Characterization of 16S rRNA methylase genes in Enterobacterales and Pseudomonas aeruginosa in Athens Metropolitan area, 2015-2016.

The aim of this study was to characterize the 16S rRNA methylase (RMT) genes in aminoglycoside-resistant Enterobacterales and Pseudomonas aeruginosa isolates in 2015-2016 in hospitals in Athens, Greece. Single-patient, Gram-negative clinical isolates resistant to both amikacin and gentamicin (n = 292) were consecutively collected during a two-year period (2015-2016) in five tertiary care hospitals in Athens. RMT genes were detected by PCR. In all RMT-producing isolates, ESBL and carbapenemase production was confirmed by PCR, and the clonal relatedness and the plasmid contents were also characterized. None of the 138 P. aeruginosa isolates harbored any of the RMT genes surveyed although some were highly resistant to aminoglycosides (MICs > = 512 mg/L). Among 154 Enterobacterales, 31 Providencia stuartii (93.9%), 42 Klebsiella pneumoniae (37.8%), six Proteus mirabilis (75%), and two Escherichia coli (100%) isolates were confirmed as highly resistant to amikacin, gentamicin, and tobramycin with MICs ≥ 512 mg/L, harboring mainly the rmtB (98.8%). All were carbapenemase producers. P. stuartii, P. mirabilis, and E. coli produced VIM-type carbapenemases. K. pneumoniae produced KPC- (n = 34, 81.0%), OXA-48 (n = 4, 9.5%), KPC- and VIM- (n = 3, 7.1%), or only VIM-type (n = 1, 2.4%) enzymes. Two groups of similar IncC plasmids were detected one harboring rmtB1, blaVEB-1, blaOXA-10, and blaTEM-1, and the other additionally blaVIM-1 and blaSHV-5. Among RMT-producing Enterobacterales, rmtB1 predominated and was associated with carbapenemase-encoding gene(s). Similar IncC plasmids carrying a multiresistant region, including ESBL genes, and in the case of VIM-producing isolates, the blaVIM-1, were responsible for this dissemination. The co-dissemination of these genes poses a public health threat.

Dissemination of International Clone II Acinetobacter baumannii Strains Coproducing OXA-23 Carbapenemase and 16S rRNA Methylase ArmA in Athens, Greece.

The aim of this study was to study the molecular epidemiology of 16S rRNA-methylase (RMT)-producing clinical Acinetobacter baumannii isolates from hospitals in Athens, Greece. Single-patient A. baumannii clinical isolates, coresistant to amikacin and gentamicin (n = 347), from five tertiary care hospitals, were submitted to minimum inhibitory concentration determination and molecular testing for carbapenemase and RMT genes. A. baumannii, resistant to amikacin and gentamicin, was isolated at participating institutions at a mean rate of 67.8%. Among them 93.7% harbored the armA. The vast majority (98.5%) of armA positive isolates were OXA-23 producers, assigned mainly (99.4%) to sequence group G1, corresponding to international clone (IC) II. Four isolates (all from the same hospital) were OXA-24 producers (1.2%), assigned to G6 corresponding to CC78 and only one isolate was OXA-58-producer, assigned to G2 (IC I). Apramycin was the most active agent inhibiting 99.7% of the isolates at ≤64 mg/L, whereas colistin, trimethoprim/sulfamethoxazole, minocycline, and tigecycline exhibited only sparse activity (S, <18%). RMT production is an emerging mechanism of resistance, capable of compromising the clinical efficacy of aminoglycosides. High prevalence of armA was observed among A. baumannii strains isolated in participating hospitals in Athens, which were mainly OXA-23 producers and belonged to IC II. Apramycin is a structurally unique aminoglycoside, currently used as a veterinary agent. Although it has not been evaluated for clinical use, apramycin appears worthy of further investigation for repurposing as a human therapeutic against difficult-to-treat pathogens.

Correction to: In vitro activity of imipenem-relebactam against non-MBL carbapenemase-producing Klebsiella pneumoniae isolated in Greek hospitals in 2015-2016.

The publisher regrets that the article has been published online on 01 March 2019 with errors in Table 1. In the originally published Table 1, the percentage of Imipenem-relebactam susceptibility was incorrectly written as 8 0, while correct data is 98.0. Also, in Meropenem row, column MIC50 (mg/L), the incorrect data 4 should be 64.

Correction to: Nationwide epidemiology of carbapenem resistant Klebsiella pneumoniae isolates from Greek hospitals, with regards to plazomicin and aminoglycoside resistance.

Following publication of the original article [1].

In vitro activity of imipenem-relebactam against non-MBL carbapenemase-producing Klebsiella pneumoniae isolated in Greek hospitals in 2015-2016.

Relebactam is a ß-lactamase inhibitor of class A and class C ß-lactamases, including carbapenemases. We evaluated the ability of relebactam to restore imipenem susceptibility against a collection of Klebsiella pneumoniae isolates from Greek hospitals. We tested 314 non-MBL carbapenemase-producing K. pneumoniae consecutive clinical strains isolated from unique patients at 18 hospitals in Greece, between November 2014 and December 2016. Susceptibility testing of imipenem, imipenem-relebactam, meropenem, doripenem, gentamicin, and colistin was performed using broth microdilution. Additionally, MICs of ceftazidime-avibactam, fosfomycin, and tigecycline were determined by MIC Test Strips. MICs were interpreted per EUCAST breakpoints. Imipenem-relebactam MICs were interpreted using the breakpoints proposed for imipenem. Carbapenemase genes were detected using PCR. Whole genome sequencing was performed for selected isolates. Imipenem-relebactam inhibited 98.0% of the KPC-producing isolates at ≤ 2 mg/L (MIC50/90, 0.25/1 mg/L) and was considerably more active than imipenem (MIC50/90, 32/> 64 mg/L). Reduced activity of imipenem-relebactam was rarely detected (2%) and was associated with chromosomal factors (ompK35 disruption and/or mutated ompK36). Only ceftazidime-avibactam showed in vitro activity comparable to imipenem-relebactam (99.6% susceptible). Relebactam provided only weak potentiation of imipenem activity against K. pneumoniae with class D OXA-48-like enzymes. Relebactam exhibited strong potential for restoring the in vitro activity of imipenem against KPC-producing K. pneumoniae, lowering the imipenem MIC50 and MIC90 from 32 to 0.25 mg/L, and from > 64 to 1 mg/L, respectively. Production of KPC carbapenemase represents the main cause of carbapenem resistance among K. pneumoniae in Greek hospitals (66.5%), and this carbapenemase appears to be very well inhibited by relebactam.

Nationwide epidemiology of carbapenem resistant Klebsiella pneumoniae isolates from Greek hospitals, with regards to plazomicin and aminoglycoside resistance.

BACKGROUND: To evaluate the in vitro activities of plazomicin and comparator aminoglycosides and elucidate the underlying aminoglycoside resistance mechanisms among carbapenemase-producing K. pneumoniae isolates collected during a nationwide surveillance study in Greek hospitals. METHODS: Three hundred single-patient carbapenemase-producing K. pneumoniae isolates were studied, including 200 KPC-, 50 NDM-, 21 VIM-, 14 KPC & VIM-, 12 OXA-48-, two NDM & OXA- and one KPC & OXA-producing isolates. Susceptibility testing was performed by broth microdilution, and minimum inhibitory concentrations (MICs) interpreted per EUCAST breakpoints. Carbapenemase-, aminoglycoside modifying enzyme- and 16S rRNA methylase- encoding genes were detected by PCR. RESULTS: Of 300 isolates tested, 5.7% were pandrug resistant and 29.3% extensively drug resistant. Plazomicin inhibited 87.0% of the isolates at ≤2 mg/L, with MIC50/MIC90 of 0.5/4 mg/L. Apramycin (a veterinary aminoglycoside) inhibited 86.7% of the isolates at ≤8 mg/L and was the second most active drug after plazomicin, followed by gentamicin (S, 43%; MIC50/MIC90, 4/> 256) and amikacin (S, 18.0%; MIC50/MIC90, 32/128). Twenty-three (7.7%) isolates (16 KPC-, 6 VIM- and one KPC & OXA-48-producers) exhibited MICs ≥64 mg/L for plazomicin, and harbored rmtB (n = 22) or armA (n = 1). AAC(6')-Іb was the most common aminoglycoside modifying enzyme (84.7%), followed by AAC(3΄)-IIa (25.3%), while those two enzymes were co-produced by 21.4% of the isolates. CONCLUSIONS: Plazomicin retains activity against most carbapenemase-producing K. pneumoniae isolated from Greek hospitals, with MICs consistently lower than those of the other aminoglycosides, even in the presence of aminoglycoside modifying enzymes. Dissemination of 16S- rRNA methylases in 8% of the isolates is an unwelcome event that needs strict infection control measures and rigorous stewardship interventions.

In vitro evaluation of double-carbapenem combinations against OXA-48-producing Klebsiella pneumoniae isolates using time-kill studies.

Purpose. The aim of this study was to evaluate the in vitro activity of double-carbapenem combinations against OXA-48-producing Klebsiella pneumoniae clinical isolates.Methodology. Double combinations of ertapenem, meropenem and imipenem were evaluated for synergy and bactericidal activity using the time-kill methodology. All antibiotics were tested at 10 mg l-1 and at a sub-inhibitory concentration of 0.5× minimum inhibitory concentration (MIC) for isolates with a carbapenem MIC≤8 mg l-1. Synergy was defined as a ≥2log10 colony-forming units (c.f.u.) ml-1 decrease of viable colonies at 24 h compared to the most active carbapenem alone.Results. Ten distinct K. pneumoniae clinical isolates were tested. All carried bla OXA-48 and bla CTX-M-15, and exhibited an MIC range of 64-128, 4-32 and 1-32 mg l-1 for ertapenem, meropenem and imipenem, respectively. Out of 48 isolate-combinations, synergy was observed in 9 (18.8 %) and cidal activity was observed in 13 (27.1 %). In vitro synergistic activity was noted for 5 out of 29 (17.2 %) ertapenem-, 6 out of 29 (20.7 %) meropenem- and 7 out of 38 (18.4 %) imipenem-containing combinations. No combination exhibited antagonism. Bactericidal activity was observed in 7 (24.1 %) ertapenem-, 8 (27.6 %) meropenem- and 11 (28.9 %) imipenem-containing combinations. Among the sub-inhibitory concentration combinations, three (15 %) ertapenem-, four (20 %) meropenem- and three (15 %) imipenem-containing ones showed synergistic interaction.Conclusion. Dual combinations of carbapenems, including those containing sub-inhibitory concentrations of antibiotics, were synergistic against multidrug-resistant (MDR) and extensively drug-resistant (XDR) K. pneumoniae isolates harbouring bla OXA-48.