Search

Being frequently exposed to foreign nucleic acids, bacteria and archaea have developed an ingenious adaptive defense system, called CRISPR-Cas. The system is composed of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array, together with CRISPR (cas)-associated genes. This system consists of a complex machinery that integrates fragments of foreign nucleic acids from viruses and mobile genetic elements (MGEs), into CRISPR arrays. The inserted segments (spacers) are transcribed and then used by cas proteins as guide RNAs for recognition and inactivation of the targets. Different types and families of CRISPR-Cas systems consist of distinct adaptation and effector modules with evolutionary trajectories, partially independent. The origin of the effector modules and the mechanism of spacer integration/deletion is far less clear. A review of the most recent data regarding the structure, ecology, and evolution of CRISPR-Cas systems and their role in the modulation of accessory genomes in prokaryotes is proposed in this article. The CRISPR-Cas system's impact on the physiology and ecology of prokaryotes, modulation of horizontal gene transfer events, is also discussed here. This system gained popularity after it was proposed as a tool for plant and animal embryo editing, in cancer therapy, as antimicrobial against pathogenic bacteria, and even for combating the novel coronavirus – SARS-CoV-2; thus, the newest and promising applications are reviewed as well.

The aim of this study was to investigate the extent of heavy metal (HM) pollution and its effect on microorganisms from rhizosphere soil in Baia Mare area (Maramures County, Romania). Two sites wit...

Selection and dissemination of resistant bacteria and antibiotic resistance genes (ARGs) require a deeper understanding since antibiotics are permanently released to the environment. The objective of this paper was to evaluate the phenotypic resistance of 499 isolates of Pseudomonas spp. from urban water sources, and the prevalence of 20 ARGs within those isolates. Resistance to penicillins, cephalosporins, carbapenems, quinolones, macrolides, and tetracyclines was mainly observed in the hospital effluent, municipal wastewater and river water downstream the city. Resistant strains were frequently identified as P. aeruginosa and P. putida. P. aeruginosa isolates were mostly resistant to cefepime, ceftazidime, imipenem, and gentamycin, while P. putida strains were especially resistant to piperacillin-tazobactam. ARGs such as blaTEM-1, blaSHV-1, blaPER-1, blaAmpC, blaVIM-1, PstS, qnrA, qnrB, ermB, tetA, tetB and tetC have been detected. The blaAmpC gene was found in P. aeruginosa, while blaTEM-1 and blaPER-1 genes were found in P. putida. Class 1 integron integrase gene was found in 6.81% of the Pseudomonas isolates.

In order to develop a proper conservation programme for several endangered, rare or endemic species of Dianhtus from Romania, molecular characterization by simple sequence repeat (SSR) markers has been accomplished. Amplification of SSR loci in individuals belonging to different populations of D. callizonus, D. glacialis ssp. gelidus, D. henteri, D. nardiformis and D. tenuifolius revealed 23 polymorphic alleles. D. callizonus and D. tenuifolius showed particular sets of SSR alleles. D. glacialis ssp. gelidus, D. henteri and D. nardiformis proved to share almost the same alleles in most of the loci. The highest genetic diversity was observed in D. glacialis ssp. gelidus and D. tenuifolius in locus MS-DINMADSBOX. Allelic patterns across Dianthus species indicate that the mean number of different alleles was highest in D. glacialis ssp. gelidus, while the number of effective alleles was highest in D. tenuifolius. There are no particular differences in individuals belonging to the same species. Genetic diversity is generally low, ranging from 0.18 (D. callizonus) to 0.44 (D. henteri). Regarding the genetic diversity within populations of the same species, no differences were revealed by the use of the SSR markers tested in the present study.

INTRODUCTION: Members of the family Enterobacteriaceae are commonly identified in the clinical laboratory, being responsible for a substantial range of infections. This study aimed to investigate phenotypic and genotypic resistance traits in pathogenic Enterobacteriaceae isolated from outpatients in Cluj-Napoca, Romania. METHODS: Pathogenic Enterobacteriaceae were isolated from urinary tract infections, wound infections and persistent diarrhea in a private laboratory from Cluj-Napoca, Romania. Bacterial strains were biochemically identified and subjected to antimicrobial susceptibility testing by disk diffusion. The carriage of antibiotic resistance genes and of class 1 integron were assessed by PCR. RESULTS: E. coli and Enterobacter spp. were the most prevalent pathogens. High levels of resistance were observed against folate pathway inhibitors (74%), fluoroquinolones (49%) and penicillins (44%). The incidence of carbapenem resistance was 3%. The strains displaying phenotypic resistance were able to produce β-lactamase enzymes encoded by bla(TEM), bla(TEM-1), bla(SHV-1) and bla(CTX-M), aminoglycoside modifying enzymes due to the carriage of aac(3)-IIIa, aac(6')-II and aac(6’)-Ie-aph(2”), to possess fluoroquinolones resistance due to qnrS DNA gyrase protection proteins and resistance to folate pathway inhibitors due to dihydropteroate synthases encoded by sul1, sul2 and sul3 genes. The high frequency of intI1 integrase was associated to sulphonamide resistance (r=0.48; p

Ex situ conservation of Dianthus giganteus subsp. banaticus was obtained by in vitro culture on different culture media supplemented with phytohormones. Assessment of somaclonal variability over a period of preservation by in vitro culture is required prior to introduction and acclimatization in natural habitats. In this study we used 2 types of molecular markers, ISSR and multilocus SSR markers, which allow comparison of different alleles, to assess the somaclonal variability of endemic taxa Dianthus giganteus subsp. banaticus after a period of 24 months of preservation by in vitro culture. The assessment of somaclonal variability of D. giganteus subsp. banaticus shows some changes in the absence/presence of certain markers in some individuals.

In vitro tissue culture protocols were tested for propagation of Echinacea purpurea, Echinacea pallida and Echinacea angustifolia in order to obtain biomass for the production of cichoric acid, which is the major active compound in the Echinacea extracts. The in vitro culture process was initiated by seed germination on half-strength Murashige and Skoog (MS) medium. Multiplication was achieved on MS medium supplemented with naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2-iso-pentenyladenine (2iP), and N6-benzyladenine (BA) in different concentrations. Shoot explants produced the highest number of shootlets on MS medium, which was supplemented with 0.1 mg/l 2iP and 0.1 mg/l IBA. RAPD markers revealed genetic polymorphism in some instances between in vitro generated plantlets such as for E. purpurea plantlets analyzed with the OPO-8 primer. RAPD markers generated with the primer 4A-29 revealed low levels of genetic variation between in vitro plantlets for all three species of Echinacea, while remaining RAPD markers revealed no variation. Content of cichoric acid in leaves, shoots, and callus was analyzed by high-performance liquid chromatography/MS and was identified in all studied samples, independent of species or tissue type. Highest levels (0.39–0.73 mg/g dw) were observed in shoots and leaves.

Efficient micropropagation and cryopreservation of Hypericum richeri ssp. transsilvanicum, an endemic species in Romania, and Hypericum umbellatum, a rare and endangered Daco-Balkan species, was achieved. The effects of type of explant and cytokinin on in vitro plant regeneration were investigated. Shoot organogenesis was achieved in all explants, but stem nodes regenerated best. Organogenesis from nodal segments was promoted by incubating these explants on Murashige and Skoog (MS) medium in the presence of cytokinins (6-benzyladenine, thidiazuron, kinetin or 6-γ,γ-dimethylallylaminopurine), each tested at four concentrations. The best morphogenic response for both Hypericum species (number of shoots per explant, shoot length, axillary branching of shoot, and frequency of shoot organogenesis) was observed when explants were incubated on MS medium containing 0.44 or 1.11 μM 6-benzyladenine. Root induction was achieved only when regenerated shoots were transferred to fresh medium with or without auxin. Maximum rooting was recorded on MS medium supplemented with 2.45 μM indole-3-butyric acid. Plantlets grown in vitro were successfully acclimatized in the greenhouse and showed normal development. Shoot tips and axillary buds excised from the in vitro regenerated plants were successfully cryopreserved in liquid nitrogen by the droplet-vitrification method. Following preculture in 0.25 M sucrose, dehydration and cryopreservation, the highest regeneration rates were obtained in both species by using axillary buds (68 % for H. richeri ssp. transsilvanicum and 71 % for H. umbellatum).

Nitrogen fixing microbial consortia from soil samples taken from five altitudinal vegetation zones (alpine, subalpine, coniferous, beech, Maleia flood plain) of Parâng Massif, Romania, were isolated and identified. Molecular characterisation of nitrogen fixing consortia was carried out by PCR and nested PCR with 7 primer sets specific to nifH genes. All nifH genes are specific to nitrogen fixation and are found within phylogenetically related organisms which have the nitrogenase enzyme complex. These molecular studies allowed the assessment of nifH gene diversity within these nitrogen fixing microbial consortia from different type of soils. At high altitude, a consortium of nitrogen fixing bacteria dominated by Azotobacter chroococcum and Azospirillum brasilense was found. Clostridium, Rhizobiales, Herbaspirillum, Frankia species were also found in different rations depending on the altitudinal vegetation zone.

Soilless culture as in vitro culture could provide important sources of genetic variability useful for new cultivar selection. The objective of our study was to establish the culture conditions for in vitro micropropagation of some grapevine cultivars and to identify if the somaclonal variations occur during in vitro culture. The following cultivars were studied: 'Feteasca Regala', 'Muscat Ottonel', 'Brumariu' and 'Radames'. In vitro culture was induced by shoots explants. Shoots explants regenerated plants on all media studied, but multiplication is generally low and it was obtained especially on MS media supplementd with BA and NAA. The in vitro plantlets were analysed by mean of RAPD markers in order to identify the somaclonal variations. The best RAPD patterns have been obtained with OPE-3, OPO-4, OPO-7 and OPO-8 primers. These markers showed differences between cultivars but the polymorphism of in vitro plantlets was not observed. Somaclonal variation could not be identified by these RAPD markers. In vitro multiplication was in fact a clonal propagation.

Soilless culture of valuable cultivars of grapevine as 'Feteasca Alba', 'Italian Riesling', 'Pinot Gris' and derived clones, was accomplished by culture of shoot tip explants on MS media supplemented with growth regulators. In vitro culture was induced after estimation of genetic polymorphism in their populations by RAPD markers. The amplification was accomplished by using 26 primers and the specific working programme for RAPD. Genetic polymorphism in the populations was oberved by RAPD markers obtained by DNA amplification with OPB-04, OPB-07, OPB-12, OP-17, OPF-04, OPA-13 primers. Plants showing the most polymorphic RAPD pattern were cultivated in vitro. Regenerated plants from shoot tip explants were obtained on MS media supplementd with 5.0 μM 2iP, but multiplication is low. Rooting of in vitro plantlets was obtained on MS media supplemented with 0.5 μM IBA.

Soilless cultures are nowadays an important tool for both (i) large scale synthesis of pharmaceutically active products, and for (ii) ex situ conservation of endemic and/or endangered plants that are also of horticultural importance. (i) Epilobium hirsutum explants were grown on both solid- and shaken liquid-medium, for multiplication and for greater biomass production, respectively. Secondary metabolites extracted from the biomass thus obtained were mainly polyphenols (galloylglucose and monomeric and dimeric ellagitannins), which are pharmaceutically important in prostate adenoma treatment, among others. Seedlings' dry biomass production increased from 1.5 g DW L -1 to 22 g DW L -1 after 15 days when nodes/inocula were grown on shaken liquid-medium, in light, and using different phytohormonal conditions. Secondary metabolites yield (polyphenols) of seedlings was equal to or higher (under some conditions) than for intact plants grown normally, in soil, under normal conditions. (ii) In vitro conservation on solid media resulted in a collection of 12 species that are endemic and/or endangered for Europe, and 7 species for Romania, respectively. Included in this collection are: Dianthus spiculifolius, D. nardiformis, D. henteri, D. glacialis subsp. gelidus, D. haematocalyx subsp. pindicola, D. seguieri, D. sternbergii, Achillea pyrenaica, Paradisea liliastrum. Dianthus petraeus subsp. simonkaianus was the species with the best multiplication rate (110 shoots/inoculum).

This study investigates the role of coliforms in the carriage of class 1 integron and biocide resistance genes in a drinking water treatment plant and explores the relationship between the carriage of such genes and the biofouling abilities of the strain. The high incidence of class 1 integron and biocide resistance genes (33.3 % of the isolates) highlights the inherent risk of genetic contamination posed by coliform populations during drinking water treatment. The association between the presence of intI1 gene and qac gene cassettes, especially qacH, was greater in biofilm cells. In coliforms recovered from biofilms, a higher frequency of class 1 integron elements and higher diversity of genetic patterns occurred, compared to planktonic cells. The coliform isolates under the study proved to mostly carry non-classical class 1 integrons lacking the typical qacEΔ1/sul1 genes or a complete tni module, but bearing the qacH gene. No link was found between the carriage of integron genes and the biofouling degree of the strain, neither in aerobic or in anaerobic conditions. Coliform bacteria isolated from established biofilms rather adhere in oxygen depleted environments, while the colonization ability of planktonic cells is not significantly affected by oxygen availability.

Micropropagation of Arnica montana L. using Murashige and Skoog (MS) medium supplemented with N6-[2-isopentenyl]adenine (2iP), zeatin and α-naphthaleneacetic acid (NAA) in different concentrations does not ensure the formation of a high number of regenerated plants; a maximum of 3.2 neoplantlets per explant were obtained. After 4 wk of culture on medium with zeatin (4.5 μM) and NAA (5.3 μM), plants were 3.06 cm in length. The following step was to improve the clonal propagation of this species. Micropropagation of Arnica montana L., initiated from nodal segments using semisolid media (4 g l−1 agar), was obtained. Explants were inoculated on MS medium supplemented with NAA (5.3 μM), 2iP (5.0 μM), maize extract (1.0 ml l−1), phloroglucinol (0.6 mM) or adenine sulfate (0.2 mM). Only 3 wk after the inoculation, plant multiplication as well as induction of roots were obtained, the optimal variant being that containing NAA (5.3 μM), 2iP (5.0 μM) and maize extract (1.0 ml l−1). Six weeks after the inoculation plants were transferred to Perlite, with 80% plant survival being obtained. By isoesterase pattern we concluded that we have obtained the clonal propagation of Arnica montana, because the pattern of several individuals belonging to different clones was the same. Only one region with esterase activity that is present in all individuals has been identified.

Biofilms are the predominant mode of microbial growth in drinking water systems. A dynamic exchange of individuals occurs between the attached and planktonic populations, while lateral gene transfer mediates genetic exchange in these bacterial communities. Integrons are important vectors for the spread of antimicrobial resistance. The presence of class 1 integrons (intI1, qac and sul genes) was assessed in biofilms occurring throughout the drinking water treatment process. Isolates from general and specific culture media, covering a wide range of environmental bacteria, fecal indicators and opportunistic pathogens were tested. From 96 isolates tested, 9.37% were found to possess genetic determinants of putative antimicrobial resistance, and these occurred in both Gram-positive and Gram-negative bacteria. Class 1 integron integrase gene was present in 8.33% of bacteria, all positive for the qacEΔ1 gene. The sul1 gene was present in 3.12% of total isolates, representing 37.5% of the class 1 integron positive cells. The present study shows that biofilm communities in a drinking water treatment plant are a reservoir of class 1 integrons, mainly in bacteria that may be associated with microbiological contamination. Eight out of nine integron bearing strains (88.8%) were identified based on 16S rRNA gene sequencing as either enteric bacteria or species that may be connected to animal and anthropogenic disturbance.

The aim of the present study was to examine soil samples from various vegetation zones in terms of physicochemical properties, microbial communities, and isolation and identification (by polymerase chain reaction and transmission electron microscopy) of bacteria producing poly-β-hydroxybutyrates (PHBs). Soil samples were analysed originating from zones with heterogeneous environmental conditions from the Romanian Carpathian Mountains (mountain zone with alpine meadow, karstic zone with limestone meadow, hill zone with xerophilous meadow, and flood plain zone with hygrophilic meadow). Different bacterial groups involved in the nitrogen cycle (aerobic mesophilic heterotrophs, ammonifiers, denitrifiers, nitrifiers, and free nitrogen-fixing bacteria from Azotobacter genus) were analysed. Soil biological quality was assessed by the bacterial indicator of soil quality, which varied between 4.3 and 4.7. A colony polymerase chain reaction technique was used for screening PHB producers. With different primers, specific bands were obtained in all the soil samples. Some wild types of Azotobacter species were isolated from the 4 studied sites. Biodegradable polymers of PHB were assessed by negative staining in transmission electron microscopy. The maximum PHB granules density was obtained in the strains isolated from the xerophilous meadow (10–18 granules/cell), which was the most stressful environment from all the studied sites, as the physicochemical and microbiological tests proved.