A mobile restriction modification system consisting of methylases on the IncA/C plasmid. - Related Documents




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449401.0000A mobile restriction modification system consisting of methylases on the IncA/C plasmid. BACKGROUND: IncA/C plasmids play important roles in the development and dissemination of multidrug resistance in bacteria. These plasmids carry three methylase genes, two of which show cytosine specificity. The effects of such a plasmid on the host methylome were observed by single-molecule, real-time (SMRT) and bisulfite sequencing in this work. RESULTS: The results showed that the numbers of methylation sites on the host chromosomes were changed, as were the sequences recognized by MTase. The host chromosomes were completely remodified by the plasmid with a methylation pattern different from that of the host itself. When the three dcm genes were deleted, the transferability of the plasmid into other Vibrio cholerae and Escherichia coli strains was lost. During deletion of the dcm genes, except for the wild-type strains and the targeted deletion strains, 18.7%~ 38.5% of the clones lost the IncA/C plasmid and changed from erythromycin-, azithromycin- and tetracycline-resistant strains to strains that were sensitive to these antibiotics. CONCLUSIONS: Methylation of the IncA/C plasmid was a new mobile restriction modification (RM) barrier against foreign DNA. By actively changing the host's methylation pattern, the plasmid crossed the barrier of the host's RM system, and this might be the simplest and most universal method by which plasmids acquire a broad host range. Elimination of plasmids by destruction of plasmid stability could be a new effective strategy to address bacterial multidrug resistance.201931182978
446510.9997Genetic 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
446720.9997PCR 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
494730.9997Use of plasmid profiles in epidemiologic surveillance of disease outbreaks and in tracing the transmission of antibiotic resistance. Plasmids are circular deoxyribonucleic acid molecules that exist in bacteria, usually independent of the chromosome. The study of plasmids is important to medical microbiology because plasmids can encode genes for antibiotic resistance or virulence factors. Plasmids can also serve as markers of various bacterial strains when a typing system referred to as plasmid profiling, or plasmid fingerprinting is used. In these methods partially purified plasma deoxyribonucleic acid species are separated according to molecular size by agarose gel electrophoresis. In a second procedure, plasmid deoxyribonucleic acid which has been cleaved by restriction endonucleases can be separated by agarose gel electrophoresis and the resulting pattern of fragments can be used to verify the identity of bacterial isolates. Because many species of bacteria contain plasmids, plasmid profile typing has been used to investigate outbreaks of many bacterial diseases and to trace inter- and intra-species spread of antibiotic resistance.19882852997
997240.9997Extensive 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
491350.9997Multiple Plasmids Contribute to Antibiotic Resistance and Macrophage Survival In Vitro in CMY2-Bearing Salmonella enterica. Multiple drug resistance (MDR) in bacteria represents a notable problem but if carried on plasmid their spread could become a significant threat to public health. Plasmids in members of the Enterobacteriaceae family and in particular Salmonella and Escherichia coli strains have been implicated in the spread of antibiotic resistance genes. However, the mechanisms involved in the transfer of plasmid-borne resistance genes are not fully understood. Here, we analyzed the ability of Salmonella enterica clinical isolates to transfer plasmid-borne MDR to E. coli. We also determined whether possession of an Inc A/C plasmid by a S. enterica isolate would confer increased fitness compared to an isolate not carrying the plasmid. Sixteen human and animal isolates of S. enterica were screened using a three-panel multiplex PCR assay, and simplex PCR for the blaCMY-2 gene. Using these data we selected a suitable strain as a plasmid donor for the construction of a new Salmonella strain with an Inc A/C plasmid. This allowed us to compare isogenic strains with and without the Inc A/C plasmid in multiple growth, fitness, and invasion assays. The results showed that possession of Inc A/C plasmid confers significant fitness advantage when tested in J774 macrophages as opposed to HEp-2 cells where no significant difference was found. In addition, stress assays performed in vitro showed that the possession of this large plasmid by Salmonella strains tested here does not appear to incur a significant fitness cost. Gaining a better understanding of molecular mechanisms of plasmid transfer between pathogenic bacteria will allow us to characterize the role of MDR in pathogenicity of bacteria and to identify methods to reduce the frequency of dissemination of multiple antibiotic resistance genes.201627070176
997460.9997Role of Plasmids in Co-Selection of Antimicrobial Resistances Among Escherichia coli Isolated from Pigs. Co-selection is thought to occur when resistance genes are located on the same mobile genetic element. However, this mechanism is currently poorly understood. In this study, complete circular plasmids from swine-derived Escherichia coli were sequenced with short and long reads to confirm that resistance genes involved in co-resistance were co-transferred by the same plasmid. Conjugative transfer tests were performed, and multiple resistance genes were transmitted. The genes possessed by the donor, transconjugant, and plasmid of the donor were highly similar. In addition, the sequences of the plasmid of the donor and the plasmid of the transconjugant were almost identical. Resistance genes associated with statistically significant combinations of antimicrobial use and resistance were co-transmitted by the same plasmid. These results suggest that resistance genes may be involved in co-selection by their transfer between bacteria on the same plasmid.202337540099
997570.9997Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli. Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.202337036197
452380.9997Mosaic structure of a multiple-drug-resistant, conjugative plasmid from Campylobacter jejuni. Partial sequence analysis of a tet(O) plasmid from a multiple-drug-resistant clinical isolate of Campylobacter jejuni revealed 10 genes or pseudogenes encoding different aminoglycoside inactivating enzymes, transposase-like genes, and multiple unknown genes from a variety of pathogenic and commensal bacteria. The plasmid could be mobilized by a P incompatibility group plasmid into Escherichia coli, where it apparently integrated into the chromosome and expressed high-level resistance to multiple aminoglycoside antibiotics. This work provides new information about both the nature of drug resistance in C. jejuni and the ability of C. jejuni to exchange genes with other bacterial species.200515917546
452690.9997The 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
420100.9996Transferable nitrofuran resistance conferred by R-plasmids in clinical isolates of Escherichia coli. A high proportion of nitrofuran-resistant strains has been found in a collection of antibiotic-resistant Gram-negative bacteria isolated from patients with urinary tract infections. Some of the Escherichia coli carried R-plasmids that conferred resistance to nitrofurantoin and nitrofurazone. The mechanism of resistance is not clear; only in lactose non-fermenting recipients was there a decrease in the nitrofuran-reducing ability of whole-cell suspensions. One of the plasmids conferred enhanced resistance to UV light on DNA repair defective mutants but not on repair efficient strains. In some resistant strains, the total resistance was apparently the result of a combination of chromosomal and plasmid-borne genes. The presence of the plasmid may allow the development of higher resistance levels by mutation of chromosomal genes.19836368515
4498110.9996A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae. Gene amplifications have been detected as a transitory phenomenon in bacterial cultures. They are predicted to contribute to rapid adaptation by simultaneously increasing the expression of genes clustered on the chromosome. However, genome amplifications have rarely been described in natural isolates. Through DNA array analysis, we have identified two Streptococcus agalactiae strains carrying tandem genome amplifications: a fourfold amplification of 13.5 kb and a duplication of 92 kb. Both amplifications were located close to the terminus of replication and originated independently from any long repeated sequence. They probably arose in the human host and showed different stabilities, the 13.5-kb amplification being lost at a frequency of 0.003 per generation and the 92-kb tandem duplication at a frequency of 0.035 per generation. The 13.5-kb tandem amplification carried the five genes required for dihydrofolate biosynthesis and led to both trimethoprim (TMP) and sulfonamide (SU) resistance. Resistance to SU probably resulted from the increased synthesis of dihydropteroate synthase, the target of this antibiotic, whereas the amplification of the whole pathway was responsible for TMP resistance. This revealed a new mechanism of resistance to TMP involving an increased dihydrofolate biosynthesis. This is, to our knowledge, the first reported case of naturally occurring antibiotic resistance resulting from genome amplification in bacteria. The low stability of DNA segment amplifications suggests that their role in antibiotic resistance might have been underestimated.200818024520
6265120.9996Fitness costs of fluoroquinolone resistance in Streptococcus pneumoniae. The fitness cost of the genes responsible for resistance to fluoroquinolones in clinical isolates of Streptococcus pneumoniae were estimated in vitro in a common genetic background. Naturally occurring parC, parE, and gyrA loci containing mutations in the quinolone-resistance-determining regions were introduced by transformation into S. pneumoniae strain R6 individually and in combinations. The fitness of these transformants was estimated by pairwise competition experiments with a common R6 strain. On average, single par and gyr mutants responsible for low-level MIC resistance (first-step resistance) impose a fitness burden of approximately 8%. Some of these mutants engender no measurable cost, while one, a parE mutant, reduces the fitness of these bacteria by more than 40%. Most interestingly, the addition of the second par or gyr mutations required for clinically significant, high-MIC fluoroquinolone resistance does not increase the fitness burden imposed by these single genes and can even reduce it. We discuss the implications of these results for the epidemiology of fluoroquinolone resistance and the evolution of acquired resistance in treated patients.200717116668
260130.9996Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species. Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.201121538255
4469140.9996Integrons: 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
4527150.9996Study 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
4528160.9996Study 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
4472170.9996Conjugative plasmids in bacteria of the 'pre-antibiotic' era. Antibiotic resistance is common in bacteria that cause disease in man and animals and is usually determined by plasmids. The prevalence of such plasmids, and the range of drugs to which they confer resistance, have increased greatly in the past 25 yr. It has become clear from work in many laboratories that plasmids have acquired resistance genes, of ultimately unknown origin, as insertions into their circular DNA. The intensive use of antibiotics since their introduction in the 1940s can explain the spread of plasmids that have acquired such genes but little is known of the incidence of plasmids in pathogenic bacteria before the widespread use of antibiotics in medicine. E.D.G. Murray collected strains of Enterobacteriaceae from 1917 to 1954; we now report that 24% of these encode information for the transfer of DNA from one bacterium to another. From at least 19% of the strains, conjugative plasmids carrying no antibiotic resistance were transferred to Escherichia coli K-12.19836835408
4499180.9996Organization of two sulfonamide resistance genes on plasmids of gram-negative bacteria. The organization of two widely distributed sulfonamide resistance genes has been studied. The type I gene was linked to other resistance genes, like streptomycin resistance in R100 and trimethoprim resistance in R388 and other recently isolated plasmids from Sri Lanka. In R388, the sulfonamide resistance gene was transcribed from a promoter of its own, but in all other studied plasmids the linked genes were transcribed from a common promoter. This was especially established with a clone derived from plasmid R6-5, in which transposon mutagenesis showed that expression of sulfonamide resistance was completely dependent on the linked streptomycin resistance gene. The type II sulfonamide resistance gene was independently transcribed and found on two kinds of small resistance plasmids and also on large plasmids isolated from clinical material.19873032095
4488190.9996The cfr and cfr-like multiple resistance genes. The Cfr methyl transferase causes an RNA methylation of the bacterial ribosomes impeding reduced or abolished binding of many antibiotics acting at the peptidyl transferase center. It provides multi-resistance to eight classes of antibiotics, most of which are in clinical and veterinary use. The cfr gene is found in various bacteria in many geographical locations and placed on plasmids or associated with transposons. Cfr-related genes providing similar resistance have been identified in Bacillales, and now also in the pathogens Clostridium difficile and Enterococcus faecium. In addition, the presence of the cfr gene has been detected in harbours and food markets.201829378339