAB and (ii) loss of LPS caused by either mutations or insertional inactivation of lipid A biosynthesis genes (2).The development of colistin resistance in clinical and laboratory-derived Cst r A. baumannii due to changes in the PmrAB system has been correlated with impaired fitness and virulence (3, 4) and lower infectivity (5, 6). Also, reduced biofilm formation has been observed in laboratory-generated Cst r A. baumannii (7,8). We previously reported the characteristics of two pairs of colistin-susceptible (Cst s )/Cst r A. baumannii clinical strains (Ab248/ Ab249 and Ab299/Ab347; colistin MICs of 0.5/128 and 0.5/32 g/ml, respectively) sequentially obtained from two patients after prolonged colistin exposure (6). Briefly, compared to Cst s strains, Cst r strains harbored a single pmrB mutation (P233S for Ab249 and P170L for Ab347) and had significantly slower growth. Strains within each pair had identical pulsed-field gel electrophoresis (PFGE) profiles, and all were assigned to the international clone 2. Important differences in the antibiotic resistance phenotypes other than colistin were not observed (6). One Cst r strain underexpressed the CsuA/B and CsuC proteins, which are involved in biofilm formation (6). In the present follow-up study, we compared these strain pairs in terms of fitness, biofilm-forming ability, and whole-genome sequencing (WGS) focusing specifically on genes involved in virulence and biofilm formation.In vitro competition assays were performed in triplicate at 5 h and 20 h (4), and relative fitness was calculated as previously described (9). To study adhesion (6 h) and biofilm formation (24 h) under static conditions, the crystal violet method was used (10) with some modifications. Briefly, inoculum was prepared by adjusting exponential cultures grown in Luria-Bertani (LB) broth to a 0.5 McFarland standard, and this was followed by a 1:10 dilution in LB broth. Then, 200-l aliquots (approximately 2 ϫ 10 6 CFU) were loaded into a 96-well polystyrene microplate (...

Colistin resistance of A. baumannii is greatly underestimated by Phoenix100/Vitek2, potentially leading to inappropriate colistin administration. Colistin AST results by automated systems within the susceptible range, particularly those at the susceptibility breakpoint (2 mg/L), need to be validated by BMD.

The rapid LRSE dissemination in Greek hospitals threatens linezolid activity. The observation that most LRSE belonged to ST22 and expressed dependence on linezolid clearly implies that the spread of linezolid resistance should have been driven by this trait, which provided the LRSE with a selective advantage under linezolid pressure.

The multi-clonal dissemination of CC5 linezolid-dependent LRSE throughout German hospitals along with the clonal expansion of ST22 linezolid-dependent LRSE in Greek hospitals is of particular concern. It is plausible that this characteristic is inherent and provides a selective advantage to CC5 LRSE under linezolid pressure, contributing to their dissemination throughout hospitals in these countries.