Plasmid最新文献

The link between E. coli strains contaminating foods and human disease is unclear, with some reports supporting a direct transmission of pathogenic strains via food and others highlighting their role as reservoirs for resistance and virulence genes. Here we take a genomics approach, analyzing a large set of fully-assembled genomic sequences from E. coli available in GenBank. Most of the strains isolated in food are more closely related to each other than to clinical strains, arguing against a frequent direct transmission of pathogenic strains from food to the clinic. We also provide strong evidence of genetic exchanges between food and clinical strains that are facilitated by plasmids. This is based on an overlapped representation of virulence and resistance genes in plasmids isolated from these two sources. We identify clusters of phylogenetically-related plasmids that are largely responsible for the observed overlap and see evidence of specialization, with some food plasmid clusters preferentially transferring virulence factors over resistance genes. Consistent with these observations, food plasmids have a high mobilization potential based on their plasmid taxonomic unit classification and on an analysis of mobilization gene content. We report antibiotic resistance genes of high clinical relevance and their specific incompatibility group associations. Finally, we also report a striking enrichment for adhesins in food plasmids and their association with specific IncF replicon subtypes. The identification of food plasmids with specific markers (Inc and PTU combinations) as mediators of horizontal transfer between food and clinical strains opens new research avenues and should assist with the design of surveillance strategies.

Horizontal gene transfer is an important mechanism in bacterial evolution and can occur at striking frequencies when mediated by mobile genetic elements. Conjugative plasmids are mobile genetic elements that are main drivers of horizontal transfer and a major facilitator in the spread of antibiotic resistance genes. However, conjugative plasmid models that readily can be genetically modified with the aim to study horizontal transfer are not currently available. The aim of this study was to develop a conjugative plasmid model where the insertion of gene cassettes such as reporter genes (e.g., fluorescent proteins) or antibiotic resistance genes would be efficient and convenient. Here, we introduced a single attTn7 site into the conjugative broad-host-range IncP-1 plasmid pKJK5 in a non-disruptive manner. Furthermore, a version with lower transfer rate and a non-conjugative version of pKJK5-attTn7 were also constructed. The advantage of having the attTn7 sites is that genes of interest can be introduced in a single step with very high success rate using the Tn7 transposition system. In addition, larger genetic fragments can be inserted. To illustrate the efficacy of the constructed pKJK5 plasmids, they were complemented with sfGFP (a gene encoding superfolder green fluorescent protein) in addition to seven different β-lactamase genes representing the four known classes of β-lactamases.

To characterize IncI1 and IncF18:A−:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate, antibiotic susceptibility testing, conjugation assays, transformation assays, S1-PFGE, and WGS analysis were performed. The 119,457-bp plasmid pEC014–1 with a multidrug-resistance region (MRR) containing four different segments interspersed with six IS26 elements, belonged to incompatibility group I1 and sequence type 71. The 154,516-bp plasmid pEC014–2 with two replicons, typed as FII-18 and FIB-1, carried 14 resistance determinants including bla_TEM-1b, bla_OXA-1, oqxAB, dfrA17, aac(6′)-Ib-cr, sul1, sul2, tet(A), floR, catB3, hph(aph(4)-Ia), aacC4(aac(3)-IV), aadA5, arr-3, and a merEDACPTR loci in MRR, and additionally encoded three virulence loci: iroNEDCB, sitABCD, and iucABCD-iutA. Plasmid stability assays showed that pEC014–1 and pEC014–2 were stable in recipient E. coli C600 for at least 15 days of passage. Competition assays were carried out to evaluate the fitness impact of pEC014–2 carriage in vitro, revealing a decrease in host fitness. Growth kinetics showed that the growth rate for pEC014–1 or/and pEC014–2 bearing cells was significantly slower than that of the E. coli C600 host strain in the exponential stage (p < 0.01), with only cells carrying pEC014–1 sustaining rapid growth after 6 h of exponential growth. Our findings highlight the mosaic structures of epidemic plasmid IncI1/ST71 and F18:A−:B1 lineages and contribute to a better understanding of the evolution and dissemination of these multidrug resistance and virulence plasmids.

pXO16, the 350 kb-conjugative plasmid from Bacillus thuringiensis sv. israelensis promotes its own transfer at high efficiency, triggers the transfer of mobilizable and non-mobilizable plasmids, as well as the transfer of host chromosomal loci. Naturally found in B. thuringiensis sv. israelensis, pXO16 transfers to various strains of Bacillus cereus sensu lato (s.l.) at a wide range of frequencies. Despite this host diversity, a paradox remains between the relatively large host spectrum and the natural occurrence of pXO16, so far restricted to B. thuringiensis sv. israelensis. Proposing first insights exploring this paradox, we investigated the behaviour of pXO16 amongst different members of the B. cereus group. We first looked at the transfer of pXO16 to two new host clusters of B. cereus s.l., Bacillus mycoides and Bacillus anthracis clusters. This examination brought to light the impairment of the characteristic rhizoidal phenotype of B. mycoides in presence of pXO16. We also explored the stability of pXO16 at different temperatures as some B. cereus group members are well-known for their psychro- or thermo-tolerance. This shed light on the thermo-sensitivity of the plasmid. The influence of pXO16 on its host cell growth and on swimming capacity also revealed no or limited impact on its natural host B. thuringiensis sv. israelensis. On the contrary, pXO16 affected more strongly both the growth and swimming capacity of other B. cereus s.l. hosts. This reinforced the running hypothesis of a co-evolution between pXO16 and B. thuringiensis sv. israelensis, enabling the plasmid maintenance without impairing the host strain development.

Giardia duodenalis, is a binuclear and microaerophilic protozoan that causes giardiasis. Up to date, several molecular approaches have been taken to understand the molecular mechanisms of diverse cellular processes in this parasitic protozoan. However, the role of many genes involved in these processes needs further analysis. The CRISPR interference (CRISPRi) system has been widely used, as a constitutive expression system for gene silencing purposes in several parasites, including Giardia. The aim of this work was to implement a tunable t-CRISPRi system in Giardia to silence abundant, moderately and low expressed genes, by constructing an optimized and inducible plasmid for the expression of both gRNA and dCas9. A doxycycline inducible pRan promoter was used to express dCas9 and each gRNA, consistently dCas9 expression and nuclear localization were confirmed by Western-blot and immunofluorescence in transfected trophozoites. The transcriptional repression was performed on α-tubulin (high expression), giardipain-1 (moderate expression) and Sir2 and Sir4 (low expression) genes. The α-tubulin gene knock-down caused by dCas9 doxycycline-induction was confirmed by a decrease in its protein expression which was of 50% and 60% at 24 and 48 h, respectively. This induced morphological alterations in flagella. The giardipain-1 knock down, showed a decrease in protein expression of 40 and 50% at 12 and 24 h, respectively, without affecting trophozoites viability, consistent with this a zymogram analysis on giardipain-1 knock down revealed a decrease in giardipain-1 protease activity. When repressing sirtuins expression, a total repression was obtained but trophozoites viability was compromised. This approach provides a molecular tool for a tailored repression to produce specific gene knockdowns.

Resistance plasmids mediate the rapid spread of antimicrobial resistance, which poses a threat to veterinary and human healthcare. This study addresses the question whether resistance plasmids from Escherichia coli isolated from foodstuffs always transfer unchanged to recipient E. coli cells, or that genetic editing can occur. Strains containing between one and five different plasmids were co-incubated with a standard recipient strain. Plasmids isolated from transconjugant strains were sequenced using short and long read technologies and compared to the original plasmids from the donor strains. After one hour of co-incubation only a single plasmid was transferred from donor to recipient strains. If the donor possessed several plasmids, longer co-incubation resulted in multiple plasmids being transferred. Transferred plasmids showed mutations, mostly in mobile genetic elements, in the conjugative transfer gene pilV and in genes involved in plasmid maintenance. In one transconjugant, a resistance cluster encoding tetracycline resistance was acquired by the IncI1 plasmid from the IncX1 plasmid that was also present in the donor strain, but that was not transferred. A single plasmid transferred twelve times back and forth between E. coli strains resulted in a fully conserved plasmid with no mutations, apart from repetitive rearrangements of pilV from and back to its original conformation in the donor strain. The overall outcome suggests that some genetic mutations and rearrangements can occur during plasmid transfer. The possibility of such mutations should be taken into consideration in epidemiological research aimed at attribution of resistance to specific sources.

Plasmids are widely involved in the dissemination of characteristics within bacterial communities. Their genomic content can be assessed by high-throughput sequencing of the whole plasmid fraction of an environment, the plasmidome. In this study, we analyzed the plasmidome of a biofilm formed in the effluents of the teaching hospital of Clermont-Ferrand (France). Our analysis discovered >350 new complete plasmids, with a length ranging from 1219 to 40,193 bp. Forty-two plasmid incompatibility (Inc) groups were found among all the plasmid contigs. Ten large plasmids, described here in detail, were reconstructed from plasmid contigs, seven of which carried antibiotic resistance genes. Four plasmids potentially confer resistance to numerous families of antibiotics, including carbapenems, aminoglycosides, colistin, and chloramphenicol. Most of these plasmids were affiliated to Proteobacteria, a phylum of Gram-negative bacteria. This study therefore illustrates the composition of an environmental mixed biofilm in terms of plasmids and antibiotic resistance genes.

In addition to tumor-inducing agrobacteria, non-pathogenic strains are often isolated from crown gall tumors. Such non-pathogenic strains sometimes contain catabolic plasmids that allow them to take advantage of the opines produced in the tumors. Here we characterize for the first time an octopine catabolic plasmid, pAtAg67, which is derived from an Agrobacterium strain isolated from a grapevine tumor in Crete. By sequence analysis, we deduce that pAtAg67 enables its host to catabolize not only octopine, but also cucumopine and agrocinopine-like compounds. We found that a highly similar set of catabolic genes was present in the Ti plasmids of tumorigenic octopine-cucumopine grapevine strains such as pTiAg57. However, the catabolic genes in octopine-cucumopine Ti plasmids were interrupted by a T-DNA segment. As no T-DNA remnants, virulence genes or border repeats were found in pAtAg67, catabolic plasmid pAtAg67 does not appear to be a degenerate octopine-cucumopine Ti plasmid. In line, plasmid pAtAg67 was found to be compatible with incRh1 octopine Ti plasmids, but to be incompatible with the incRh2 agropine Ti plasmid pTiBo542, forming cointegrates by recombination in the homologous trb genes.

Gateway system is one of the most known cloning systems, which makes it compatible with several expression vectors, including those used for Yeast Two-Hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC) assays. However, this system is laborious and expensive due to its two-step cloning. In this research, we developed a new cloning strategy named Brick into the Gateway (BiG). This approach uses GoldenBraid/Gate assemblies to create a DNA fragment of interest flanked by attL sites, which can be directly recombined into Gateway destination vectors. BiG method showed a high recombination efficiency and ensured the correct reading frame, which was successfully tested in Y2H and BiFC assays. BiG has proven to be a rapid, low-cost, reusable, and directional cloning method which allows the merged use of systems.

Acinetobacter baumannii RepAci1-RepAci10 plasmids pA388 from a global clone 1 (GC1) isolate from Greece, and pACICU1 and variant pACICU1b from an Italian GC2 isolate were found to share a common ancestor. The ancestor formed via recombination between pdif sites in the widely-distributed RepAci1 plasmid pA1–1 and in a RepAci10 plasmid carrying the oxa58 carbapenem-resistance gene in a dif module. Each plasmid includes copies of IS26 and multiple dif modules surrounded by 28 bp pdif sites resembling the chromosomal dif site, including one carrying the oxa58 gene. Plasmid sequences were compared to identify factors driving their evolution and divergence. IS26-mediated events, recombination between pdif sites and homologous recombination have all contributed. A translocatable unit that includes oxa58, generated by an IS26-mediated adjacent deletion, had been re-inserted by IS26 adjacent to an IS26 in pACICU1b to create the oxa58 gene duplication previously found in pACICU1. The oxa58 duplication has been lost from pACICU1b and the Tn6020 variant carrying the aphA1 (kanamycin, neomycin resistance) gene in pA388 has been lost from pACICU1/1b via recombination between directly-oriented IS26 copies. Two dif modules located between directly-oriented pdif sites in pA388 have been lost from pACICU1/1b and the product of this and other deletion events as well as inversion of dif modules located between inversely-oriented pdif sites were detected experimentally in pA388 DNA by PCR. Also, the new junctions were detected in a minority of reads in pA388 long-read sequence data. Inversion and deletion were only detected when the spacers in the pdif sites were identical and equivalent events involving mismatched spacers were not detected.