Plasmid sequence
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The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014 bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion sequence) elements and transposons. Additional 14 ORFs code for proteins with similarities to known proteins (4 ORFs) or for hypothetical proteins with unknown function (10 ORFs). The differences in G+C content of individual ORFs and gene clusters of pMccC7-H22 indicate a mosaic structure for the plasmid, resulting from recombination events. Real-time PCR quantification was applied to measure the copy number of pMccC7-H22. Escherichia coli H22 carries approximately 5 copies of pMccC7-H22 per chromosome and thus pMccC7-H22 belongs to the group of relatively low-copy-number plasmids. Following 360 generations, all bacterial colonies (out of 100 tested) synthesized microcin C7 indicating that pMccC7-H22 is stably maintained in E. coli H22. Screening of 105 E. coli strains isolated from human fecal samples revealed 2 (1.9%) strains that produced microcin C7.
The complete 98,192bp nucleotide sequence was determined for plasmid pA81, which is harbored by the haloaromatic acid-degrading bacterium Achromobacter xylosoxidans A8. The majority of the 103 open reading frames identified on pA81 could be categorized as either "backbone" genes, genes encoding (halo)aromatic compound degradation, or heavy metal resistance determinants. The backbone genes controlled conjugative transfer, replication and plasmid stability, and were well conserved with other IncP1-beta plasmids. Genes encoding (halo)aromatic degradation were clustered within a type I transposon, TnAxI, and included two ring-hydroxylating oxygenases (ortho-halobenzoate oxygenase, salicylate 5-hydroxylase) and a modified ortho-cleavage pathway for chlorocatechol degradation. The cluster of heavy metal resistance determinants was contained within a Type II transposon TnAxII, and included a predicted P-type ATPase and cation diffusion facilitator system. Genes identical to those carried by TnAxI and TnAxII were identified on other biodegradative/resistance plasmids and genomic islands, indicating an evolutionary relationship between these elements. Collectively, these insights further our understanding of how mobile elements, and interactions between mobile elements affect the fate of organic and inorganic toxicants in the environment.
- MeSH
- Achromobacter denitrificans genetika MeSH
- aromatické uhlovodíky metabolismus MeSH
- bakteriální geny MeSH
- DNA bakterií genetika chemie MeSH
- financování organizované MeSH
- molekulární evoluce MeSH
- molekulární sekvence - údaje MeSH
- multigenová rodina MeSH
- otevřené čtecí rámce MeSH
- oxygenasy se smíšenou funkcí genetika MeSH
- oxygenasy genetika MeSH
- plazmidy genetika chemie MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA MeSH
- těžké kovy metabolismus MeSH
- transpozibilní elementy DNA MeSH
Extended-spectrum-beta-lactamase (ESBL)-producing, AmpC beta-lactamase-producing, and plasmid-mediated quinolone resistance (PMQR) gene-positive strains of Escherichia coli were investigated in wintering rooks (Corvus frugilegus) from eight European countries. Fecal samples (n = 1,073) from rooks wintering in the Czech Republic, France, Germany, Italy, Poland, Serbia, Spain, and Switzerland were examined. Resistant isolates obtained from selective cultivation were screened for ESBL, AmpC, and PMQR genes by PCR and sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were performed to reveal their clonal relatedness. In total, from the 1,073 samples, 152 (14%) cefotaxime-resistant E. coli isolates and 355 (33%) E. coli isolates with reduced susceptibility to ciprofloxacin were found. Eighty-two (54%) of these cefotaxime-resistant E. coli isolates carried the following ESBL genes: blaCTX-M-1 (n = 39 isolates), blaCTX-M-15 (n = 25), blaCTX-M-24 (n = 4), blaTEM-52 (n = 4), blaCTX-M-14 (n = 2), blaCTX-M-55 (n = 2), blaSHV-12 (n = 2), blaCTX-M-8 (n = 1), blaCTX-M-25 (n = 1), blaCTX-M-28 (n = 1), and an unspecified gene (n = 1). Forty-seven (31%) cefotaxime-resistant E. coli isolates carried the blaCMY-2 AmpC beta-lactamase gene. Sixty-two (17%) of the E. coli isolates with reduced susceptibility to ciprofloxacin were positive for the PMQR genes qnrS1 (n = 54), qnrB19 (n = 4), qnrS1 and qnrB19 (n = 2), qnrS2 (n = 1), and aac(6')-Ib-cr (n = 1). Eleven isolates from the Czech Republic (n = 8) and Serbia (n = 3) were identified to be CTX-M-15-producing E. coli clone B2-O25b-ST131 isolates. Ninety-one different sequence types (STs) among 191 ESBL-producing, AmpC-producing, and PMQR gene-positive E. coli isolates were determined, with ST58 (n = 15), ST10 (n = 14), and ST131 (n = 12) predominating. The widespread occurrence of highly diverse ESBL- and AmpC-producing and PMQR gene-positive E. coli isolates, including the clinically important multiresistant ST69, ST95, ST117, ST131, and ST405 clones, was demonstrated in rooks wintering in various European countries.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální geny MeSH
- bakteriální léková rezistence * MeSH
- cefalosporiny farmakologie MeSH
- Escherichia coli klasifikace účinky léků genetika izolace a purifikace MeSH
- fluorochinolony farmakologie MeSH
- genotyp MeSH
- multilokusová sekvenční typizace MeSH
- plazmidy MeSH
- polymerázová řetězová reakce MeSH
- pulzní gelová elektroforéza MeSH
- vrány mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
Multidrug resistant (MDR) Gram-negative bacteria have been increasingly reported in humans, companion animals and farm animals. The growing trend of plasmid-mediated resistance to antimicrobial classes of critical importance is attributed to the emergence of epidemic plasmids, rapidly disseminating resistance genes among the members of Enterobacteriaceae family. The use of antibiotics to treat humans and animals has had a significant impact on the environment and on wild animals living and feeding in human-influenced habitats. Wildlife can acquire MDR bacteria selected in hospitals, community or livestock from diverse sources, including wastewater, sewage systems, landfills, farm facilities or agriculture fields. Therefore, wild animals are considered indicators of environmental pollution by antibiotic resistant bacteria, but they can also act as reservoirs and vectors spreading antibiotic resistance across the globe. The level of resistance and reported plasmid-mediated resistance mechanisms observed in bacteria of wildlife origin seem to correlate well with the situation described in humans and domestic animals. Additionaly, the identification of epidemic plasmids in samples from different human, animal and wildlife sources underlines the role of horizontal gene transfer in the dissemination of resistance genes. The present review focuses on reports of plasmid-mediated resistance to critically important antimicrobial classes such as broad-spectrum beta-lactams and colistin in Enterobacteriaceae isolates from samples of wildlife origin. The role of plasmids in the dissemination of ESBL-, AmpC- and carbapenemase-encoding genes as well as plasmid-mediated colistin resistance determinants in wildlife are discussed, and their similarities to plasmids previously identified in samples of human clinical or livestock origin are highlighted. Furthermore, we present features of completely sequenced plasmids reported from wildlife Enterobacteriaceae isolates, with special focus on genes that could be associated with the plasticity and stable maintenance of these molecules in antibiotic-free environments.
- MeSH
- antibakteriální látky terapeutické užití MeSH
- bakteriální proteiny genetika MeSH
- beta-laktamasy genetika MeSH
- beta-laktamy terapeutické užití MeSH
- gramnegativní bakterie účinky léků genetika patogenita MeSH
- lidé MeSH
- mnohočetná bakteriální léková rezistence genetika MeSH
- plazmidy genetika MeSH
- přenos genů horizontální genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.
- MeSH
- Enterococcaceae * MeSH
- larva mikrobiologie MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- Paenibacillus larvae * genetika MeSH
- Paenibacillus * genetika MeSH
- plazmidy genetika MeSH
- transpozibilní elementy DNA MeSH
- včely genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Metallosupramolecular chemistry was used to design a new class of synthetic agents, namely, tetracationic supramolecular cylinders, that bind strongly and noncovalently in the major groove of DNA. To gain additional information on interactions of the cylinders with DNA we explored DNA unwinding and sequence-specific binding properties, as well as DNA photonuclease activity of ruthenium(II) metallosupramolecular cylinder [Ru(2)L(3)](4+), where L is a bis-pyridylimine ligand. We found that [Ru(2)L(3)](4+) unwinds negatively supercoiled plasmid DNA and exhibits binding preference to regular alternating purine-pyrimidine sequences in a similar way to the [Fe(2)L(3)](4+) analogue. Photocleavage studies showed that, unlike [Fe(2)L(3)](4+), [Ru(2)L(3)](4+) induces single-strand breaks on irradiation by visible and UVA light and cleaves DNA mainly at guanine residues contained preferentially in regularly alternating purine-pyrimidine nucleotides. As [Ru(2)L(3)](4+) binds and cleaves DNA in a sequence-dependent manner, it may provide a useful tool for basic and applied biology, such as for controlled manipulation of the genome.
