Phthalic acid isomers are the monomers of phthalate molecules, also known as phthalic acid esters, widely employed in the plastics industry. This study aims to investigate the biodegradation of phthalic acid (PA) and terephthalic acid (TPA) by five industry-borne Comamonas testosteroni strains: 3APTOL, 3ABBK, 2B, 3A1, and C8. To assess the ability of C. testosteroni strains to biodegrade phthalic acid isomers in fermentation media, an analytical method was employed, consisting of high-performance liquid chromatography (HPLC) analyses. Subsequently, molecular screening of the genomic and plasmid DNA was conducted to identify the degradative genes responsible for the breakdown of these chemicals. The genes of interest, including ophA2, tphA2, tphA3, pmdA, and pmdB, were screened by real-time PCR. The five C. testosteroni strains effectively degraded 100% of 100 mg/L PA (p = 0.033) and TPA (p = 0.0114). Molecular analyses indicated that all C. testosteroni strains contained the pertinent genes at different levels within their genomes and plasmids, as reflected in the threshold cycle (Ct) values. Additionally, DNA temperature of melting (Tm) analyses uncovered minor differences between groups of genes in genomic and plasmid DNA. C. testosteroni strains could be excellent candidates for the removal of phthalic acid isomers from environmental systems.
- Klíčová slova
- Comamonas testosteroni, Biodegradation, Gene detection, Phthalic acid isomers, Real-time PCR,
- MeSH
- biodegradace MeSH
- Comamonas testosteroni * genetika metabolismus MeSH
- fermentace MeSH
- kyseliny ftalové * metabolismus MeSH
- plazmidy genetika MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- kyseliny ftalové * MeSH
- phthalic acid MeSH Prohlížeč
- terephthalic acid MeSH Prohlížeč
A laser-plasma source emitting photons with energies in the water window spectral range has been used to reveal the radiation chemical yields of single-strand breaks in plasmid DNA as a function of ·OH radical scavenger concentration. Direct and indirect effects were investigated separately using DNA samples with various levels of hydration. We experimentally determined the value of the efficiency factor for strand cleavage in DNA caused by the reaction with ·OH radicals at 0.11, which was previously found in the theoretical studies. Additionally, the radiation chemical yield of ·OH radicals specific to the water window radiation emission of the source was determined by comparison with the gamma radiation-induced strand break yields. The ·OH radical yield determined using the plasmid DNA samples as a model was similar to the yield found using sensitive fluorescent dosimeters in previous experiments.
- Klíčová slova
- Gamma radiation, OH radical, Plasmid DNA, Radical scavengers, Soft X-rays,
- MeSH
- DNA * chemie MeSH
- hydroxylový radikál * chemie MeSH
- jednořetězcové zlomy DNA MeSH
- plazmidy chemie MeSH
- rentgenové záření MeSH
- scavengery volných radikálů chemie MeSH
- voda * chemie MeSH
- záření gama MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA * MeSH
- hydroxylový radikál * MeSH
- scavengery volných radikálů MeSH
- voda * MeSH
BACKGROUND: Antibiotic resistance stands as a critical medical concern, notably evident in commonly prescribed beta-lactam antibiotics. The imperative need for expeditious and precise early detection methods underscores their role in facilitating timely intervention, curbing the propagation of antibiotic resistance, and enhancing patient outcomes. RESULTS: This study introduces the utilization of surface-enhanced Raman spectroscopy (SERS) in tandem with machine learning (ML) for the sensitive detection of characteristic gene fragments responsible for antibiotic resistance appearance and spreading. To make the detection procedure close to the real case, we used bacterial plasmids as starting biological objects, containing or not the characteristic gene fragment (up to 1:10 ratio), encoding beta-lactam antibiotics resistance. The plasmids were subjected to enzymatic digestion and without preliminary purification or isolation the created fragments were captured by functional SERS substrates. Based on subsequent SERS measurements, a database was created for the training and validation of ML. Method validation was performed using separately measured spectra, which did not overlap with the database used for ML training. To check the efficiency of recognising the target fragment, control experiments involved bacterial plasmids containing different resistance genes, the use of inappropriate enzymes, or the absence of plasmid. SIGNIFICANCE: SERS-ML allowed express detection of bacterial plasmids containing a characteristic gene fragment up to the 10-7 concentration of the initial plasmid, despite the complex composition of the biological sample, including the presence of interfering plasmids. Our approach offers a promising alternative to existing methods for monitoring antibiotic-resistant bacteria, characterized by its simplicity, low detection limit, and the potential for rapid and straightforward analysis.
- Klíčová slova
- Bacterial plasmid, Beta-lactam antibiotics resistance, Detection, Logistic regression, Machine learning, SERS,
- MeSH
- antibakteriální látky farmakologie MeSH
- beta-laktamová antibiotika MeSH
- beta-laktamová rezistence genetika MeSH
- beta-laktamy farmakologie MeSH
- Escherichia coli genetika účinky léků izolace a purifikace MeSH
- plazmidy * genetika MeSH
- povrchové vlastnosti MeSH
- Ramanova spektroskopie * metody MeSH
- strojové učení * MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
- beta-laktamová antibiotika MeSH
- beta-laktamy MeSH
Complete plastid loss seems to be very rare among secondarily non-photosynthetic eukaryotes. Leukarachnion sp. PRA-24, an amoeboid colourless protist related to the photosynthetic algal class Synchromophyceae (Ochrophyta), is a candidate for such a case based on a previous investigation by transmission electron microscopy. Here, we characterize this organism in further detail and describe it as Leucomyxa plasmidifera gen. et sp. nov., additionally demonstrating it is the first known representative of a broader clade of non-photosynthetic ochrophytes. We recovered its complete plastid genome, exhibiting a reduced gene set similar to plastomes of other non-photosynthetic ochrophytes, yet being even more extreme in sequence divergence. Identification of components of the plastid protein import machinery in the L. plasmidifera transcriptome assembly corroborated that the organism possesses a cryptic plastid organelle. According to our bioinformatic reconstruction, the plastid contains a unique combination of biosynthetic pathways producing haem, a folate precursor and tocotrienols. As another twist to its organellar biology, L. plasmidifera turned out to contain an unusual long insertion in its mitogenome related to a newly discovered mitochondrial plasmid exhibiting unprecedented features in terms of its size and coding capacity. Combined, our work uncovered further striking outcomes of the evolutionary course of semiautonomous organelles in protists.
- Klíčová slova
- Leukarachnion, mitochondrial plasmids, non-photosynthetic plastid, plastid evolution, plastid genome, stramenopiles,
- MeSH
- fylogeneze * MeSH
- genom mitochondriální MeSH
- genom plastidový * MeSH
- mitochondrie genetika metabolismus MeSH
- molekulární evoluce MeSH
- plastidy * genetika metabolismus MeSH
- plazmidy * genetika MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Wastewaters are considered as important players in the spread of antimicrobial resistance, thus affecting the health of humans and animals. Here, we focused on wastewaters as a possible source of carbapenemase-producing Enterobacterales for the environment. METHODS: A total of 180 presumptive coliforms from hospital and municipal wastewaters, and a river in the Czech Republic were obtained by selective cultivation on meropenem-supplemented media and tested for presence of carbapenemase-encoding genes by PCR. Strains carrying genes of interest were characterized by testing antimicrobial susceptibility, carbapenemase production and combination of short- and long- read whole-genome sequencing. The phylogenetic tree including publicly available genomes of Enterobacter asburiae was conducted using Prokka, Roary and RAxML. RESULTS: Three VIM-producing Enterobacter asburiae isolates, members of the Enterobacter cloacae complex, were detected from hospital and municipal wastewaters, and the river. The blaVIM-1 gene was located within a class 1 integron that was carried by different F-type plasmids and one non-typeable plasmid. Furthermore, one of the isolates carried plasmid-borne colistin-resistance gene mcr-10, while in another isolate chromosomally located mcr-9 without colistin resistance phenotype was detected. In addition, the analysis of 685 publicly available E. asburiae genomes showed they frequently carry carbapenemase genes, highlighting the importance of this species in the emergence of resistance to last-line antibiotics. CONCLUSION: Our findings pointed out the important contribution of hospital and community wastewaters in transmission of multi-drug resistant pathogens.
- Klíčová slova
- mcr, Antimicrobial resistance, Carbapenemase, Environment,
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální léková rezistence genetika MeSH
- bakteriální proteiny genetika MeSH
- beta-laktamasy * genetika MeSH
- Enterobacter * genetika účinky léků izolace a purifikace MeSH
- fylogeneze MeSH
- kolistin * farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- odpadní voda * mikrobiologie MeSH
- plazmidy genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antibakteriální látky MeSH
- bakteriální proteiny MeSH
- beta-laktamasy * MeSH
- carbapenemase MeSH Prohlížeč
- kolistin * MeSH
- odpadní voda * MeSH
- VIM-1 metallo-beta-lactamase MeSH Prohlížeč
This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.
- Klíčová slova
- Dishevelled, DNA cloning, In vitro DNA assembly, Mutagenesis, PCR, Plasmid-based cloning, Site-directed mutagenesis,
- MeSH
- DNA genetika MeSH
- klonování DNA * metody MeSH
- mutageneze cílená * metody MeSH
- plazmidy * genetika MeSH
- polymerázová řetězová reakce metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- DNA MeSH
UNLABELLED: Lyme disease, caused by spirochetes in the Borrelia burgdorferi sensu lato clade within the Borrelia genus, is transmitted by Ixodes ticks and is currently the most prevalent and rapidly expanding tick-borne disease in Europe and North America. We report complete genome sequences of 47 isolates that encompass all established species in this clade while highlighting the diversity of the widespread human pathogenic species B. burgdorferi. A similar set of plasmids has been maintained throughout Borrelia divergence, indicating that they are a key adaptive feature of this genus. Phylogenetic reconstruction of all sequenced Borrelia genomes revealed the original divergence of Eurasian and North American lineages and subsequent dispersals that introduced B. garinii, B. bavariensis, B. lusitaniae, B. valaisiana, and B. afzelii from East Asia to Europe and B. burgdorferi and B. finlandensis from North America to Europe. Molecular phylogenies of the universally present core replicons (chromosome and cp26 and lp54 plasmids) are highly consistent, revealing a strong clonal structure. Nonetheless, numerous inconsistencies between the genome and gene phylogenies indicate species dispersal, genetic exchanges, and rapid sequence evolution at plasmid-borne loci, including key host-interacting lipoprotein genes. While localized recombination occurs uniformly on the main chromosome at a rate comparable to mutation, lipoprotein-encoding loci are recombination hotspots on the plasmids, suggesting adaptive maintenance of recombinant alleles at loci directly interacting with the host. We conclude that within- and between-species recombination facilitates adaptive sequence evolution of host-interacting lipoprotein loci and contributes to human virulence despite a genome-wide clonal structure of its natural populations. IMPORTANCE: Lyme disease (also called Lyme borreliosis in Europe), a condition caused by spirochete bacteria of the genus Borrelia, transmitted by hard-bodied Ixodes ticks, is currently the most prevalent and rapidly expanding tick-borne disease in the United States and Europe. Borrelia interspecies and intraspecies genome comparisons of Lyme disease-related bacteria are essential to reconstruct their evolutionary origins, track epidemiological spread, identify molecular mechanisms of human pathogenicity, and design molecular and ecological approaches to disease prevention, diagnosis, and treatment. These Lyme disease-associated bacteria harbor complex genomes that encode many genes that do not have homologs in other organisms and are distributed across multiple linear and circular plasmids. The functional significance of most of the plasmid-borne genes and the multipartite genome organization itself remains unknown. Here we sequenced, assembled, and analyzed whole genomes of 47 Borrelia isolates from around the world, including multiple isolates of the human pathogenic species. Our analysis elucidates the evolutionary origins, historical migration, and sources of genomic variability of these clinically important pathogens. We have developed web-based software tools (BorreliaBase.org) to facilitate dissemination and continued comparative analysis of Borrelia genomes to identify determinants of human pathogenicity.
- Klíčová slova
- Borrelia burgdorferi, Lyme disease, evolution, genome diversification, plasmids, recombination,
- MeSH
- Borrelia burgdorferi komplex genetika klasifikace MeSH
- Borrelia burgdorferi genetika klasifikace MeSH
- Borrelia genetika klasifikace MeSH
- fylogeneze * MeSH
- genetická variace MeSH
- genom bakteriální * MeSH
- interakce mikroorganismu a hostitele genetika MeSH
- klíště mikrobiologie MeSH
- lidé MeSH
- lipoproteiny * genetika MeSH
- lymeská nemoc * mikrobiologie přenos MeSH
- molekulární evoluce MeSH
- plazmidy genetika MeSH
- rekombinace genetická * MeSH
- sekvenování celého genomu MeSH
- selekce (genetika) * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Evropa MeSH
- Severní Amerika MeSH
- Názvy látek
- lipoproteiny * MeSH
Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales, but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii. The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3, blaKPC-2, and blaVIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3-harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C. freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales, affecting even low-virulence pathogens, as C. freundii.
- Klíčová slova
- Citrobacter freundii, carbapenemases, fosA3 gene, fosfomycin, fosfomycin resistance,
- MeSH
- antibakteriální látky * farmakologie MeSH
- bakteriální proteiny * genetika metabolismus MeSH
- beta-laktamasy * genetika metabolismus MeSH
- Citrobacter freundii * genetika enzymologie účinky léků MeSH
- enterobakteriální infekce * mikrobiologie MeSH
- fosfomycin * farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mnohočetná bakteriální léková rezistence genetika MeSH
- plazmidy genetika MeSH
- sekvenování celého genomu MeSH
- transpozibilní elementy DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Itálie epidemiologie MeSH
- Názvy látek
- antibakteriální látky * MeSH
- bakteriální proteiny * MeSH
- beta-laktamasy * MeSH
- carbapenemase MeSH Prohlížeč
- fosfomycin * MeSH
- transpozibilní elementy DNA MeSH
Our study highlights the escalating issue of beta-lactam resistance in nosocomial pathogens, driven by the broad spectrum of antibiotic-degrading enzymes and plasmid exchange. We catalogued known beta-lactamases across 230 bacterial genera, identified 2349 potential beta-lactamases across over 673 genera, and anticipate discovering many new types, underscoring the need for targeted gene analysis in combating resistance. This study also elucidates the complex relationship between the diversity and frequency of beta-lactamase genes across bacterial genera, highlighting the need for genus-specific approaches in combating antibiotic resistance and emphasizing these genes' significant global distribution and host-specific prevalence. We report many transcriptional regulators, transposases and other factors in the genomes of 20 different bacterial isolates, some of which are consistent with the ability of these species to adapt to different environments. Although we could not determine precisely which factors regulate the presence of beta-lactamases in specific bacteria, we found that the proportion of regulatory genes, the size of the genome, and other factors are not decisive. Further studies are needed to elucidate key aspects of this process.
- Klíčová slova
- Bacteria, Beta-lactamases, Genome, Regulatory genes, Resistance,
- MeSH
- antibakteriální látky farmakologie MeSH
- Bacteria * genetika účinky léků enzymologie klasifikace MeSH
- beta-laktamasy * genetika metabolismus MeSH
- beta-laktamová rezistence genetika MeSH
- fylogeneze MeSH
- genom bakteriální MeSH
- lidé MeSH
- plazmidy genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antibakteriální látky MeSH
- beta-laktamasy * MeSH
PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Liběchov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.
- Klíčová slova
- Subretinal injection, large animal, non-viral gene vector, pars plana vitrectomy, safety,
- MeSH
- genetická terapie * metody MeSH
- genetické vektory * aplikace a dávkování MeSH
- injekce nitrooční MeSH
- miniaturní prasata * MeSH
- modely nemocí na zvířatech MeSH
- pilotní projekty MeSH
- plazmidy aplikace a dávkování MeSH
- prasata MeSH
- retina MeSH
- studie proveditelnosti * MeSH
- technika přenosu genů * MeSH
- vitrektomie * metody MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- zelené fluorescenční proteiny MeSH
