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.

In vitro activity of ceftolozane/tazobactam alone and in combination with amikacin against MDR/XDR Pseudomonas aeruginosa isolates from Greece.

OBJECTIVES: We evaluated the in vitro activity of ceftolozane/tazobactam and comparator agents against MDR non-MBL Pseudomonas aeruginosa isolates collected from nine Greek hospitals and we assessed the potential synergistic interaction between ceftolozane/tazobactam and amikacin. METHODS: A total of 160 non-MBL P. aeruginosa isolates collected in 2016 were tested for susceptibility to ceftolozane/tazobactam and seven comparator agents including ceftazidime/avibactam. Time-kill assays were performed for synergy testing using ceftolozane/tazobactam 60 or 7.5 mg/L, corresponding to the peak and trough concentrations of a 1.5 g q8h dose, respectively, in combination with 69 mg/L amikacin, corresponding to the free peak plasma concentration. Synergy was defined as a ≥2 log10 cfu/mL reduction compared with the most active agent. RESULTS: Overall, ceftolozane/tazobactam inhibited 64.4% of the P. aeruginosa strains at ≤4 mg/L. Colistin was the most active agent (MIC50/90, 0.5/2 mg/L; 96.3% susceptible) followed by ceftazidime/avibactam (MIC50/90, 4/16 mg/L; 80.6% susceptible). GES-type enzymes were predominantly responsible for ceftolozane/tazobactam resistance; 81.6% of the non-producers were susceptible. MICs for the P. aeruginosa isolates selected for synergy testing were 2-32 mg/L ceftolozane/tazobactam and 2-128 mg/L amikacin. The combination of ceftolozane/tazobactam with amikacin was synergistic against 85.0% of all the isolates tested and against 75.0% of the GES producers. No antagonistic interactions were observed. CONCLUSIONS: Ceftolozane/tazobactam demonstrated good in vitro activity against MDR/XDR P. aeruginosa clinical isolates, including strains with co-resistance to other antipseudomonal drugs. In combination with amikacin, a synergistic interaction at 24 h was observed against 85.0% of P. aeruginosa strains tested, including isolates with ceftolozane/tazobactam MICs of 32 mg/L or GES producers.

Pore-water nutrient concentrations variability under different oxygen regimes: A case study in Elefsis Bay, Greece.

Anthropogenic pressures considerably affect coastal areas, increasing nitrogen and phosphorous loads that lead to eutrophication. Eutrophication sometimes results in hypoxic and/or anoxic conditions near the bottom water. Dissolved oxygen (DO) concentrations influence redox-sensitive nutrients, which can alter the benthic flux of nutrients. We retrieved sediment cores from two sites in the eastern and western parts of Elefsis Bay, a semi-enclosed area of the Eastern Mediterranean, Greece, during winter and summer. In the western part, seasonally hypoxic or anoxic conditions occurred. We analysed pore-water samples under normoxic, hypoxic and anoxic bottom water conditions to study the pore-water nutrient concentrations variability under different oxygen regimes. Ex situ incubation experiments were conducted at the site experiencing oxygen deficiency by manipulating the DO concentrations. The pore-water nutrient concentrations showed higher variability at the site experiencing oxygen deficiency. Notably, elevated ammonium concentrations were observed in the pore water during anoxic conditions, in the 2-20-cm sediment layer. However, the benthic fluxes of ammonium and phosphate at the 0-2-cm sediment layer were comparable under hypoxic and anoxic conditions. The results of the incubation experiments demonstrate a direct decrease in nitrate concentrations as the DO concentrations diminished in the overlying water. The incubations after re-oxygenating the overlying water show that phosphate was more efficiently scavenged when anoxic conditions prevailed in the bottom water. The incubation experiments indicate the rapid response of the seafloor to oxygen availability, particularly concerning processes that influence nitrate and phosphate concentrations. These observations highlight the dynamic nature of nutrient cycling in shallow, seasonally anoxic environments, such as Elefsis Bay, and emphasise the sensitivity of the seafloor ecosystem to changes in bottom water oxygen availability.

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.