scholarly journals IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Christopher J. Harmer ◽  
Ruth M. Hall
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
High Frequency ◽  
Antibiotic Resistance Genes ◽  
Kanamycin Resistance ◽  
Gram Negative Bacteria ◽  
Critical Determinant ◽  
Gram Negative ◽  
Precise Excision ◽  
Replicative Transposition

ABSTRACTWe 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 IS26from a compound transposon bounded by IS26. In arecAmutant strain, Tn4352, a kanamycin resistance transposon carrying theaphA1agene, 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 IS26transposase Tnp26. However, the Tnp26 of only one IS26in Tn4352B was required, specifically the IS26downstream of theaphA1agene, 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 IS26that is upstream of theaphA1agene. Transcription oftnp26was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end.IMPORTANCEResistance to antibiotics limits treatment options. In Gram-negative bacteria, IS26plays 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, IS26can use a conservative movement mechanism in which an incoming IS26targets 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 IS26by examining whether the reverse precise excision reaction is also catalyzed by the IS26transposase.

scholarly journals IS26-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes

mSphere ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Christopher J. Harmer ◽  
Ruth M. Hall
Keyword(s):  
Antibiotic Resistance ◽  
Homologous Recombination ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Class I ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type ◽  
Movement Mechanism

ABSTRACT 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. 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.

scholarly journals Movement of IS26-Associated Antibiotic Resistance Genes Occurs via a Translocatable Unit That Includes a Single IS26 and Preferentially Inserts Adjacent to Another IS26

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Christopher J. Harmer ◽  
Robert A. Moran ◽  
Ruth M. Hall
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Gene ◽  
Resistance Genes ◽  
Antibiotic Resistance Gene ◽  
Antibiotic Resistance Genes ◽  
Class I ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Unusual Structure ◽  
Gram Negative

ABSTRACTThe insertion sequence IS26plays 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 IS26movement 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 IS26such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26at each end and between each unique segment. This model requires that IS26recognizes another IS26as 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 IS26or 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 IS26movement when a target IS26was 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 IS26target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency.IMPORTANCEResistance 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, IS26is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS26copies, 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 IS26movement do not resemble those of the IS and class I transposons studied to date. IS26uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called “translocatable units.” The IS26mechanism 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.

Extended Spectrum Beta Lactamase (ESBL), bla TEM,bla SHVand bla CTX-M,resistance genes in Community and Healthcare Associated Gram Negative Bacteria from Osun State, Nigeria

2020 ◽  
Vol 20 ◽  
Author(s):  
Ganiyat Shitta ◽  
Olufunmilola Makanjuola ◽  
Olusolabomi Adefioye ◽  
Olugbenga Adekunle Olowe
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Gram Negative Bacteria ◽  
Beta Lactamase ◽  
Multiple Antibiotic Resistance ◽  
Esbl Production ◽  
Gram Negative ◽  
Extended Spectrum Beta Lactamase ◽  
Healthcare Associated ◽  
Esbl Producers

Background: Extended Spectrum Beta Lactamase (ESBL) production in gram negative bacteria confers multiple antibiotic resistance, adversely affecting antimicrobial therapy in infected individuals. ESBLs result from mutations in β-lactamases encoded mainly by the bla TEM,bla SHVand bla CTX-Mgenes. The prevalence of ESBL producing bacteria has been on the increase globally especially its upsurge among isolates from community-acquired infections. Aim: To determine ESBL prevalence and identify ESBL genes among clinical isolates in Osun State, Nigeria. Material and Methods: A cross-sectional study was carried out from August 2016 –July 2017 in Osun State, Nigeria. Three hundred and sixty Gram negative bacteria recovered from clinical samples obtained from both community and healthcare associated infections were tested. They included147 Escherichia coli(40.8%), 116 Klebsiella spp(32.2%), 44 Pseudomo-nas aeruginosa(12.2%) and23 Proteus vulgaris (6.4%) isolates. Others were Acinetobacter baumannii, Serratia rubidae, Citrobacter spp, Enterobacter spp and Salmonella typhi. Disk diffusion antibiotic susceptibility testing was carried out, isolates were screened for ESBL production and confirmed using standard laboratory procedures. ESBLs resistance genes were identified by Polymerase Chain Reaction (PCR). Results: All isolates demonstrated multiple antibiotic resistance. Resistance to ampicillin, amoxicillin with clavulanate and erythromycin was 100%, whereas resistance to Imipenem was very low (5.0%). : Overall prevalence of ESBL producers was 41.4% with Klebsiellaspp as the highest ESBL producing Enterobacteriacaea. ESBL producers were more prevalent among the hospital pathogens than community pathogens, 58% vs 29.5% (p=0.003). ESBL genes were detected in all ESBL producers with the blaCTX-Mgene predominating (47.0%) followed by blaTEM(30.9%) and blaSHVgene was the least, 22.1%. The blaCTX-Mgene was also the most prevalent in the healthcare pathogens (62%) but it accounted for only 25% in those of community origin. Conclusion: A high prevalence of ESBL producing gram negative organisms occurs both in healthcare and in the community in our environment with the CTX-M variant predominating. Efforts to control spread of these pathogens should be addressed.

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