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Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type β-lactamases are often reported as resistant to available β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non β-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-β-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the β-lactam antibiotic meropenem by four-fold. publishedVersion

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type β-lactamases are often reported as resistant to available β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non β-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-β-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the β-lactam antibiotic meropenem by four fold.

Background and aim: A multicentre nationwide surveillance study was conducted in Greek hospitals to evaluate epidemiology of carbapenemase-producing Klebsiella pneumoniae clinical isolates, and their susceptibilities to last-line antibiotics. Methods: Minimum inhibitory concentrations (MICs) were evaluated by Etest, colistin MICs were also evaluated by broth microdilution SensiTest (now known as ComASP) Colistin. Carbapenemase genes were detected by PCR. Clonal relatedness was assessed by PFGE. Isolates were prospectively collected between November 2014 and April 2016, from 15 hospitals. Results: Among 394 isolates, K. pneumoniae carbepenemase (KPC) remained the most prevalent carbapenemase (66.5%). NDM was the second most prevalent (13.7%), identified in 12 hospitals, followed by VIM (8.6%). OXA-48- and double carbapenemase-producers remained rare (3.6%, 6.3%, respectively). Carbapenemase-producing K. pneumoniae isolates showed high resistance to last-line antibiotics. Gentamicin and colistin were the most active in vitro with 61.9% and 59.6% of the isolates to be inhibited at

Purpose To evaluate the in vitro efficacy of several anti-staphylococcal agents against a nationwide collection of contemporary Staphylococcus aureus clinical isolates from several healthcare centres in Greece. Methods Thirty hospitals throughout Greece (18 in Attica) provided all clinical isolates of S.aureus from April 2012 to May 2013 to a central lab to be re-submitted to susceptibility testing. The MICs were evaluated by Vitek (R) 2 with the exception of ceftaroline (OXOID M.I.C. Evaluator). Vancomycin and daptomycin MICs were also evaluated by Etest (R). Heterogeneously vancomycin-intermediate strains (hVISA) were detected by the Etest (R) GRD. VISA phenotype was confirmed by PAP-AUC. Results A total of 1005 isolates (39% MRSA) were studied. Susceptibility rates were: erythromycin 66.5%, clindamycin 79.2%, SXT 98.9%, rifampicin 97.3%, fusidic acid 67%, moxifloxacin 78.8%, vancomycin 99.9%, ceftaroline 92.9% and linezolid, tigecycline and daptomycin 100%. For mupirocin, high level resistance could be excluded for 98.9% of isolates. Vancomycin Etest (R) MIC50/90 were 1.5/1.5mg/L, 58.5% of isolates exhibited a MIC>1 and 8.7% a MIC of 2mg/L, while Vitek (R) MIC50/90 were 1/1 and 3.1% showed MIC>1mg/L. One VISA strain was detected. Among the selected 175 isolates that were screened for hVISA phenotype, six (3.4%) were positive. In 315 bloodstream isolates, 64.1% had a vancomycin Etest (R) MIC>1mg/L. Conclusions This multi-centre surveillance study revealed that a significant percentage of contemporary S.aureus isolates from Greek patients have a vancomycin MIC (>1mg/L) that may compromise the clinical efficacy of the drug for the treatment of serious infections. The in vitro activity of SXT, rifampicin, mupirocin, linezolid, tigecycline, daptomycin and ceftaroline remains excellent.