Identification and Characterization of Type II Toxin-Antitoxin Systems in the Opportunistic Pathogen Acinetobacter baumannii

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-copy-number 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.     

Identification andin silico characterisation of putative conjugative transfer genes onGeobacillus stearothermophilus plasmids

We repor the first data demonstrating the presence of putative conjugative transfer genes on plasmids of the speciesGeobacillus stearothermophilus. Partial sequence analysis of the plasmid pGS18 fromG. stearothermophilus 18 was determined. It contained eleven complete open reading frames. Five of them encoded proteins which are homologous toBacillus megaterium pBM300 Mob/TraA,Lactococcus lactis pMRC01 TrsD and TrsE,Staphylococcus aureus pGO1 TrsG andS. aureus subsp.aureus pUSA03 TraL, the proteins that are associated with conjugative plasmid transfer. Southern hybridizations were performed on two other plasmids isolated fromG. stearothermophilus 3 andG. stearothermophilus 19 strains using the most homologous parts of those five genes as probes. Data from different hybridization patterns show a close homology of putative conjugative transfer genes between pGS18 and pGS3 hypothesizing a similar molecular organization of putative conjugative plasmid transfer region of both plasmids.     

The impacts of heavy metals on oxidative stress and growth of spring barley

Oxidative stress is accepted to play a significant role in stress symptoms, caused by different stressors in a variety of organisms. In this study seedlings of spring barley (Hordeum vulgare L.) were exposed to a wide range of copper, zinc, chromium, nickel, lead and cadmium concentrations in order to determine the relationships between heavy metals-induced oxidative stress and plant growth inhibition. All investigated heavy metals induced an essential increase in lipid peroxidation and a reduction of dry biomass along with an increase in metal concentration in the nutrient solution. A very close and statistically significant exponential relationship between lipid peroxidation and growth inhibition was detected in this study. According to the results of analysis of variance (ANOVA), the intensity of nonspecific oxidative stress is identified as the main factor of barley growth inhibition, explaining 75% of total variance. Almost 10% of growth inhibition is attributed to the specific impact of heavy metals. The most pronounced increase of malondialdehyde content and growth inhibition was observed in Cu and Cd treatments, whereas the lowest changes in observed indicators were detected after exposure to Zn and Pb.     

Geostrategic Supply Risk and Economic Importance as Drivers for Implementation of Industrial Ecology Measures in a Nitrogen Fertilizer Production Company

Among other concerns, safeguarding the supply chains of raw materials is an important task for industrial companies. Therefore, not surprisingly, the number of scientific publications concerning the evaluation of resource criticality has increased in recent years. However, it was noticed that currently published methodologies are too complex to be applied by industrial companies on a daily basis. For this reason, the need to develop a methodology that would allow not only assessing resource criticality, but could also be integrated into widely applied methodological frameworks as an additional driver to improve resource efficiency was identified. Geostrategic supply risk and economic importance were chosen as key indicators to analyze and assess relative resource criticality. The developed methodology was field tested by applying it to a resource‐intensive nitrogen fertilizer production company. Five scenarios for resource efficiency improvements, consisting of cleaner production and industrial symbiosis measures, were investigated. If all the proposed measures were implemented, consumption of natural gas would decrease by 3.552 million cubic meters per year (0.3% of the total consumption). However, not all identified measures contribute to a reduction of the overall criticality of resources for the production company. Nevertheless, the integration of criticality assessments into the widely applied methodologies for development and implementation of resource efficiency innovations is a valuable addition and should be included in the analysis for sustainable innovations and development.     

Genetic and biochemical diversity of Gardnerella vaginalis strains isolated from women with bacterial vaginosis

Gardnerella vaginalis is considered a substantial player in the progression of bacterial vaginosis (BV). We analysed 17 G. vaginalis strains isolated from the genital tract of women diagnosed with BV to establish a potential link between genotypes/biotypes and the expression of virulence factors, vaginolysin (VLY) and sialidase, which are assumed to play a substantial role in the pathogenesis of BV. Amplified ribosomal DNA restriction analysis revealed two G. vaginalis genotypes. Gardnerella vaginalis isolates of genotype 2 appeared more complex than genotype 1 and were subdivided into three subtypes. Biochemical typing allowed us to distinguish four different biotypes. A great diversity of the level of VLY production among the isolates of G. vaginalis may be related to a different cytotoxicity level of the strains. We did not find any correlation between VLY production level and G. vaginalis genotype/biotype. In contrast, a link between G. vaginalis genotype and sialidase production was established. Our findings on the diversity of VLY expression level in different clinical isolates and linking sialidase activity with the genotype of G. vaginalis could help to evaluate the pathogenic potential of different G. vaginalis strains.     

Identification of <Emphasis Type="Italic">Aeromonas salmonicida</Emphasis> in European perch from North Lithuanian rivers during mass mortalities in 2008

The investigation was carried out to ascertain the cause of mass mortalities in European perch (Perca fluviatilis L.) in North Lithuanian rivers in 2008 that reached a maximum in mid-autumn at a water temperature of 6°C. Marked changes were detected in morphophysiological parameters (spleen-, gill-, liver- and heart-somatic indices), which corresponded to changes in haematological parameters (leukocyte count was significantly elevated or reduced) of the infected fish. The viable bacterial counts in the gill, liver and kidney of infected live fish samples ranged from 2.3±0.3 × 10³ to 6.3×00B1;0.4 × 10³ c.f.u./g. The BOX-PCR fingerprinting technique was used for characterization and identification of the isolated bacterial strains from liver and kidney of infected perch. The bacterial isolates were identified by sequence analysis of the 16S rRNA gene. Pathogenic Aeromonas salmonicida subsp. salmonicida has been identified as a possible causative agent of the observed mortality in European perch.     

Determination of Plasma Heparin Level Improves Identification of Systemic Mast Cell Activation Disease

Diagnosis of mast cell activation disease (MCAD), i.e. systemic mastocytosis (SM) and idiopathic systemic mast cell activation syndrome (MCAS), usually requires demonstration of increased mast cell (MC) mediator release. Since only a few MC mediators are currently established as biomarkers of MCAD, the sensitivity of plasma heparin level (pHL) as an indicator of increased MC activation was compared with that of serum tryptase, chromogranin A and urinary N-methylhistamine levels in 257 MCAD patients. Basal pHL had a sensitivity of 41% in MCAS patients and 27% in SM patients. Non-pharmacologic stimulation of MC degranulation by obstruction of venous flow for 10 minutes increased the sensitivity of pHL in MCAS patients to 59% and in SM patients to 47%. In MCAS patients tryptase, chromogranin A, and N-methylhistamine levels exhibited low sensitivities (10%, 12%, and 22%, respectively), whereas sensitivities for SM were higher (73%, 63%, and 43%, respectively). Taken together, these data suggest pHL appears more sensitive than the other mediators for detecting systemic MC activity in patients with MCAS. The simple, brief venous occlusion test appears to be a useful indicator of the presence of pathologically irritable MCs, at least in the obstructed compartment of the body.     

Acclimation effect on fish behavioural characteristics: determination of appropriate acclimation period for different species

In the present study, the authors investigated the effect of acclimation duration (up to 4 h) on behavioural characteristics of taxonomically and functionally different fish species, i.e., the migratory rheophilic salmonids rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar), and the non-migratory eurytopic European perch (Perca fluviatilis) and three-spined stickleback (Gasterosteus aculeatus). Specifically, the authors explored fish behavioural patterns based on specific endpoints (average, maximum and angular velocity) during the acclimation period, and determined the acclimation period suitable for the tested fish species. The performed behavioural data analysis showed that the minimum time needed to adjust fish activity to a more stable (baseline) level should be at least 2 h for O. mykiss and S. salar and 1 h for G. aculeatus. Nonetheless, P. fluviatilis behaviour did not show significant changes during the 4 h acclimation. The results of this study revealed that the effect of the acclimation duration on such rheophilic species as O. mykiss and S. salar was greater than that on the eurytopic species P. fluviatilis and G. aculeatus, indicating that acclimation period is important in managing fish stress before behavioural observations. For all species, the highest variability was found in the endpoint of maximum velocity, and the lowest in that of angular velocity. This study showed that before starting actual toxicity testing experiments, it is important to determine an appropriate, species-specific acclimation period.     

Identification of the genus Geobacillus using genus-specific primers, based on the 16S-23S rRNA gene internal transcribed spacer

The aim of this study was to develop an easy and accurate technique for the identification of the genus Geobacillus. For this purpose, Geobacillus genus-specific primers GEOBAC (GEOBAC-F and GEOBAC-R) based on the 16S-23S rRNA gene internal transcribed spacer (ITS) region sequences have been designed. In total, 52 sequences from three species of the genus Geobacillus (Geobacillus stearothermophilus, Geobacillus kaustophilus and Geobacillus lituanicus) were examined for the design of these primers. Analysis of the sequences revealed three highly conservative regions common to these species: 5' and 3' end regions of 16S-23S rRNA gene ITSs and box A. Some sequences possessed two additional conservative regions - genes of tRNA(Ile) and tRNA(Ala). These particular sequences were chosen for the construction of the primers. The designed primers targeted the gene of tRNA(Ile) and the 3' end region of ITSs. This technique was validated with both the reference strains of the genus Geobacillus and the thermophilic aerobic endospore-forming environmental isolates. Different Geobacillus species could be grouped according to the number and size of GEOBAC-PCR products and identified on the basis of the AluI and TaqI restriction analysis of these products.