Distribution and expression of the aac(6')-Im (aacA16) aminoglycoside resistance gene.

BACKGROUND: The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. OBJECTIVES: To identify sources for the aac(6')-Im fragment found in A. baumannii. METHODS: MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. RESULTS: Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different. CONCLUSIONS: The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.

Aminoglycoside resistance genes in early members of the Acinetobacter baumannii ST78A (SMAL, Italian clone) reside in an IS26-bounded island in the chromosome.

BACKGROUND: The Acinetobacter baumannii isolate called SMAL, previously used to determine the structures of capsular polysaccharide and lipooligosaccharide, was recovered in Pavia, Italy in 2002 among the collection of aminoglycoside-resistant isolates designated as SMAL type. This type was later called the Italian clone, then ST78. ST78 isolates are now widely distributed. OBJECTIVES: To establish the resistance gene complement and the location and structure of acquired resistance regions in early members of the Italian/ST78 clone. METHODS: The draft genome of SMAL2002 was assembled from Illumina MiSeq reads. Contigs containing resistance genes were joined and located in the chromosome using PCR with custom primers. The resistance profile was determined using disc diffusion. RESULTS: SMAL2002 is an ST78A isolate and includes three aminoglycoside resistance genes, aadB (gentamicin, kanamycin, tobramycin) aphA1 (kanamycin, neomycin) and aac(6')-Ian (amikacin, kanamycin, tobramycin). The aadB gene cassette is incorporated at a secondary site in a relative of the aphA1-containing, IS26-bounded pseudo-compound transposon, PTn6020. The aac(6')-Ian gene is in an adjacent IS26-bounded structure that includes sul2 (sulphonamide) and floR (florfenicol) resistance genes. The two pseudo-compound transposons overlap and are in the chromosomal hutU gene flanked by an 8 bp target site duplication. Although aac(6')-Ian was not noticed previously, the same genes and structures were found in several available draft genomes of early ST78A isolates. CONCLUSIONS: This study highlights the importance of correlating resistance profiles with resistance gene content. The location of acquired resistance genes in the SMAL2002 chromosome represents the original location in the ST78A lineage of ST78.

IS26 and the IS26 family: versatile resistance gene movers and genome reorganizers.

SUMMARYIn Gram-negative bacteria, the insertion sequence IS26 is highly active in disseminating antibiotic resistance genes. IS26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS26 can also enhance expression of adjacent potential resistance genes. IS26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS26. TU formed by homologous recombination between the bounding IS26s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS26 is the best characterized IS in the IS26 family, which includes IS257/IS431, ISSau10, IS1216, IS1006, and IS1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens.

Acinetobacter baumannii GC2 Sublineage Carrying the aac(6')-Im Amikacin, Netilmicin, and Tobramycin Resistance Gene Cassette.

The aminoglycoside antibiotics amikacin, gentamicin, and tobramycin are important therapeutic options for Acinetobacter iinfections. Several genes that confer resistance to one or more of these antibiotics are prevalent in the globally distributed resistant clones of Acinetobacter baumannii, but the aac(6')-Im (aacA16) gene (amikacin, netilmicin, and tobramycin resistance), first reported in isolates from South Korea, has rarely been reported since. In this study, GC2 isolates (1999 to 2002) from Brisbane, Australia, carrying aac(6')-Im and belonging to the ST2:ST423:KL6:OCL1 type were identified and sequenced. The aac(6')-Im gene and surrounds have been incorporated into one end of the IS26-bounded AbGRI2 antibiotic resistance island and are accompanied by a characteristic 70.3-kbp deletion of adjacent chromosome. The compete genome of the 1999 isolate F46 (RBH46) includes only two copies of ISAba1 (in AbGRI1-3 and upstream of ampC) but later isolates, which differ from one another by <10 single nucleotide differences (SND), carry two to seven additional shared copies. Several complete GC2 genomes with aac(6')-Im in an AbGRI2 island (2004 to 2017; several countries) found in GenBank and two additional Australian A. baumannii isolates (2006) carry different gene sets, KL2, KL9, KL40, or KL52, at the capsule locus. These genomes include ISAba1 copies in a different set of shared locations. The distribution of SND between F46 and AYP-A2, a 2013 ST2:ST208:KL2:OCL1 isolate from Victoria, Australia, revealed that a 640-kbp segment that includes KL2 and the AbGRI1 resistance island replaces the corresponding region in F46. Over 1,000 A. baumannii draft genomes also include aac(6')-Im, indicating that it is currently globally disseminated and significantly underreported. IMPORTANCE Aminoglycosides are important therapeutic options for treatment of Acinetobacter infections. Here, we show that a little-known aminoglycoside resistance gene, aac(6')-Im (aacA16), that confers amikacin, netilmicin, and tobramycin resistance has been circulating undetected for many years in a sublineage of A. baumannii global clone 2 (GC2), generally with a second aminoglycoside resistance gene, aacC1, which confers resistance to gentamicin. These two genes are commonly found together in GC2 complete and draft genomes and globally distributed. One isolate appears to be ancestral, as its genome contains few ISAba1 copies, providing insight into the original source of this insertion sequence (IS), which is abundant in most GC2 isolates. Tracking ISAba1 spread can provide a simple means to track the development and ongoing evolution as well as the dissemination of specific lineages and detect the formation of many sublineages. The complete ancestral genome will provide an essential base point for tracking this process.

The RuvABC Holliday Junction Processing System Is Not Required for IS26-Mediated Targeted Conservative Cointegrate Formation.

The insertion sequence IS26 plays a key role in the spread of antibiotic resistance genes in Gram-negative bacteria. IS26 and members of the IS26 family are able to use two distinct mechanisms to form cointegrates made up of two DNA molecules linked via directly oriented copies of the IS. The well-known copy-in (formerly replicative) reaction occurs at very low frequency, and the more recently discovered targeted conservative reaction, which joins two molecules that already include an IS, is substantially more efficient. Experimental evidence has indicated that, in the targeted conservative mode, the action of Tnp26, the IS26 transposase, is required only at one end. How the Holliday junction (HJ) intermediate generated by the Tnp26-catalyzed single-strand transfer is processed to form the cointegrate is not known. We recently proposed that branch migration and resolution via the RuvABC system may be needed to process the HJ; here, we have tested this hypothesis. In reactions between a wild-type and a mutant IS26, the presence of mismatched bases near one IS end impeded the use of that end. In addition, evidence of gene conversion, potentially consistent with branch migration, was detected in some of the cointegrates formed. However, the targeted conservative reaction occurred in strains that lacked the recG, ruvA, or ruvC genes. As the RuvC HJ resolvase is not required for targeted conservative cointegrate formation, the HJ intermediate formed by the action of Tnp26 must be resolved by an alternate route. IMPORTANCE In Gram-negative bacteria, the contribution of IS26 to the spread of antibiotic resistance and other genes that provide cells with an advantage under specific conditions far exceeds that of any other known insertion sequence. This is likely due to the unique mechanistic features of IS26 action, particularly its propensity to cause deletions of adjacent DNA segments and the ability of IS26 to use two distinct reaction modes for cointegrate formation. The high frequency of the unique targeted conservative reaction mode that occurs when both participating molecules include an IS26 is also key. Insights into the detailed mechanism of this reaction will help to shed light on how IS26 contributes to the diversification of the bacterial and plasmid genomes it is found in. These insights will apply more broadly to other members of the IS26 family found in Gram-positive as well as Gram-negative pathogens.

Identification of an Outbreak Cluster of Extensively Antibiotic-Resistant GC1 Acinetobacter baumannii Isolates in U.S. Military Treatment Facilities.

An outbreak involving an extensively antibiotic-resistant Acinetobacter baumannii strain in three military treatment facilities was identified. Fifty-nine isolates recovered from 30 patients over a 4-year period were found among a large collection of isolates using core genome multilocus sequence typing (MLST). They differed by only 0 to 18 single nucleotide polymorphisms (SNPs) and carried the same resistance determinants except that the aphA6 gene was missing in 25 isolates. They represent a novel sublineage of GC1 lineage 1 that likely originated in Afghanistan. IMPORTANCE A. baumannii is recognized as one of the most important nosocomial pathogens, and carbapenem-resistant strains pose a particularly difficult treatment challenge. Outbreaks linked to this pathogen are reported worldwide, particularly during periods of societal upheaval, such as natural disasters and conflicts. Understanding how this organism enters and establishes itself within the hospital environment is key to interrupting transmission, but few genomic studies have examined these transmissions over a prolonged period. Though historical, this report provides an in-depth analysis of nosocomial transmission of this organism across continents and within and between different hospitals.

Characterisation of an early South African multiply antibiotic-resistant global clone 1 Acinetobacter baumannii isolate.

OBJECTIVES: The aim of this study was to characterise an early clinical multiply antibiotic resistant Acinetobacter baumannii global clone 1 (GC1) isolate from Africa. METHODS: The draft genome sequence was determined using short-read (Illumina MiSeq) sequence data and compared to other early GC1 isolates. Resistance genes and other features were identified using various bioinformatics tools. Plasmids were visualised. RESULTS: LUH6050, recovered in South Africa between January 1997 and January 1999, is ST1IP:ST231Ox:KL1:OCL1. Several antibiotic resistance genes (aacC1, aadA2, aphA1, catA1, sul1, and tetA(A)) reside in AbaR32. LUH6050 also includes the plasmid pRAY*, carrying the aadB gentamicin and tobramycin resistance gene, and a 29.9 kb plasmid, pLUH6050-3, carrying the msrE-mphE (macrolide resistance) and dfrA44 (trimethoprim resistance) genes and a small cryptic Rep_1 plasmid. Plasmid pLUH6050-3, a cointegrate of pA1-1 (R3-T1; RepAci1) with an R3-T33 type plasmid encoding a different Rep_3 family Rep, carries 15 pdif sites and 13 dif modules, including those that carry the mrsE-mphE and dfrA44 genes and three that include toxin-antitoxin gene pairs. The closest relative of pLUH6050-3 found in GenBank was from an unrelated 2013 Tanzanian A. baumannii isolate. The chromosome has an AbaR0-type region in comM and includes no ISAba1 copies. Similar features were found in most other sequenced lineage 1 GC1 isolates recovered prior to 2000. CONCLUSION: LUH6050 represents an early form of the GC1 lineage 1, supplementing limited information about early isolates and isolates from Africa. These data contribute to the understanding of the emergence, evolution, and dissemination of the A. baumannii GC1 clonal complex.

Insertion sequences related to ISAjo2 target pdif and dif sites and belong to a new IS family, the IS1202 family.

Several insertion sequences (IS) found in various Acinetobacter species exhibit target specificity. They are found, in the same orientation, 5 bp from the XerC binding site of the pdif sites associated with dif modules in Acinetobacter plasmids, and searches revealed they are also found near chromosomal dif sites of Acinetobacter species. These IS are 1.5 kb long, bounded by 24-26 bp imperfect terminal inverted repeats (TIRs) and encode a large transposase of 441-457 aa. They generate 5 bp target site duplications (TSDs). Structural predictions of the ISAjo2 transposase, TnpAjo2, modelled on TnsB of Tn7 revealed two N-terminal HTH domains followed by an RNaseH fold (DDE domain), a ß barrel and a C-terminal domain. Similar to Tn7, the outer IS ends are 5'-TGT and ACA-3', and an additional Tnp binding site, corresponding to the internal portion of the IR, is found near each end. However, the Acinetobacter IS do not encode further proteins related to those required by Tn7 for targeted transposition, and the transposase may interact directly with XerC bound to a dif-like site. We propose that these IS, currently in the IS1202 group in the not characterized yet (NCY) category in ISFinder, are part of a distinct IS1202 family. Other IS listed as in the IS1202 group encode transposases related to TnpAjo2 (25-56 % amino acid identity) and have similar TIRs but fall into three groups based on the TSD length (3-5, >15, 0 bp). Those with 3-5 bp TSDs may also target dif-like sites but targets were not found for the other groups.

IS26-mediated loss of the translocatable unit from Tn4352B requires the presence of the recA1 allele.

The pseudo-compound transposon Tn4352B is unusual in that the translocatable unit (TU) consisting of one of the bounding IS26 copies and the central portion containing the aphA1a gene has been found to be readily lost in the Escherichia coli strains used as host. Rapid loss required the presence of an additional 2 G residues adjacent to the internal end of one of the IS26 that flank the central portion and an active Tnp26 transposase. However, Tn4352B was found to be stable in wild-type Klebsiella pneumoniae strains. Though it was concluded that the difference may be due to the species background, the E. coli strains used were recombination-deficient. Here, we have further investigated the requirements for TU loss in E. coli and found that Tn4352B was stable in recombination-proficient strains. Among several recombination-deficient strains examined, rapid loss occurred only in strains that carry the recA1 allele but not in strains carrying different recA alleles, recA13 and a novel recA allele identified here, that also render the strain deficient in homologous recombination. Hence, it appears that a specific property of the RecA1 protein underlies the observed TU loss from Tn4352B.

Mechanisms of IS26-Mediated Amplification of the aphA1 Gene Leading to Tobramycin Resistance in an Acinetobacter baumannii Isolate.

Enhanced levels of resistance to antibiotics arising from amplification of an antibiotic resistance gene that impact therapeutic options are increasingly observed. Amplification can also disclose novel phenotypes leading to treatment failure. However, the mechanism is poorly understood. Here, the route to amplification of the aphA1 kanamycin and neomycin resistance gene during tobramycin treatment of an Acinetobacter baumannii clinical isolate, leading to tobramycin resistance and treatment failure, was investigated. In the tobramycin-susceptible parent isolate, MRSN56, a single copy of aphA1 is present in the pseudocompound transposon PTn6020, bounded by directly oriented copies of IS26. For two clinical resistant isolates, new long-read sequence data were combined with available short-read data to complete the genomes. Comparison to the completed genome of MRSN56 revealed that, in both cases, IS26 had generated a circular translocatable unit (TU) containing PTn6020 and additional adjacent DNA. In one case, this TU was reincorporated into the second product generated by the deletion that formed the TU via the targeted conservative route and amplified about 7 times. In the second case, the TU was incorporated at a new location via the copy-in route and amplified about 65 times. Experimental amplification ex vivo by subjecting MRSN56 to tobramycin selection pressure yielded different TUs, which were incorporated at either the original location or a new location and amplified many times. The outcomes suggest that when IS26 is involved, amplification occurs via rolling circle replication of a newly formed TU coupled to the IS26-mediated TU formation or reincorporation step. IMPORTANCE Heteroresistance, a significant issue that is known to impact antibiotic treatment outcomes, is caused by the presence of spontaneously arising cells with elevated levels of resistance to therapeutically important antibiotics in a population of susceptible cells. Gene amplification is one well-documented cause of heteroresistance, but precisely how extensive amplification occurs is not understood. Here, we establish the case for the direct involvement of IS26 activity in the amplification of the aphA1 gene to disclose resistance to tobramycin. The aphA1 gene is usually found associated with IS26 in Gram-negative pathogens and is commonly found in extensively resistant Acinetobacter baumannii strains. IS26 and related IS cause adjacent deletions, forming a nonreplicating circular molecule known as a translocatable unit (TU), and amplification via a rolling circle mechanism appears to be coupled to either IS26-mediated TU formation or reincorporation. Related IS found in Gram-positive pathogens may play a similar role.

Complete genome of the extensively antibiotic-resistant GC1 Acinetobacter baumannii isolate MRSN 56 reveals a novel route to fluoroquinolone resistance.

OBJECTIVES: To examine the causes of antibiotic resistance in the extensively resistant global clone 1 (GC1) Acinetobacter baumannii isolate MRSN 56 recovered at a US military treatment facility. METHODS: MRSN 56 was sequenced using MinION (Oxford Nanopore) and the reads combined with available Illumina MiSeq data using Unicycler. Acquired resistance genes were identified using ABRicate and their environment examined. ISAba1 and ISAba125 copies were located. RESULTS: MRSN 56 is ST1IP:ST231Ox:KL1:OCL1 and the complete genome includes four small plasmids, none of which carry resistance genes. The acquired resistance genes were found at four locations in the chromosome in addition to AbaR28 (aphA1, aacC1, aadA1, sul1) in comM. Tn2006 (oxa23, carbapenem resistance) was both in AbaR4 and alone elsewhere. Two copies of Tn7 (dfrA1, sat, aadA1) were identified. One was associated with a 22 852 bp adjacent segment [tetA(B), sul2] derived from the AbGRI1 island, and this novel configuration was designated Tn7+. Tn7+ was incorporated in the position preferred by Tn7, downstream of glmS, by transposition using a sequence in AbGRI1 resembling the Tn7 terminal inverted repeats. Tn7 was found at a secondary site. Fluoroquinolone resistance appears to involve a mutation in gyrA combined with inactivation by ISAba1 of the marR gene in the mar operon and constitutive expression of marA from the promoter internal to ISAba1. CONCLUSIONS: MRSN 56 represents a new sublineage of GC1 lineage 1 with novel features that had not been detected previously. The involvement of the mar operon in fluoroquinolone resistance has not been noted previously.

Evolution of Acinetobacter baumannii plasmids carrying the oxa58 carbapenemase resistance gene via plasmid fusion, IS26-mediated events and dif module shuffling.

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.

Variants of Tn6924, a Novel Tn7 Family Transposon Carrying the blaNDM Metallo-ß-Lactamase and 14 Copies of the aphA6 Amikacin Resistance Genes Found in Acinetobacter baumannii.

Carbapenem resistance in Acinetobacter baumannii is primarily due to the global spread of two main clones that carry oxa23, oxa24, and oxa58. However, new carbapenem-resistant clones are emerging that are also resistant to a wide range of antibiotics. Strains belonging to ST85IP (Institut Pasteur) carry the blaNDM metallo-ß-lactamase carbapenem resistance gene. Here, we completed the genome sequence of an ST85IP strain, Cl300, recovered in 2015 in Lebanon, using a combination of Illumina MiSeq and Oxford Nanopore sequencing and a hybrid assembly approach. Cl300 is highly resistant to meropenem and amikacin, and consistent with this, a copy of the blaNDM carbapenem and 14 copies of the aphA6 amikacin resistance genes were found in the genome. Cl300 also contains the sul2 sulfonamide and the msr(E) macrolide resistance genes. All aphA6 copies and blaNDM are in a novel 76-kb Tn7 family transposon designated Tn6924. Like Tn7, Tn6924 is bounded by 29-bp inverted repeats with additional TnsB binding sites at each end. Several variants of Tn6924 were found in a set of diverse strains, including ST85IP strains as well as members of global clones 1 and 2. sul2 and msr(E) are in a 13.0-kb pseudocompound transposon (PCT) bounded by IS1008. ST85s represent a diverse group of strains, particularly in their antibiotic resistance gene content and the K and OC surface polysaccharide loci. Acquisition of Tn6924 by members of global clones indicates the significance of this transposon in spreading two clinically significant resistance genes, blaNDM and aphA6. IMPORTANCE To date, efforts to study the resistance mechanisms of carbapenem-resistant Acinetobacter baumannii have been largely focused on the two major globally distributed clones (GC1 and GC2). ST85 is an emerging sequence type, and unlike other clones, it is associated with the carriage of the blaNDM gene. Here, we completed the genome sequence of an ST85 strain and showed that blaNDM and 14 copies of the aphA6 amikacin resistance genes are in Tn6924, a novel Tn7 family transposon. Analysis of all publicly available ST85s predicted that all strains in the main lineage carry a variant of Tn6924. Variants of Tn6924 were also found in other clones, including GC1 and GC2. Tn6924 is an important mobile element given that it carries two clinically important resistance genes (blaNDM and aphA6) and has spread to other clones. Therefore, outbreaks caused by ST85s should be studied and tracked.

Characterization of the specific DNA-binding properties of Tnp26, the transposase of insertion sequence IS26.

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1-P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix-turn-helix domain (amino acids I13-R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1-D12) did not bind. The N-terminal M1-P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1-M234) and Tnp26 M1-P56 fragment. These findings indicate that the helix-turn-helix and disordered domains of Tnp26 play a role in Tnp26-TIR complex formation. Both domains are conserved in all members of the IS26 family.

HI1 and I1 Resistance Plasmids from Salmonella enterica Serovar Typhimurium Strain SRC27 Are Epidemic.

Conjugative plasmids are a major contributor to the global spread of antibiotic resistance determinants, but the tracking of their evolutionary history is often neglected. Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SRC27 was isolated from an equine infection in Australia in 1999. SRC27 was known to carry conjugative HI1 and I1 resistance plasmids. In this study, SRC27 was sequenced to determine the relationship between these HI1 and I1 resistance plasmids it was known to carry and HI1 and I1 resistance plasmids circulating worldwide. The resistance genes in the HI1 plasmid, pSRC27-H, are all located in a single complex 34.7 kb resistance region. The backbone sequence and location of the pSRC27-H resistance island were used to identify the most closely related HI1 plasmids among the >90 that have been sequenced since 2011. This defined a sublineage of 20 type 2 HI1 plasmids that have been circulating in Europe, Asia, North America, and Australia since at least 1993. The overall resistance gene content of these HI1 plasmids differs, indicating extensive evolution in situ through the acquisition of additional transposons and deletion or replacement of ancestral regions. The I1 plasmid contains a complete copy of Tn5393a, containing the strAB genes that confer resistance to streptomycin. The precise location of Tn5393a in the backbone also defined a globally disseminated sublineage of I1 plasmids, many of which have also acquired additional resistance determinants. The sequence revealed that SRC27 also carried two additional plasmids, the pSLT-type FIB(S):FII(S) virulence plasmid and a small cryptic theta-replicating Col156 plasmid.

IS26 cannot move alone.

BACKGROUND: IS26 plays a major role in the dissemination of antibiotic resistance determinants in Gram-negative bacteria. OBJECTIVES: To determine whether insertion sequence IS26 is able to move alone (simple transposition) or if it exclusively forms cointegrates. METHODS: A two-step PCR using outward-facing primers was used to search for circular IS26 molecules. Gibson assembly was used to clone a synthetic IS26 containing a catA1 chloramphenicol resistance gene downstream of the tnp26 transposase gene into pUC19. IS activity in a recA-Escherichia coli containing the non-conjugative pUC19-derived IS26::catA1 construct and the conjugative plasmid R388 was detected using a standard mating-out assay. Transconjugants were screened for resistance. RESULTS: Circular IS26 molecules that would form with a copy-out route were not detected by PCR. The synthetic IS26::catA1 construct formed CmRTpR transconjugants (where CmR and TpR stand for chloramphenicol resistant and trimethoprim resistant, respectively), representing an R388 derivative carrying the catA1 gene at a frequency of 5.6 × 10-7 CmRTpR transconjugants per TpR transconjugant, which is comparable to the copy-in activity of the unaltered IS26. To test for simple transposition of IS26::catA1 (without the plasmid backbone), 1200 CmRTpR colonies were screened and all were resistant to ampicillin, indicating that the pUC19 backbone was present. Hence, IS26::catA1 had only formed cointegrates. CONCLUSIONS: IS26 is unable to move alone and cointegrates are the exclusive end products of the reactions mediated by the IS26 transposase Tnp26. Consequently, when describing the formation of complex resistance regions, simple 'transposition' of a single IS26 should not be invoked.

Targeted Conservative Cointegrate Formation Mediated by IS26 Family Members Requires Sequence Identity at the Reacting End.

IS26 forms cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target and a targeted conservative mechanism involving two ISs in different DNA molecules. In this study, the ability of IS26 and some close relatives, IS1006, IS1008, and a natural hybrid, IS1006/IS1008, which are found predominantly in Acinetobacter spp., to interact was examined. IS1006/1008 consists of 175 bp from IS1006 at the left end, with the remainder from IS1008. These ISs all have the same 14-bp terminal inverted repeats, and the Tnp26, Tnp1006, and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognize each other's ends. In a recA-negative Escherichia coli strain, IS1006, IS1008, and IS1006/1008 each formed cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS26 However, using mixed pairs, targeted cointegration was detected only when IS1008 was paired with the IS1006/1008 hybrid, which also encodes Tnp1008, and the targeted cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS26 paired with IS26::catA1, an artificially constructed IS26 derivative that includes the catA1 gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalyzed strand transfer, followed by migration and resolution of the Holliday junction formed.IMPORTANCE The IS26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS26 seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS26 family members. However, the mechanism underlying the recently discovered targeted conservative mode of cointegrate formation mediated by IS26, IS257/IS431, and IS1216, which is unlike any previously studied IS movement mechanism, is not well understood. An important question is what features of the IS and the transposase are key to allowing IS26 family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS26 and naturally occurring close relatives of IS26 that are found near resistance genes in Acinetobacter baumannii and are widespread in Acinetobacter species.

Structures bounded by directly-oriented members of the IS26 family are pseudo-compound transposons.

Antibiotic resistance genes are often found in structures bounded by copies of IS26, IS257/IS431 or IS1216 that resemble compound (or composite) transposons. However, because of the mechanisms used by IS26 family members, namely that they form cointegrates but cannot resolve them, none of these structures can move together as a coherent single unit. Apparent transposition of these structures is possible via a 2-step process but only if the IS are in direct orientation. An intermolecular reaction catalysed by the IS-encoded transposase and an intramolecular homologous recombination step can occur in either order. In one route, one of the IS bounding the structure forms a cointegrate between the DNA molecule carrying it and a target molecule. Cointegrates formed by either copy-in or targeted conservative routes contain three directly-oriented IS copies and can be resolved by homologous recombination between specific pairs of IS, with one pair leading to apparent transposition of the whole structure. In the other route, homologous recombination first forms a circular intermediate, a translocatable unit or TU, which is incorporated by the transposase either at a random site or adjacent to another IS copy in a target molecule. We therefore conclude that the transposon-like structures are not compound (or composite) transposons and the nomenclature for them should be revised. We propose that the term "pseudo compound transposon" (PCT), first coined in 1989, should be used to describe those structures where the IS are in direct orientation. Structures with the IS in opposite orientation should not be named as transposons.

IS26 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.