Filogenetsko istraživanje djelomičnih sekvencija gena G terenskih izolata virusa bjesnoće u Litvi.

The objective of this study was to evaluate the molecular epidemiology of rabies virus G-gene partial sequences in rabies positive field samples, isolated in different regions of Lithuania, and to compare the sequences phylogenetically with various rabies virus isolates of wild carnivores from the Baltic Sea region, Europe and Russia. Twenty brain samples of rabid animals, collected during the period 2006-2011, were investigated. Multiple alignments of 358 nt G gene (nt 3324-3682) sequences were performed using ClustalW with default settings. Phylogenetic and molecular evolutionary analyses were conducted using the Neighbor-Joining method (NJ) in MEGA version 4. The phylogenetic investigations of rabies virus glycoprotein indicate that the Lithuanian rabies virus isolates’ G-sequences were closely related and showed 89.8-98.9 % of nt identity. The G-sequences between Lithuanian raccoon dog and red fox rabies virus isolates were more conservative (97.2 % nt identity) than the rabies virus isolates inside the raccoon dog group (95.1 % nt identity). Two Lithuanian raccoon dog rabies virus isolate G-sequences were rather divergent (90.8 and 91.2 % nt identity) and were closely associated with rabies virus isolate G-sequences from Estonia (92.5 % nt identity), Russia (91.0 %) and Poland (89.3 %) respectively. The comparative investigation of G-sequences between the Lithuanian rabies virus isolates and different isolates from Germany, Slovenia and France indicated 84.2 % nt identity, whereas the G-sequences of Lithuanian rabies virus isolates and rabies virus isolate G-sequences from Russia were 88.1 % nt identical.Svrha ovog rada bila je odrediti molekularnu epidemiologiju djelomičnih sekvencija gena G virusa bjesnoće u terenskim uzorcima pozitivnima na bjesnoću iz različitih područja Litve te ih filogenetski usporediti s različitim izolatima virusa bjesnoće iz divljih mesojeda na području Baltičkog jezera, Europe i Rusije. Pretraženo je 20 uzoraka tkiva mozga bijesnih životinja prikupljenih u razdoblju od 2006. do 2011. Višestruka poravnanja sekvencija od 358 nukleotida gena G (nt 3324-3682) učinjena su pomoću programa ClustalW. Filogenetska i molekularno evolucijska analiza provedena je metodom združivanja genetski najsličnijih sojeva (engl. Neighbour-joining method) u MEGA verziji 4. Filogenetska istraživanja glikoproteina virusa naznačuju da su G-sekvencije izolata litavskoga virusa bjesnoće usko srodne i pokazuju 89,8-98,9 % nt identičnosti. G-sekvencije izolata virusa bjesnoće iz litavskog rakunskog psa i crvenih lisica bile su sačuvanije (97,2 % nt identičnosti) nego izolati virusa bjesnoće izdvojeni iz rakunskog psa (95,1 % nt identičnosti). G-sekvencije dvaju izolata iz litavskih rakunskih pasa bile su prilično divergentne (90,8 i 91,2 % nt identičnosti) i usko srodne s G-sekvencijama izolata virusa bjesnoće iz Estonije (92,5 % nt identičnosti), Rusije (91,0 %) i Poljske (89,3 %). Komparativno istraživanje G-sekvencije litavskih izolata virusa bjesnoće i različitih izolata iz Njemačke, Slovenije i Francuske pokazalo je 84,2 % nt identičnosti, dok je identičnost između G-sekvencije između litavskih izolata virusa bjesnoće i ruskih izolata bila na razini 88,1 % nt identičnosti

Microbial Diversity and Antimicrobial Resistance Profile in Microbiota From Soils of Conventional and Organic Farming Systems

Soil is one of the biggest reservoirs of microbial diversity, yet the processes that define the community dynamics are not fully understood. Apart from soil management being vital for agricultural purposes, it is also considered a favorable environment for the evolution and development of antimicrobial resistance, which is due to its high complexity and ongoing competition between the microorganisms. Different approaches to agricultural production might have specific outcomes for soil microbial community composition and antibiotic resistance phenotype. Therefore in this study we aimed to compare the soil microbiota and its resistome in conventional and organic farming systems that are continually influenced by the different treatment (inorganic fertilizers and pesticides vs. organic manure and no chemical pest management). The comparison of the soil microbial communities revealed no major differences among the main phyla of bacteria between the two farming styles with similar soil structure and pH. Only small differences between the lower taxa could be observed indicating that the soil community is stable, with minor shifts in composition being able to handle the different styles of treatment and fertilization. It is still unclear what level of intensity can change microbial composition but current conventional farming in Central Europe demonstrates acceptable level of intensity for soil bacterial communities. When the resistome of the soils was assessed by screening the total soil DNA for clinically relevant and soil-derived antibiotic resistance genes, a low variety of resistance determinants was detected (resistance to β-lactams, aminoglycosides, tetracycline, erythromycin, and rifampicin) with no clear preference for the soil farming type. The same soil samples were also used to isolate antibiotic resistant cultivable bacteria, which were predominated by highly resistant isolates of Pseudomonas, Stenotrophomonas, Sphingobacterium and Chryseobacterium genera. The resistance of these isolates was largely dependent on the efflux mechanisms, the soil Pseudomonas spp. relying mostly on RND, while Stenotrophomonas spp. and Chryseobacterium spp. on RND and ABC transporters

Identification of colistin resistance gene origin in Escherichia coli isolated from migratory bird

Introduction: As antimicrobial resistance is becoming a major threat to global health, colistin remains as one of the very few antibiotics that can still be effectively used against multi-drug resistant bacteria. However, an outbreak of plasmid-mediated colistin resistance, conferred by mcr-1 gene, was recently reported. Since the mcr-1 plasmids can be transferred between different species of bacteria, it causes concern of the emergence of bacteria resistant to all known antibiotics. Therefore, to identify and understand the mechanisms of mcr-1 spread is crucial for reducing fatalities caused by untreatable bacterial infections. Aim: To identify the origin of mcr-1 from E. coli isolated from faeces of Larus argentatus in Kaunas city dump. To the best of our knowledge, it is the first known occurrence of mcr-1 in Lithuania. Materials and Methods: To ensure that the mcr-1 is located in a plasmid, it was transferred to another strain via conjugation. PCR was performed to confirm the presence of mcr-1 and to find other antibiotic resistance genes in the plasmid. NCBI GenBank Database was screened for occurrences of mcr-1 gene using BLAST (100% identity and coverage cut-off). Potential plasmids were evaluated by restriction analysis. Results: The mcr-1 was successfully transferred into E. coli K-12 laboratory strain, proving that it is located in a plasmid. Any other antibiotic resistance genes were not found in the plasmid. 143 complete plasmid sequences from NCBI GenBank database contained identical mcr-1 sequence. By data acquired from restriction analysis of the extracted target plasmid, we can conclude, that it belongs to a group of 23 highly similar plasmids. Conclusion: The mcr-1 gene in E. coli isolated from migratory bird was found to be located in a plasmid widely spread across the world and shared between both animal vectors and human hospital patients

Functional ccreening of antibiotic resistance genes in soil Chryseobacterium spp. genomic DNA libraries

Background: Species of the genus Chryseobacterium are abundantly found in soil and water and have recently emerged as opportunistic nosocomial pathogens in humans. Due to multidrug-resistant phenotype, displayed by some species, and well documented examples of gene exchange between the environmental and the clinical strains, the more detailed picture of Chryseobacterium resistance mechanisms is needed. Therefore, functional genomic screening was applied in order to identify antibiotic resistance determinants in environmental Chryseobacterium spp. isolates. Materials and Methods: Functional screening of Chryseobacterium spp. genomic DNA library from soil isolates were carried out with clinically important antibacterial agents which include: aminoglycosides (gentamicin, kanamycin, streptomycin), beta-lactam antibiotics (imipenem, cefuroxime), tetracycline, chloramfenicol, benzalkonium chloride and ciprofloxacin. Minimum inhibitory concentrations of clones were determined and interpreted according to EUCAST breakpoints. The inserts displaying resistance phenotype were sequenced and analyzed using bioinformatic approach. Results: Selections with streptomycin, tetracycline and cefuroxime resulted in single resistant clone per antibiotic. Two resistant clones were identified using imipenem and the same resistant clone was identified in separate selections with kanamycin or gentamycin. Analysis of sequences revealed that in 4 out of 6 inserts contained genes coding directly antibiotic modifying enzymes- streptomycin adenylyltransferase, tetracycline MFS transporter, metallo-like beta-lactamase and carbapenem-hydrolyzing IND beta-lactamase. The remaining inserts did not contain obvious resistance genes. Resistance to imipenem may be determined by the GNAT family N-acetyltransferase or monooxygenase, while the lipid A biosynthesis acyltransferase may be responsible for the resistance to gentamycin and kanamycin. Conclus[...]

E. coli paplitimas mažmeninės prekybos realizuojamose kepenėlėse, jų atsparumas antimikrobinėms medžiagoms

Tyrimų tikslas – ištirti E. coli paplitimą mažmeninės prekybos realizuojamose vištų kepenėlėse, kaip viename iš populiariausių vištienos subproduktų, ir nustatyti šių bakterijų atsparumą antimikrobinėms medžiagoms. Iš skirtingų mažmeninės prekybos vietų surinkti įvairių Lietuvos gamintojų šviežių žalių vištų kepenėlių mėginiai (240 mėginių) ir atlikti E. coli bakteriologiniai tyrimai. Iš mėginių išskirtas vienas šimtas E. coli padermių (41,7 proc.) ir nustatytas jų atsparumas antimikrobinėms medžiagioms. Tyrimams taikytas mikroskiedimų metodas plokštelėse, naudojant 14 skirtingų antimikrobinių medžiagų. Rezultatai vertinti pagal ribines jautrumo reikšmes, nurodytas Europos antimikrobinio jautrumo tyrimų komiteto (EUCAST) duomenų bazėje. Nustatyta, jog dažniausiai tirtosios E. coli padermės pasižymėjo atsparumu streptomicinui (100 proc.), ampicilinui (60 proc.), nalidikso rūgščiai (50 proc.), ciprofloksacinui (47 proc.) ir tetraciklinui (45 proc.). Atsparių amikacinui padermių nenustatyta. Nustatytas retas atsparumas cefoksitinui (2 proc.), ceftiofurui (7 proc.), chloramfenikoliui (10 proc.), amoksicilino ir klavulano rūgšties kombinacijai (15 proc.). Mažiausios slopinamosios koncentracijos, didesnės nei plokštelėse esančios antimikrobinių medžiagų koncentracijos, nustatytos visoms tirtoms medžiagoms, išskyrus amikaciną. Rasta daug E. coli padermių, kurioms inaktyvuoti reikėjo didelio MSK ampicilino, nalidikso rūgšties, sulfonamidų ir tepraciklino kiekio. Tyrimų duomenys rodo potencialią riziką vartotojams (užsikrėsti atspariomis bakterijomis ar jų perduodamais atsparumo veiksniais per maisto gamybos grandinę), nes šios antimikrobinės medžiagos naudojamos tiek žmonių, tiek ir veterinarinėje medicinojeThe objective of this study was to estimate the prevalence and the antimicrobial resistance of E. coli that contaminates raw chicken liver as one of the most popular poultry sub-product sold in retail markets. Two hundred and forty samples of fresh raw chicken liver were obtained from national poultry producers in different retail marketing sites and tested for the presence of E. coli. One hundred E. coli strains (41.7%) were isolated and tested for antimicrobial susceptibility. The MICs of 14 antimicrobial agents were determined for each of the isolates using the broth microdilution method with custom-made microtitre plates. EUCAST cut-off values were used for the interpretation of susceptibility of isolated bacteria to antimicrobial agents. The most frequent resistances were demonstrated to streptomycin (100 %), ampicillin (60%), nalidixic acid (50%), ciprofloxacin (47%) and tetracycline (45%). No resistant strains were found to amikacin. Law percentage of resistant strains was recorded to cefoxitin (2%), ceftiofur (7%), chloramphenicol (10%) and amoxicillin/clavulanic acid (15%). MIC’s values above dilution ranges were found to all antimicrobials except amikacin. The highest numbers of resistant strains that demonstrated resistance to the highest concentrations of antimicrobial agents were found to ampicillin, nalidixic acid, sulphonamides and tetracycline. The data demonstrate potential risk during food preparation for consumers in the context of resistant E. coli as abovementioned antimicrobial agents are used in veterinary and human medicine as wellLietuvos sveikatos mokslų universitetasMatematikos ir statistikos katedraVytauto Didžiojo universiteta

Detection of antibiotic resistance determinants in bacteria isolated from fish

Introduction: For decades antibiotic therapy was the most efficient treatment of infectious diseases. However, microbes reacted to antimicrobial agents by developing antibiotics resistance (AR). Spreading of multidrug-resistant bacteria is a worldwide problem. The clinically relevant bacterial AR genes are constantly spreading to the environment from human and animal sources. Specific DNA elements integrons enable the spreading of AR genes between different bacterial species. Aim: The aim of this study was to examine the prevalence of genetic determinants responsible for antibiotic resistance in bacteria isolated from wild and farmed fish. Materials and methods: A total of 115 bacterial isolates from fish obtained from fish farming (95) and natural waters (20) were examined for the genes conferring resistance to clinically important antibiotics (aminoglycosides, β-lactams, fluoroquinolones, chloramphenicol, tetracyclines, macrolides, glycopeptides), biocides and for the carriage of class 1 and 2 integrons. Detection has been performed using PCR with specific primers, integron structure was accessed by DNA sequencing and bioinformatic analysis. Results: Genes conferring resistance to aminoglycosides (aph (6) Id, ant (3 ‚‘) Ib, aac (3) IIa), β-lactams including 3th generation of cephalosporins (oxa1, ctx-M), fluoroquinolones (qnrS) and biocides (qacE) were found. Bacterial isolates from fish obtained in natural water pond and river were distinguished by multiple antibiotic resistance profile, whereas bacteria isolated from fish obtained in breeding farms and supermarkets harbored genes responsible for biocide resistance. Integrons were rare and most of them carried no gene cassettes. Class 1 integron with integrated gene cassettes was found in two bacterial isolates from wild fish (Nemunas river). Integrons carried aminoglycoside adenylyltransferase aadA2 gene and dihydrofolate reductase dfrA12 gene responsible

Lietuvoje išskirtų Escherichia coli ir Salmonella enterica padermių atsparumas chinolonams

Lithuanian isolates of Escherichia coli and Salmonella enterica from animals and humans were examined for resistance to quinolones, fluoroquinolones and for resistance-associated mutations. 9% of S. enterica from animals and 4% of isolates from clinical samples of humans were resistant to nalidixic acid and susceptible to fluoroquinolones. DNA analysis of nalidixic acid-resistant S. enterica strains from animals revealed a single mutation at codon 83 (Ser→Phe) in gyrA gene, whereas resistant clinical strains contained a single gyrA mutation at codon 87 (Asp→Tyr). 10% of human isolates of E. coli were resistant to nalidixic acid and ciprofloxacin. 22% of E. coli isolates from calves were resistant to nalidixic acid. 40% and 20% of E. coli isolates from pigs were resistant to nalidixic acid and to fluoroquinolones, respectively. E. coli isolates of animal and human origin analyzed for nalidixic acid resistance-associated mutations carried single mutations at codon 83 (Ser→Leu) or at codon 87 (Asp→Tyr) in gyrA gene. Fluoroquinolone-resistant E. coli isolates from calves and humans carried multiple mutations within gyrA (83Ser→Leu, 87Asp→Gly or Asn) and parC (80Ser→Ile or Arg, 84Glu→Val or Lys) genes