Acinetobacter baumannii is an opportunistic pathogen that causes nosocomial infections. Due to the ability to persist in the clinical environment and rapidly acquire antibiotic resistance, multidrug-resistant A. baumannii clones have spread in medical units in many countries in the last decade. The molecular basis of the emergence and spread of the successful multidrug-resistant A. baumannii clones is not understood. Bacterial toxin-antitoxin (TA) systems are abundant genetic loci harbored in low-copynumber plasmids and chromosomes and have been proposed to fulfill numerous functions, from plasmid stabilization to regulation of growth and death under stress conditions. In this study, we have performed a thorough bioinformatic search for type II TA systems in genomes of A. baumannii strains and estimated at least 15 possible TA gene pairs, 5 of which have been shown to be functional TA systems. Three of them were orthologs of bacterial and archaeal RelB/RelE, HicA/HicB, and HigB/HigA systems, and others were the unique SplT/SplA and CheT/CheA TA modules. The toxins of all five TA systems, when expressed in Escherichia coli, inhibited translation, causing RNA degradation. The HigB/HigA and SplT/SplA TA pairs of plasmid origin were highly prevalent in clinical multidrug-resistant A. baumannii isolates from Lithuanian hospitals belonging to the international clonal lineages known as European clone I (ECI) and ECII.

Bacterial toxin-antitoxin (TA) systems are operons that code for a stable toxic protein and a labile antitoxin. TA modules are widespread on the chromosomes of free-living Bacteria and Archaea, where they presumably act as stress response elements. The chromosome of Escherichia coli K-12 encodes four known TA pairs, as well as the dinJ-yafQ operon, which is hypothesized to be a TA module based on operon organization similar to known TA genes. Induction of YafQ inhibited cell growth, but its toxicity was counteracted by coexpression of dinJ cloned on a separate plasmid. YafQ(His)(6) and DinJ proteins coeluted in Ni(2+)-affinity and gel filtration chromatography, implying the formation of a specific and stable YafQ-DinJ protein complex with an estimated molecular mass of c. 37.3 kDa. Induction of YafQ reduced protein synthesis up to 40% as judged by incorporation of [(35)S]-methionine, but did not influence the rates of DNA and RNA synthesis. Structure modelling of E. coli YafQ revealed its structural relationship with bacterial toxins of known structure suggesting that it might act as a sequence-specific mRNA endoribonuclease.

The aim of this research was to assess interactions between metals at low exposure concentrations (Maximum-Permissible-Concentrations accepted for the inland waters in EU) and to assess possible influence of background exposure (10-times reduced concentration of a single metal) on toxicological significance of selected biomarkers in Salmo salar after treatment with metal mixture (Zn - 0.1, Cu - 0.01, Ni - 0.01, Cr - 0.01, Pb - 0.005 and Cd - 0.005 mg/L). The tissue-specific bioaccumulation, genotoxicity and cytotoxicity responses (erythrocytic nuclear abnormalities assay) in peripheral blood, kidneys, gills and liver erythrocytes of fish to metal mixtures were assessed after 14 days treatment. Treatment with primary mixture (MIX) or two variants of this mixture (Cr↓ (10 times reduced Cr concentration) and Cu↓ (10 times reduced Cu concentration)) induced the strongest responses in genotoxicity and cytotoxicity endpoints. Exposure to these mixtures highly affected Zn, Cu and Cd bioaccumulation in liver tissue. The highest amount of Ni accumulated was measured after Cd↓ treatment in all tissues. Treatments with reduced concentration of non-essential metal resulted in an increased accumulation of Pb, Ni, or Cd; treatments with reduced concentration of essential metal resulted in a reduced accumulation of certain metals (especially Cd and Pb) in tissues compared between treatments. Glucose content in blood and behavioural endpoints were evaluated after short-term exposure to metal mixtures (MIX, Cr↓, Cu↓). Significant increase in blood glucose concentration was measured after all treatments. These metal mixtures elicit significant behavioural alterations in fish. Consequently, this research revealed a significant influence of background exposure considering mixture toxicity.

BackgroundWe have identified a novel class 1 integron (1503 bp), named In671 in a clinical Pseudomonas aeruginosa isolate. Integron sequence analysis revealed two gene cassettes, one coding for a new OXA-type β-lactamase designated as OXA-205 and the other coding for the aadB gene that is responsible for aminoglycoside resistance. The 266 amino acid sequence of OXA-205 revealed that this β-lactamase belongs to the Ambler class D showing highest sequence homology to the OXA-2 sub-lineage. Our objective was to purify and characterize β-lactamase OXA-205.MethodsEscherichia coli cells were transformed with a plasmid containing cloned blaOXA-205 gene from P. aeruginosa. Purification of overproduced OXA-205 consisted of a single ion-exchange chromatography step. SDS-PAGE and isoelectric focusing were performed to determine the molecular mass and pI, respectively. Size-exclusion chromatography was undertaken to determine the OXA-205 oligomerization state. Substrate hydrolysis reactions were employed to assess enzyme kinetic parameters.ResultsPurification of OXA-205 yielded the enzyme with >95 % purity (as verified by SDS-PAGE). Approximate yield of the protein was estimated to be 20 mg per liter of culture. OXA-205 had a pI at 8.1, molecular mass of 26 kDa and a monomeric native structure. Kinetic analysis revealed that OXA-205 hydrolyzed narrow spectrum substrates, including ampicillin, carbenicillin, oxacillin, penicillin G, cefazolin and cefuroxime. Additionally, we observed a substrate inhibition profile towards carbenicillin and oxacillin, but not with ampicillin or penicillin G. Our results also show that OXA-205 conferred unusually high (among class D β-lactamases) resistance towards inhibition by NaCl.ConclusionsOXA-205 can be considered a narrow spectrum monomeric β-lactamase that demonstrates unusually high resistance profile towards inhibition by NaCl.Electronic supplementary materialThe online version of this article (doi:10.1186/s12941-015-0113-1) contains supplementary material, which is available to authorized users.

Background and objective. The ongoing search for the enhancement of efficacy of photodynamic therapy stimulates the interest in molecular mechanisms of the response to the treatment. Looking for the cell line suitable for investigation of cellular response both in vivo and in vitro, we evaluated phototoxicity of m-tetrakis-(3-hydroxyphenyl)-chlorin (mTHPC) on viability of Lewis lung carcinoma (LLC1) cells in vitro, growth of murine transplantable tumor, and mice survival.Material and methods. LLC1 cell culture and male C57BL/6 mice bearing Lewis lung carcinoma were used for the experiments. Photodynamic treatment was mediated by m-tetrakis-(3hydroxyphenyl)-chlorin as a photosensitizer. Light emitting diode array was used for illumination. The effect of the photodynamic treatment was evaluated by comparison of viability of control and treated cells, growth of tumors, and survival of the control and treated mice.Results. In vitro, a cytotoxic dose inducing a reduction in viability of LLC1 cells by 50% was achieved at 60 mJ/cm 2 and approximately 400 ng/mL of the photosensitizer, or 30 mJ/cm 2 and 600 ng/mL of mTHPC. Both the concentration of the photosensitizer and duration of light exposure were significant determinants of cytotoxic effect. In vivo, an injection of 0.25 mg/kg of mTHPC to mice bearing Lewis lung tumor and illumination at 120 J/cm 2 taking place after 24 h significantly inhibited tumor growth and prolonged mice survival. However, the tumors regained their growth potential after 9 days.Conclusions. Photodynamic treatment mediated by m-tetrakis-(3-hydroxyphenyl)-chlorin had a significant effect on LLC1 cells in vitro and growth of Lewis lung carcinoma in vivo.