IS26 Family Members IS257 and IS1216 Also Form Cointegrates by Copy-In and Targeted Conservative Routes. - Related Documents




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300601.0000IS26 Family Members IS257 and IS1216 Also Form Cointegrates by Copy-In and Targeted Conservative Routes. IS26 has been shown to form cointegrates both by a copy-in mechanism involving one insertion sequence (IS) and a target and by a targeted conservative mechanism involving two ISs. IS26 is the flagship of a group of 65 bacterial ISs in the recently redefined IS6/IS26 family. Here, whether other family members can also use two mechanisms was examined using members of the IS257/IS431 and IS1216 isoform groups, which are associated with antibiotic resistance genes in staphylococci and enterococci, respectively. Transposases Tnp257 and Tnp1216 have 39% and 47% amino acid identities, respectively, with Tnp26 and are 62% identical to one another. Using a novel transposition assay, pUC-based plasmids carrying these ISs integrated into the chromosome of a temperature-sensitive polAEscherichia coli strain grown at the restrictive temperature. In the cointegrates, the plasmid carrying IS257 was flanked by various 8-bp target site duplications, consistent with random target selection. However, in a mating-out assay, only the targeted conservative reaction was detectable at a low frequency in a recA-negative E. coli strain, indicating that IS257 is at least 100-fold less active than IS26 For IS1216, in mating-out assays, both copy-in and targeted conservative cointegrate formation were detectable at frequencies similar to those observed for IS26 Duplication of various 8-bp target sites was detected for the copy-in route. For both IS257 and IS1216, when both of the plasmids carried an IS, the targeted conservative route occurred at a significantly higher frequency than the copy-in route, and only cointegrates formed by the conservative route were detected.IMPORTANCE IS26 differs from other studied ISs in the reactions that it can undertake. The differences make IS26 uniquely suited to its key role in the recruitment and spread of antibiotic resistance genes in Gram-negative bacteria. However, whether other ISs in the IS6/IS26 family can perform the same reactions is not known. IS257/IS431 and IS1216 isoforms found associated with antibiotic resistance genes in the Gram-positive bacteria staphylococci, enterococci, streptococci, and clostridia are related to IS26 However, the way that they move had not been investigated, limiting interpretation of their role in resistance gene dissemination and in the formation of cointegrates and complex resistance regions in staphylococci and enterococci. Here, they are shown to share the broad catalytic capabilities of IS26, demonstrating that it is likely that all members of the redefined IS6/IS26 family of bacterial ISs likewise are able to use both the copy-in and conservative routes.202031915227
300510.9998Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26. The insertion sequence IS26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS26. A model presented for a second mode of IS26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26 at each end and between each unique segment. This model requires that IS26 recognizes another IS26 as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS26. This reaction was conservative, with no additional IS26 or target site duplication generated, and orientation specific as the IS26s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS26 movement when a target IS26 was not present. Intact transposase genes in both IS26s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency. Importance: Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram-negative bacteria, IS26 is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS26 copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS26-flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS26 movement do not resemble those of the IS and class I transposons studied to date. IS26 uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called "translocatable units." The IS26 mechanism also explains the properties of IS257 (IS431) and IS1216, which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.201425293759
300420.9998IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit. We recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end. IMPORTANCE: Resistance to antibiotics limits treatment options. In Gram-negative bacteria, IS26 plays a major role in the acquisition and dissemination of antibiotic resistance. IS257 (IS431) and IS1216, which belong to the same insertion sequence (IS) family, mobilize resistance genes in staphylococci and enterococci, respectively. Many different resistance genes are found in compound transposons bounded by IS26, and multiply and extensively antibiotic-resistant Gram-negative bacteria often include regions containing several antibiotic resistance genes and multiple copies of IS26. We recently showed that in addition to replicative transposition, IS26 can use a conservative movement mechanism in which an incoming IS26 targets a preexisting one, and this reaction can create these regions. This mechanism differs from that of all the ISs examined in detail thus far. Here, we have continued to extend understanding of the reactions carried out by IS26 by examining whether the reverse precise excision reaction is also catalyzed by the IS26 transposase.201526646012
300330.9997IS26-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes. The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. Recently, Tnp26 was shown to catalyze an additional intermolecular, conservative reaction between two preexisting copies of IS26 in different plasmids. Here, we have investigated the relative contributions of homologous recombination and Tnp26-catalyzed reactions to the generation of a transposon from a TU. Circular TUs containing the aphA1a kanamycin and neomycin resistance gene or the tet(D) tetracycline resistance determinant were generated in vitro and transformed into Escherichia coli recA cells carrying R388::IS26. The TU incorporated next to the IS26 in R388::IS26 forms a transposon with the insertion sequence (IS) in direct orientation. Introduction of a second TU produced regions containing both the aphA1a gene and the tet(D) determinant in either order but with only three copies of IS26. The integration reaction, which required a preexisting IS26, was precise and conservative and was 50-fold more efficient when both IS26 copies could produce an active Tnp26. When both ISs were inactivated by a frameshift in tnp26, TU incorporation was not detected in E. coli recA cells, but it did occur in E. coli recA (+) cells. However, the Tnp-catalyzed reaction was 100-fold more efficient than RecA-dependent homologous recombination. The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded transposons in the resistance regions found in Gram-negative bacteria. IMPORTANCE In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds transposons and brings additional transposons into resistance regions.201627303727
41740.9995Site-specific integration of genes into hot spots for recombination flanking aadA in Tn21 transposons. Tn21-related transposons are widespread among bacteria and carry various resistance determinants at preferential sites, hs1 and hs2. In an in vivo integrative recombination assay it was demonstrated that these hot spots direct the integration of aminoglycoside resistance genes like aadB from Klebsiella pneumoniae and aacAI from Serratia marcescens, in a recA- background. The maximum required recognition sequence which must be present in both the donor and recipient plasmids is 5' CTAAAACAAAGTTA 3' (hs2). The double-site-specific recombination occurred with a frequency of 10(-5)-10(-6). The resulting structures include not only replicon fusion products but also more complex structures carrying two copies of the donor plasmid or simply the donor gene flanked by hs elements. hs1 and hs2 are thought to act as recognition sites for a transacting site-specific recombinase. By the use of Tn21 deletion derivatives, it has been shown that the recombinase is not encoded by Tn21. This new integrative recombination system is involved in the acquisition of new genes by Tn21-related transposons and their spread among bacterial populations.19911654505
446850.9995Mobile gene cassettes and integrons: moving antibiotic resistance genes in gram-negative bacteria. In Gram-negative pathogens, multiple antibiotic resistance is common and many of the known resistance genes are contained in mobile gene cassettes. Cassettes can be integrated into or deleted from their receptor elements, the integrons, or infrequently may be integrated at other locations via site-specific recombination catalysed by an integron-encoded recombinase. As a consequence, arrays of several different antibiotic resistance genes can be created. Over 40 gene cassettes and three distinct classes of integrons have been identified to date. Cassette-associated genes conferring resistance to beta-lactams, aminoglycosides, trimethoprim, chloramphenicol, streptothricin and quaternary ammonium compounds used as antiseptics and disinfectants have been found. In addition, most members of the commonest family of integrons (class 1) include a sulfonamide resistance determinant in the backbone structure. Integrons are themselves translocatable, though most are defective transposon derivatives. Integron movement allows transfer of the cassette-associated resistance genes from one replicon to another or into another active transposon which facilitates spread of integrons that are transposition defective. Horizontal transfer of the resistance genes can be achieved when an integron containing one or more such genes is incorporated into a broad-host-range plasmid. Likewise, single cassettes integrated at secondary sites in a broad-host-range plasmid can also move across species boundaries.19979189642
997360.9995Spread and Persistence of Virulence and Antibiotic Resistance Genes: A Ride on the F Plasmid Conjugation Module. The F plasmid or F-factor is a large, 100-kbp, circular conjugative plasmid of Escherichia coli and was originally described as a vector for horizontal gene transfer and gene recombination in the late 1940s. Since then, F and related F-like plasmids have served as role models for bacterial conjugation. At present, more than 200 different F-like plasmids with highly related DNA transfer genes, including those for the assembly of a type IV secretion apparatus, are completely sequenced. They belong to the phylogenetically related MOB(F12)A group. F-like plasmids are present in enterobacterial hosts isolated from clinical as well as environmental samples all over the world. As conjugative plasmids, F-like plasmids carry genetic modules enabling plasmid replication, stable maintenance, and DNA transfer. In this plasmid backbone of approximately 60 kbp, the DNA transfer genes occupy the largest and mostly conserved part. Subgroups of MOB(F12)A plasmids can be defined based on the similarity of TraJ, a protein required for DNA transfer gene expression. In addition, F-like plasmids harbor accessory cargo genes, frequently embedded within transposons and/or integrons, which harness their host bacteria with antibiotic resistance and virulence genes, causing increasingly severe problems for the treatment of infectious diseases. Here, I focus on key genetic elements and their encoded proteins present on the F-factor and other typical F-like plasmids belonging to the MOB(F12)A group of conjugative plasmids.201830022749
982270.9995Molecular mechanisms for transposition of drug-resistance genes and other movable genetic elements. Transposition is proposed to be responsible for the rapid evolution of multiply drug-resistant bacterial strains. Transposons, which carry the genes encoding drug resistance, are linear pieces of DNA that range in size from 2.5 to 23 kilobase pairs and always contain at their ends nucleotide sequences repeated in inverse order. In some transposons the terminal inverted repeat sequences are capable of independent movement and are called insertion sequences. Transposons carry a gene that encodes transposase(s), the enzyme(s) responsible for recombination of the transposon into another DNA molecule. Studies on transposable genetic elements in bacteria have not only given insight into the spread of antibiotic resistance but also into the process of DNA movement.19873035697
446680.9995Antibiotic resistance in gram-negative bacteria: the role of gene cassettes and integrons. Resistance of gram-negative organisms to antibiotics such as beta-lactams, aminoglycosides, trimethoprim and chloramphenicol is caused by many different acquired genes, and a substantial proportion of these are part of small mobile elements known as gene cassettes. A gene cassette consists of the gene and a downstream sequence, known as a 59-base element (59-be), that acts as a specific recombination site. Gene cassettes can move into or out of a specific receptor site (attl site) in a companion element called an integron, and integration or excision of the cassettes is catalysed by a site-specific recombinase (Intl) that is encoded by the integron. At present count there are 40 different cassette-associated resistance genes and three distinct classes of integron, each encoding a distinct Intl integrase. The same cassettes are found in all three classes of integron, indicating that cassettes can move freely between different integrons. Integrons belonging to class I often contain a further antibiotic resistance gene, sull, conferring resistance to sulphonamides. The sull gene is found in a conserved region (3'-CS) that is not present in all members of this class. Class I integrons of the sull type are most prevalent in clinical isolates and have been found in many different organisms. Even though most of them are defective transposon derivatives, having lost at least one of the transposition genes, they are none the less translocatable and consequently found in many different locations. The transposon Tn7 is the best known representative of class 2 integrons, and Tn7 and relatives are also found in many different species.199816904397
446990.9995Integrons: an antibiotic resistance gene capture and expression system. Bacteria can transfer genetic information to provide themselves with protection against most antibiotics. The acquisition of resistance gene arrays involves genetic mobile elements like plasmids and transposons. Another class of genetic structures, termed integrons, have been described and contain one or more gene cassettes located at a specific site. Integrons are defined by an intl gene encoding an integrase, a recombination site attl and a strong promoter. At least six classes of integrons have been determined according to their intl gene. Classes 1, 2 and 3 are the most studied and are largely implicated in the dissemination of antibiotic resistance. A gene cassette includes an open reading frame and, at the 3'-end, a recombination site attC. Integration or excision of cassettes occur by a site-specific recombination mechanism catalyzed by the integrase. However, insertion can occur, albeit rarely, at non-specific sites leading to a stable situation for the cassette. Cassettes are transcribed from the common promoter located in the 5'-conserved segment and expression of distal genes is reduced by the presence of upstream cassettes. Most gene cassettes encode antibiotic resistant determinants but antiseptic resistant genes have also been described. Integrons seem to have a major role in the spread of multidrug resistance in gram-negative bacteria but integrons in gram-positive bacteria were described recently. Moreover, the finding of super-integrons with gene-cassettes coding for other determinants (biochemical functions, virulence factors) in Vibrio isolates dating from 1888 suggests the likely implication of this multicomponent cassette-integron system in bacterial genome evolution before the antibiotic era and to a greater extent than initially believed.200010987194
4467100.9994PCR mapping of integrons reveals several novel combinations of resistance genes. The integron is a new type of mobile element which has evolved by a site-specific recombinational mechanism. Integrons consist of two conserved segments of DNA separated by a variable region containing one or more genes integrated as cassettes. Oligonucleotide probes specific for the conserved segments have revealed that integrons are widespread in recently isolated clinical bacteria. Also, by using oligonucleotide probes for several antibiotic resistance genes, we have found novel combinations of resistance genes in these strains. By using PCR, we have determined the content and order of the resistance genes inserted between the conserved segments in the integrons of these clinical isolates. PCR mapping of integrons can be a useful epidemiological tool to study the evolution of multiresistance plasmids and transposons and dissemination of antibiotic resistance genes.19957695304
4465110.9994Genetic analyses of sulfonamide resistance and its dissemination in gram-negative bacteria illustrate new aspects of R plasmid evolution. In contrast to what has been observed for many other antibiotic resistance mechanisms, there are only two known genes encoding plasmid-borne sulfonamide resistance. Both genes, sulI and sulII, encode a drug-resistant dihydropteroate synthase enzyme. In members of the family Enterobacteriaceae isolated from several worldwide sources, plasmid-mediated resistance to sulfonamides could be identified by colony hybridization as being encoded by sulI, sulII, or both. The sulI gene was in all cases found to be located in the newly defined, mobile genetic element, recently named an integron, which has been shown to contain a site-specific recombination system for the integration of various antibiotic resistance genes. The sulII gene was almost exclusively found as part of a variable resistance region on small, nonconjugative plasmids. Colony hybridization to an intragenic probe, restriction enzyme digestion, and nucleotide sequence analysis of small plasmids indicated that the sulII gene and contiguous sequences represent an independently occurring region disseminated in the bacterial population. The sulII resistance region was bordered by direct repeats, which in some plasmids were totally or partially deleted. The prevalence of sulI and sulII could thus be accounted for by their stable integration in transposons and in plasmids that are widely disseminated among gram-negative bacteria.19911952855
4528120.9994Study on the excision and integration mediated by class 1 integron in Streptococcus pneumoniae. As a novel antibiotic resistance mobile element, integron was recognized as a primary source of antibiotic genes among Gram-positive organisms for its excision and integration of exogenous genes. In this study, Streptococcus pneumoniae was subjected to investigate the excision and integration of class 1 integron with eight different plasmids. As the results indicated, excision in both att site and gene cassettes were successfully observed, which was further confirmed by integration assays and PCR amplification. The observation of class 1 integron mediated excision and integration of various exogenous antibiotics resistance genes may raise the attention of integrons as novel antibiotic resistance determinant in Gram-positive bacteria, especially in Streptococcus.201728923604
9888130.9994Evolution and typing of IncC plasmids contributing to antibiotic resistance in Gram-negative bacteria. The large, broad host range IncC plasmids are important contributors to the spread of key antibiotic resistance genes and over 200 complete sequences of IncC plasmids have been reported. To track the spread of these plasmids accurate typing to identify the closest relatives is needed. However, typing can be complicated by the high variability in resistance gene content and various typing methods that rely on features of the conserved backbone have been developed. Plasmids can be broadly typed into two groups, type 1 and type 2, using four features that differentiate the otherwise closely related backbones. These types are found in many different countries in bacteria from humans and animals. However, hybrids of type 1 and type 2 are also occasionally seen, and two further types, each represented by a single plasmid, were distinguished. Generally, the antibiotic resistance genes are located within a small number of resistance islands, only one of which, ARI-B, is found in both type 1 and type 2. The introduction of each resistance island generates a new lineage and, though they are continuously evolving via the loss of resistance genes or introduction of new ones, the island positions serve as valuable lineage-specific markers. A current type 2 lineage of plasmids is derived from an early type 2 plasmid but the sequences of early type 1 plasmids include features not seen in more recent type 1 plasmids, indicating a shared ancestor rather than a direct lineal relationship. Some features, including ones essential for maintenance or for conjugation, have been examined experimentally.201830081066
4526140.9994The tetracycline resistance gene tet(M) exhibits mosaic structure. Tetracycline resistance genes of the M class, tet(M), are typically found on mobile genetic elements as the conjugative transposons of gram-positive bacteria. By comparing the sequences of eight different tet(M) genes (from Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Ureaplasma urealyticum, and Neisseria), a mosaic structure was detected which could be traced to two distinct alleles. The two alleles displayed a divergence of 8% and a different G/C content. The block structure of these genes provides evidence for the contribution of homologous recombination to the evolution and the heterogeneity of the tet(M) locus. Unlike described cases of chromosomally located mosaic loci, tet(M) is a relatively recently acquired determinant in the species examined and it would appear that mosaic structure within tet(M) has evolved after acquisition of the gene by the mobile genetic elements upon which it is located.19968812782
9867150.9994Mosaic plasmids are abundant and unevenly distributed across prokaryotic taxa. Mosaic plasmids, plasmids composed of genetic elements from distinct sources, are associated with the spread of antibiotic resistance genes. Transposons are considered the primary mechanism for mosaic plasmid formation, though other mechanisms have been observed in specific instances. The frequency with which mosaic plasmids have been described suggests they may play an important role in plasmid population dynamics. Our survey of the confirmed plasmid sequences available from complete and draft genomes in the RefSeq database shows that 46% of them fit a strict definition of mosaic. Mosaic plasmids are also not evenly distributed over the taxa represented in the database. Plasmids from some genera, including Piscirickettsia and Yersinia, are almost all mosaic, while plasmids from other genera, including Borrelia, are rarely mosaic. While some mosaic plasmids share identical regions with hundreds of others, the median mosaic plasmid only shares with 8 other plasmids. When considering only plasmids from finished genomes (51.6% of the total), mosaic plasmids have significantly higher proportions of transposase and antibiotic resistance genes. Conversely, only 56.6% of mosaic fragments (DNA fragments shared between mosaic plasmids) contain a recognizable transposase gene, and only 1.2% of mosaic fragments are flanked by inverted repeats. Mosaic fragments associated with the IS26 transposase gene are 3.8-fold more abundant than any other sequence shared between mosaic plasmids in the database, though this is at least partly due to overrepresentation of Enterobacteriaceae plasmids. Mosaic plasmids are a complicated trait of some plasmid populations, only partly explained by transposition. Though antibiotic resistance genes led to the identification of many mosaic plasmids, mosaic plasmids are a broad phenomenon encompassing many more traits than just antibiotic resistance. Further research will be required to determine the influence of ecology, host repair mechanisms, conjugation, and plasmid host range on the formation and influence of mosaic plasmids. AUTHOR SUMMARY: Plasmids are extrachromosomal genetic entities that are found in many prokaryotes. They serve as flexible storage for genes, and individual cells can make substantial changes to their characteristics by acquiring, losing, or modifying a plasmid. In some pathogenic bacteria, such as Escherichia coli, antibiotic resistance genes are known to spread primarily on plasmids. By analyzing a database of 8592 plasmid sequences we determined that many of these plasmids have exchanged genes with each other, becoming mosaics of genes from different sources. We next separated these plasmids into groups based on the organism they were isolated from and found that different groups had different fractions of mosaic plasmids. This result was unexpected and suggests that the mechanisms and selective pressures causing mosaic plasmids do not occur evenly over all species. It also suggests that plasmids may provide different levels of potential variation to different species. This work uncovers a previously unrecognized pattern in plasmids across prokaryotes, that could lead to new insights into the evolutionary role that plasmids play.201930797764
9972160.9994Extensive antimicrobial resistance mobilization via multicopy plasmid encapsidation mediated by temperate phages. OBJECTIVES: To investigate the relevance of multicopy plasmids in antimicrobial resistance and assess their mobilization mediated by phage particles. METHODS: Several databases with complete sequences of plasmids and annotated genes were analysed. The 16S methyltransferase gene armA conferring high-level aminoglycoside resistance was used as a marker in eight different plasmids, from different incompatibility groups, and with differing sizes and plasmid copy numbers. All plasmids were transformed into Escherichia coli bearing one of four different lysogenic phages. Upon induction, encapsidation of armA in phage particles was evaluated using qRT-PCR and Southern blotting. RESULTS: Multicopy plasmids carry a vast set of emerging clinically important antimicrobial resistance genes. However, 60% of these plasmids do not bear mobility (MOB) genes. When carried on these multicopy plasmids, mobilization of a marker gene armA into phage capsids was up to 10000 times more frequent than when it was encoded by a large plasmid with a low copy number. CONCLUSIONS: Multicopy plasmids and phages, two major mobile genetic elements (MGE) in bacteria, represent a novel high-efficiency transmission route of antimicrobial resistance genes that deserves further investigation.202032719862
9889170.9994Evolution and dissemination of L and M plasmid lineages carrying antibiotic resistance genes in diverse Gram-negative bacteria. Conjugative, broad host-range plasmids of the L/M complex have been associated with antibiotic resistance since the 1970s. They are found in Gram-negative bacterial genera that cause human infections and persist in hospital environments. It is crucial that these plasmids are typed accurately so that their clinical and global dissemination can be traced in epidemiological studies. The L/M complex has previously been divided into L, M1 and M2 subtypes. However, those types do not encompass all diversity seen in the group. Here, we have examined 148 complete L/M plasmid sequences in order to understand the diversity of the complex and trace the evolution of distinct lineages. The backbone sequence of each plasmid was determined by removing translocatable genetic elements and reversing their effects in silico. The sequence identities of replication regions and complete backbones were then considered for typing. This supported the distinction of L and M plasmids and revealed that there are five L and eight M types, where each type is comprised of further sub-lineages that are distinguished by variation in their backbone and translocatable element content. Regions containing antibiotic resistance genes in L and M sub-lineages have often formed by initial rare insertion events, followed by insertion of other translocatable elements within the inceptive element. As such, islands evolve in situ to contain genes conferring resistance to multiple antibiotics. In some cases, different plasmid sub-lineages have acquired the same or related resistance genes independently. This highlights the importance of these plasmids in acting as vehicles for the dissemination of emerging resistance genes. Materials are provided here for typing plasmids of the L/M complex from complete sequences or draft genomes. This should enable rapid identification of novel types and facilitate tracking the evolution of existing lineages.202132781088
4527180.9994Study on the excision and integration mediated by class 1 integron in Enterococcus faecalis. Recognized as a mobile genetic element, integron is correlated to the excision and integration of exogenous genes, especially bacterial resistance genes. However, most of the investigations focused on Gram-positive bacteria with few exceptions. In this study, Enterococcus faecalis was selected to investigate the excision and integration of class 1 integron. A total of eight plasmids were subjected to establish the transformants for excision and integration test. As results showed, positive excision assay was observed, which had been confirmed by the further integration assays and PCR amplification. The observation of class 1 integron mediated excision and integration of various exogenous antibiotics resistance genes should raise the attention of integrons as novel antibiotic resistance determinant in Gram-positive bacteria, especially in Enterococcus.201728390978
3043190.9994The role of insertions, deletions, and substitutions in the evolution of R6 related plasmids encoding aminoglycoside transferase ANT-(2"). In 7% of gram-negative bacteria resistance to gentamicin is mainly mediated by plasmid-encoded aminoglycoside transferase ANT-(2"). The genome organization of 15 aadB plasmids (42-110 kb) was analyzed by restriction and hybridization techniques. They appeared to be IncFII-like replicons but were distinct from R6 by virtue of small substitutions in the transfer region. Aminoglycoside resistance genes aadB and aadA were located on Tn21 related elements. Only one of them was able to transpose its resistance genes mer sul aadA and aadB ( Tn4000 ), the other elements were naturally occurring defective transposons. In some of these structures deletions were identified at the termini, at sul, aadA , mer or transposition function--insertions adjacent to aadA or mer. The mode of these rearrangements and their site-specificity were considered with respect to the evolution of the Tn21 transposon family.19846328217