An update on polymyxin susceptibility testing methods for Acinetobacter baumannii.

Introduction: Carbapenem-resistant Acinetobacter baumannii (CRAB) currently comprises >50% of A. baumannii isolates in Europe and the USA and commonly are extensively or pandrug resistant. They cause severe hospital infections, which according to CDC have very high mortality. Polymyxins are universally recognized as the last-line choice against CRAB infections. The role of polymyxin susceptibility testing (ST) for these pathogens remains a subject of debate, although it is critical for selection of appropriate antimicrobial therapy. There are significant discrepancies between in vitro ST methods and important shortcomings for diffusion and automated methods have been repeatedly verified. The joint CLSI-EUCAST Working Group has recently recommended broth microdilution (BMD) without additives as the reference method for polymyxin ST. Areas covered: This review, focused on this threatening pathogen, summarizes the current available ST methods for polymyxins and discusses the challenges encountered by clinical microbiological laboratories in obtaining and interpreting valid susceptibility results. Studies on polymyxin ST methods for Acinetobacter spp., retrieved from Pubmed database until May 2019, were evaluated. Expert opinion: Currently, no single commercial ST method for polymyxins is providing fully accurate results. Further optimization and standardization of the ST methods and international harmonization of guidelines should be achieved to facilitate the accurate detection of polymyxin-resistant A. baumannii.

Dissemination of linezolid-dependent, linezolid-resistant Staphylococcus epidermidis clinical isolates belonging to CC5 in German hospitals.

Objectives: Linezolid-resistant Staphylococcus epidermidis (LRSE) and linezolid-dependent ST22 strains have been shown to predominate in tertiary care facilities all over Greece. We report herein the dissemination of ST22 but also ST2, ST5 and ST168 linezolid-dependent LRSE clones in four unrelated German hospitals. Methods: Fourteen LRSE clinical isolates recovered during 2012-14 from five distantly located German hospitals were tested by for MIC determination broth microdilution and Etest, PCR/sequencing for cfr and for mutations in 23S rRNA, rplC, rplD and rplV genes, MLST, PFGE and growth curves without and with linezolid at 16 and 32 mg/L. Results: Most (11, 78.6%) isolates had linezolid MICs >256 mg/L. Five isolates carried the cfr gene. Eight isolates belonged to ST22, two isolates each to ST168 and ST2 and one isolate each to ST5 and ST23. Ten isolates [seven belonging to ST22 and one to each of ST2, ST5 and ST168; all these STs belong to clonal complex (CC) 5] exhibited linezolid-dependent growth, growing significantly faster in linezolid-containing broth. Four isolates were non-dependent (one belonging to each of ST22, ST2, ST23 and ST168). Four isolates came from three different hospitals, whereas four and six isolates were recovered during outbreaks of LRSE in two distinct hospitals. Conclusions: 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.

Evaluation of two automated systems for colistin susceptibility testing of carbapenem-resistant Acinetobacter baumannii clinical isolates.

Background: Colistin is commonly needed for the treatment of infections due to carbapenem-resistant Acinetobacter baumannii (CRAB) and the determination of its in vitro activity is obviously important. However, the accurate routine antimicrobial susceptibility testing (AST) of colistin is still challenging. The only acceptable method for colistin AST is broth microdilution (BMD); disc and gradient diffusion assays are inappropriate and the performance of semi-automated systems has not been validated. Objectives: In the present study, two commonly used semi-automated systems were evaluated for colistin AST of contemporary CRAB clinical isolates. Methods: A total of 117 single-patient CRAB isolates collected randomly during 2015 from distinct tertiary hospitals located throughout Greece were tested. Colistin MICs were determined using the semi-automated systems Phoenix100 and Vitek2 and also agar dilution (AD), compared with the reference BMD. Results: Colistin resistance rates for Phoenix100/Vitek2/AD/BMD were 15.4%/16.2%/35.9%/24.8%. The essential/categorical agreement rates were as follows: Phoenix100, 91.5%/88.9%; Vitek2, 88.9%/89.7%; and AD, 93.2%/87.2%. Alarmingly high rates of very major errors (VMEs) were observed for Phoenix100 (41.4%) and Vitek2 (37.9%), while major errors (MEs) were limited (1.1% by both systems); VMEs were much more common for isolates with MICs of 2 mg/L than for isolates with MICs of ≤ 1 mg/L, as determined by automated methods. AD produced considerably higher colistin MICs, yielding MEs of 15.9%. Conclusions: 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.

Predominance of international clone 2 OXA-23-producing-Acinetobacter baumannii clinical isolates in Greece, 2015: results of a nationwide study.

In a previous nationwide study in Greece, OXA-58 was the sole carbapenemase present among carbapenem-resistant Acinetobacter baumannii (CRAB) isolated between 2000 and 2009. In this study, the antibiotic resistances, carbapenemase gene content and clonal relatedness of 194 single-patient CRAB clinical isolates collected randomly during 2015 from 11 tertiary hospitals located throughout Greece were investigated. Antimicrobial susceptibility was determined using commercial and dilution methods. PCR assays for carbapenemase genes were performed. Clonality was tested by a scheme based on two multiplex PCRs and single-locus blaOXA-51-like sequence-based typing. Furthermore, Pasteur's multilocus sequence typing (MLST) scheme and pulsed-field gel electrophoresis (PFGE) were applied to 31 selected representative isolates. The most active antibiotics were trimethoprim/sulfamethoxazole (SXT) (34.6% of isolates susceptible), minocycline (71.6%), colistin (72.7%) and tigecycline (MIC50/90 values, 1/2 mg/L). The blaOXA-23-like gene was identified in 188 isolates (96.9%), blaOXA-23-like together with blaOXA-58-like in 3 isolates (1.5%), blaOXA-58-like in 2 isolates (1.0%) and blaOXA-40-like in 1 isolate (0.5%). ISAba1 was found upstream of the blaOXA-23-like gene in all isolates. International clone (IC) 2 comprised 157 isolates (80.9%), IC1 comprised 36 isolates (18.6%) and ST78 comprised 1 isolate (0.5%). All IC2 and IC1 isolates tested by MLST were ST2 and ST1, respectively. Seven PFGE types were detected. IC2 isolates were resistant to more antibiotics than IC1, except for SXT. This nationwide study showed that CRAB isolates in Greek hospitals currently produce almost uniformly the OXA-23 carbapenemase and belong mainly to IC2 and, to a lesser extent, IC1. Of particular concern, colistin susceptibility is recently severely reduced.

Colistin-Resistant Acinetobacter baumannii Clinical Strains with Deficient Biofilm Formation.

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.

Comparative Evaluation of Colistin Susceptibility Testing Methods among Carbapenem-Nonsusceptible Klebsiella pneumoniae and Acinetobacter baumannii Clinical Isolates.

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.

Single-locus-sequence-based typing of blaOXA-51-like genes for rapid assignment of Acinetobacter baumannii clinical isolates to international clonal lineages.

Single-locus blaOXA-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 blaOXA-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 blaOXA-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.

Growth retardation, reduced invasiveness, and impaired colistin-mediated cell death associated with colistin resistance development in Acinetobacter baumannii.

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.

The indoleamine 2,3-dioxygenase inhibitor 1-methyl-tryptophan suppresses mitochondrial function, induces aerobic glycolysis and decreases interleukin-10 production in human lymphocytes.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. It is known that IDO induces T-cell differentiation to regulatory T-cells (Treg) through tryptophan depletion and/or kynurenine pathway products. CD4+ effector T-cells require distinct metabolic programs in order to support their function as compared to Treg cells. Furthermore, glucose metabolism is also known to affect B-cell survival and function. The effect of IDO on glucose metabolism of lymphocytes was evaluated by using its inhibitor 1-methyl-DL-tryptophan (1-MT). METHODS: Ten healthy volunteers vaccinated against tetanus. Peripheral blood mononuclear cells (PBMC) were cultured with or without tetanus toxoid and/or 1-MT. Cell proliferation was assessed by optical microscopy, glucose uptake by measuring its concentration in the supernatant, aerobic glycolysis by assessing lactate concentration in the supernatant, mitochondrial function by XTT assay, and finally production of Tregs' signature cytokine IL-10 by means of ELISA. RESULTS: Primarily, IDO decreases glucose uptake by stimulated lymphocytes. Secondly, IDO increases mitochondrial function in stimulated lymphocytes. In addition, IDO decreases aerobic glycolysis in stimulated lymphocytes. Finally, IDO induces the production of the immunosuppressive cytokine IL-10 by stimulated lymphocytes. CONCLUSION: Considering that cell metabolism plays a significant role in lymphocyte differentiation and function, IDO may exert its immunomodulatory effect by interfering with cell metabolism.