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The predominant type of plasmids found in Acinetobacter species encode a Rep_3 initiation protein and many of these carry their accessory genes in dif modules. Here, available sequences of the 14 members of the group of Rep_3 plasmids typed as R3-T33, using a threshold of 95% identity in the repA gene, were compiled and compared. These plasmids were from various Acinetobacter species. The pdif sites were identified allowing the backbone and dif modules to be defined. As for other Rep_3 plasmids carrying dif modules, orfX encoding a protein of unknown function was found downstream of repA followed by a pdif site in the orientation XerC binding site-spacer-XerD binding site. Most backbones (n = 12) also included mobA and mobC genes but the two plasmids with the most diverged repA and orfX genes had different backbone contents. Although the gene content of the plasmid backbone was largely conserved, extensive recombinational exchange was detected and only two small groups carried identical or nearly identical backbones. Individual plasmids were associated with 1 to 13 dif modules. Many different dif modules were identified, including ones containing antibiotic or chromate resistance genes and several toxin/antitoxin gene pairs. In some cases, modules carrying the same genes were significantly diverged. Generally, the orientation of the pdif sites alternated such that C modules (XerC binding sites internal) alternated with D modules (XerD binding sites internal). However, fusions of two dif modules via mutational inactivation or loss of a pdif site were also detected., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The complete genome of RBH2, a sporadic, carbapenem resistant ST111 Acinetobacter baumannii isolate from Brisbane, Australia was determined and analysed. RBH2 is extensively resistant and the chromosome includes two transposons carrying antibiotic resistance genes, AbaR4 (oxa23 in Tn2006) and Tn7::Tn2006 (dfrA1, sat2, aadA1, oxa23). The chromosome also includes two copies of Tn6175, a transposon carrying putative copper resistance genes, and 1-17 copies of six different insertion sequences. RBH2 has six plasmids ranging in size from 6 kb - 141 kb, four carrying antibiotic resistance genes. Plasmids pRBH2-1 (aadB) and pRBH2-2 (aphA6 in TnaphA6) were found to be essentially identical to known plasmids pRAY*-v1 and pS21-1, respectively. The largest plasmids, pRBH2-5 (oxa23 in AbaR4) and pRBH2-6 (oxa23 in AbaR4::ISAba11 and sul2, tet(B), strA and strB in Tn6172) have known transfer-proficient relatives. pRBH2-5, an RP-T1 (RepAci6) plasmid, also carries a different putative copper resistance transposon related to Tn6177 found in pS21-2. The backbone of pRBH2-5 is related to those of previously described RepAci6 plasmids pAb-G7-2 and pA85-3 but has some distinctive features. Three different RepAci6 backbone types were distinguished, Type 1 (pAb-G7-2), Type 2 (pA85-3) and Type 3 (pRBH2-5 and pS21-2). pRBH2-6 is closely related to pAB3 and their backbones differ by only 5 SNPs. Plasmids pRBH2-3 and pRBH2-4 do not carry antibiotic resistance genes. pRBH2-3 does not include an identifiable rep gene and is a novel plasmid type. pRBH2-4 is of the R3-T3 type and includes segments of the larger pABTJ2 that heads this group. Other ST111 genomes carry different plasmids., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

An IncC or IncA plasmid is needed to enable transfer of SGI1 type integrative mobilisable elements but an IncC plasmid does not stably co-exist with SGI1. However, the plasmid is stably maintained with SGI1-K, a natural SGI1 deletion variant that lacks the sgaDC genes (S007 and S006) and the upstream open reading frame (S008) found in the SGI1 backbone. Here, the effect of the sgaDC genes and S008 on the stability of an IncC plasmid in an Escherichia coli strain with or without SGI1-K was examined. Co-transcription of the S008 open reading frame with the downstream sgaDC genes was established. When a strain containing SGI1-K complemented with a pK18 plasmid that included S008-sgaDC or sgaDC expressed from the constitutive pUC promoter was grown without antibiotic selection, the resident IncC plasmid was rapidly lost but loss was slower when S008 was present. In contrast, SGI1-K and the S008-sgaDC or sgaDC plasmid were quite stably maintained for >100 generations. However, the high copy number plasmids carrying the SGI1-derived S008-sgaDC or sgaDC genes constitutively expressed could not be introduced into an E. coli strain carrying the IncC plasmid but without SGI1-K. Using equivalent plasmids with S008-sgaDC or sgaDC genes controlled by an arabinose-inducible promoter, under inducing conditions the IncC plasmid was stable but the plasmid containing the SGI1-derived genes was rapidly lost. This unexpected observation indicates that there are multiple interactions between the IncC plasmid and SGI1 in which the transcriptional activator genes sgaDC play a role. These interactions will require further investigation., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Though IncC and IncA plasmids are compatible, they exert high level exclusion on one another. Here, the question of whether the presence of an SGI1 family element in the donor can overcome the exclusion of an IncC plasmid exerted by an IncC or IncA plasmid in the recipient was investigated. The transfer of the integrative mobilizable element SGI1 and its many variant forms into a new host is dependent on transfer machinery supplied by IncC or IncA plasmids. SGI1 elements include the determinants of a mobilization system and three genes that encode homologues of transfer proteins including TraG. Exclusion of a complete IncC plasmid by a complete IncA or IncC plasmid in the recipient was not ameliorated by an SGI1 element in the donor. However, transfer of the SGI was unaffected indicating that a functional mating apparatus was formed. The presence of only the plasmid-derived eexC or eexA gene in the recipient exerted high level exclusion on an incoming IncC plasmid and this was overcome by an SGI1 variant in the donor. Hence, the SGI affects only entry exclusion and additional plasmid features must influence other routes to plasmid exclusion., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The sequence of a conjugative plasmid, pSRC22-2, found in a multiply antibiotic resistant Salmonella enterica serovar Ohio isolate SRC22 originally cultured from swine in 1999, was determined. Plasmid pSRC22-2 has a copy number of approximately 40 and transfers tetracycline resistance at very high frequency. It was typed as IncX1 using the three typing schemes proposed for X-type plasmids, which utilize the replication region, iteron region and taxC conjugation gene and pSRC22-2 belongs to the X1α subgroup. The plasmid backbone, derived by removing mobile elements, is shared with pOLA52, which was the first fully sequenced IncX1 plasmid, and five other X1α plasmids. The pSRC22-2 backbone is interrupted by a complete copy of an IS903 isoform, partial copies of IS1 and IS903 on either side of a 5930 bp IS26-bounded pseudo-compound transposon (PCT), and a novel 256 bp miniature inverted repeat transposable element (MITE). The MITE belongs to the Tn3 family and was named MITESen1. The PCT, which carries a tet(C) tetracycline resistance determinant, is bounded by copies of a novel IS26 variant, IS26-v4, and was designated PTn6184. Comparison of PTn6184 with other tet(C)-carrying PCTs revealed that it can be derived from the largest, PTntet(C), via a two-step process that re-orders the central fragment and involves both an IS26-mediated event and homologous recombination. IS26-v4, which encodes a variant transposase, Tnp26 G184D, has appeared in only 46 entries in the GenBank non-redundant database., (Copyright © 2021. Published by Elsevier Inc.)

A large plasmid, pCERC14, found in an antibiotic resistant commensal Escherichia coli isolate recovered from a healthy adult was sequenced. pCERC14 was 162,926 bp and carried FII-18 and FIB-1 replicons and an F-like transfer region as well as several virulence determinants, some of which are involved in the uptake of iron which would be advantageous for the commensal lifestyle. The plasmid backbone is interrupted in 11 places by complete IS (IS1 (4 copies), IS2 (2), IS629 (2) and single IS100, IS186, ISEc33) and in three places by partial IS copies. The antibiotic resistance genes were found in two IS26-bounded pseudo-compound transposons (PCT). One contained a remnant of a class 1 integron that includes a dfrA5 gene cassette and the sul1 gene conferring resistance to trimethoprim and sulphonamides, respectively. The second, named PTntet(C)-var, contained a 4828 bp DNA segment that includes the tet(C) tetracycline resistance determinant. As tet(C) is relatively rare in E. coli and other Gram-negative bacterial isolates, the structure and evolution of tet(C)-containing PCT in available sequences was examined. The largest identified was PTntet(C), a close relative of PTntet(C)-var, and a potential progenitor for these PCT. Most PCT shared one internal boundary with PTntet(C) but the length of the central tet(C)-containing segment was shorter due to IS26-mediated deletions. The most abundant variant form, previously named Tn6309, was widely distributed and, in a derivative of it, most of the tetA(C) gene has been replaced by the tetA(A) gene presumably by homologous recombination., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Several groups of integrative mobilizable elements (IMEs) that harbour a class 1 integron carrying antibiotic resistance genes have been found at the 3'-end of the chromosomal trmE gene. Here, a new IME, designated SGI0, was found in trmE in the sequenced and assembled genome of a French clinical, multiply antibiotic resistant Proteus mirabilis strain, Pm1LENAR. SGI0 shares the same gene content as the backbones of SGI1 and SGI2 (overall 97.6% and 97.7% nucleotide identity, respectively) but it lacks a class 1 integron. However, SGI0 is a mosaic made up of segments with >98.5% identity to SGI1 and SGI2 interspersed with segments sharing 74-95% identity indicating that further diverged backbone types exist and that recombination between them is occurring. The structure of SGI1-V, here re-named SGI-V, which lacks two SGI1 (S023 and S024) backbone genes and includes a group of additional genes in the backbone, was re-examined. In regions shared with SGI1, the backbones shared 97.3% overall identity with the differences distributed in patches with various levels of identity. The class 1 integron is also in a slightly different position with the target site duplication AAATT instead of ACTTG for SGI1 and variants, indicating that it was acquired independently. The Pm1LENAR resistance genes are in the chromosome, in Tn7 and an ISEcp1-mobilised segment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The IncB/O multiresistance plasmid R16, recovered in 1956 and used in experimental studies of B/O plasmid features, was sequenced. The resistance genes are all within a class 1 integron closely related to In1 containing the cassette array oxa2-aadA1-oxa2-orfD and the sul1 gene in the 3'-conserved segment (3'-CS), with Tn10, containing tetA(B), inserted just inside the 3'-CS. The integron is part of a 25,144 bp region inserted in the plasmid backbone, bounded on only one side by a truncated Tn6018 and flanked by a 4 bp duplication. Most of the 82,026 bp R16 backbone is almost identical to that of the B/O plasmid R805a. However, two short regions containing genes of unknown function are <95% identical to the corresponding regions of R805a, and were likely acquired from a related plasmid. The insertion in R16 is related to one in the I1 plasmid pSE69-3861-1, which is embedded at the same position in an almost 11 kb segment of R16 backbone. In pSE69-3861-1, Tn6018 is complete, and two regions previously seen only in AbaR type islands of GC1 Acinetobacter baumannii are present. One contains a top topoisomerase gene and the second contains a resX resolvase gene. These regions are identical to the corresponding parts of AbaR islands. Thus, the complete sequence of R16 sheds light on the role of homologous recombination in the evolution of plasmid backbones and the acquisition of antibiotic resistance genes by I-complex plasmids, as well as on the formation of the AbaR islands of GC1 A. baumannii., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Most Acinetobacter plasmids are genus specific but their properties have not been investigated. Small plasmids with Rep&#95;3 family replication initiation proteins and iterons are common in Acinetobacter baumannii and often carry antibiotic resistance genes and toxin-antitoxin systems. A RepAci1 plasmid, carrying the carbapenem resistance gene oxa23 in Tn2006 and a RepAci2 plasmid carrying the amikacin (kanamycin and neomycin) resistance gene aphA6 in TnaphA6 were identified. These two plasmids have related rep regions; the consensus 22 bp iteron repeats differ only at three positions and the RepA proteins are 84% identical. However, they were shown to be compatible, whereas the RepAci1 plasmid displaced another RepAci1 plasmid demonstrating that they were incompatible. Despite encoding no mobilisation proteins, the RepAci1 plasmid was transferred to a new host at low frequency when a conjugatively proficient RepAci6 plasmid was present, whereas the RepAci2 plasmid carrying mobA and mobC mobilisation genes was not. Comparison of the sequences of the mobilised and mobilising plasmids revealed a short region of high similarity that is upstream of the predicted mobilisation genes in the RepAci6 plasmid, and has an organisation similar to that of F-type oriT transfer origins. The segment carrying the oriT-like region is present in many RepAci1 plasmids, including ones carrying the cabarpenem resistance genes oxa24 or oxa58 in dif modules, and in some RepAci2 or other Rep&#95;3 plasmids of further types, including one carrying the tet39 tetracycline resistance determinant. These plasmids are also likely to be mobilised, spreading resistance., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Two B/O plasmids were sequenced. The kanamycin resistance plasmid R805a was found in a Salmonella Typhi strain from a 1972 typhoid fever outbreak in Mexico City. pCERC6, which confers resistance to ampicillin, streptomycin and sulphamethoxazole, was found in a commensal E. coli isolated from a healthy adult in Sydney in 2008. Both plasmids contain the same gene for RNAI, the incompatibility determinant, as the reference B/O plasmid pMU707 and the recently reported plasmid p838B-R, which came from a commensal E. coli isolated in Sydney in 2004. However, three different repA alleles are associated with the IncB/O rnaI. Whereas the repA gene of p838B-R is identical to that of pMU707, R805a and pCERC6 each carry a different repA. Comparison of the plasmid backbones revealed that R805a has a different oriT-nikAB region to that in pCERC6 and p838B-R, encoding different NikA relaxosome accessory factors and NikB relaxases. The different nikA genes were associated with oriT sites that differed in the sequences of their long, imperfect inverted repeats. A further 11 publicly available complete plasmid sequences contain the same B/O rnaI, with either the repA of pMU707/p838B-R or of R805a, and the oriT-nikAB region of p838B-R/pCERC6 or of R805a. All four possible combinations were seen. Extensive variation was also observed in the traY-excA entry exclusion region, and in the locations of mobile elements, including ones carrying antibiotic resistance genes. Plasmids that contain the same rnaI gene would be considered the same by a number of typing methods, but this study has unveiled previously unnoticed mosaicism in the backbones of one such group. This highlights the importance of considering entire plasmid backbones for the typing and tracking of specific lineages., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The four plasmids in E. coli 39R861 have been used as size standards for closed-circular plasmid DNA since the 1980s. However, their sizes had only been estimated. Here, the complete sequence of the 61.3 kb cryptic FII plasmid p39R861-3 is reported, and the sizes of p39R861-4, pSa and NTP168 were determined to be 155.8, 40.1 and 6.8 kb, respectively. An improved size standard, 39R861+, was created by adding two sequenced, compatible plasmids to 39R861 via conjugation. The 94.8 kb conjugative B/O plasmid p838B-R mobilised the cryptic, 2.0 kb pBuzz. The six plasmids in 39R861+ are all stably maintained., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ampicillin, streptomycin and sulphamethoxazole resistant commensal E. coli 838-3B contains five plasmids that range in size from >90 kb to <2 kb. The resistance genes bla TEM (ampicillin), strA (streptomycin) and sul2 (sulphamethoxazole) transferred along with a B/O plasmid named p838B-R. However, three plasmids smaller than 7 kb were also found in transconjugants, suggesting that they could be mobilised by the B/O plasmid. The complete sequences of p838B-R and pBuzz, a small plasmid mobilised by p838B-R with 70% efficiency, were determined. p838B-R is 94,803 bp and contains an 8400 bp resistance island that includes the three antibiotic resistance genes. The p838B-R backbone contains a complete conjugative transfer region, including an oriT site upstream of nikAB that resembles the experimentally-defined oriT of R64. The 1982 bp pBuzz contains a rep gene and sites associated with replication that resemble those of pC194/pUB110 family rolling-circle plasmids. It also contains two, inversely oriented copies of an 84 bp sequence that differs from the oriT region in p838B-R at just 6 positions. These oriT-like sites likely explain the ability of pBuzz to co-transfer with the B/O plasmid using the NikB relaxase and NikA accessory protein encoded by p838B-R, i.e. pBuzz utilises relaxase-in trans mobilisation. Several rolling-circle plasmids related to pBuzz were found in the GenBank non-redundant nucleotide database. They contain diverse potential oriTs, including sequences similar to known oriTs found in conjugative plasmids of I-complex (I1, B/O, K, Z and I2), L or M types., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In an early study, IncA and IncC plasmids that were reported to be compatible were grouped as the "A-C complex" based on similarities and on strong entry exclusion. However, recently, the term IncA/C has been used frequently to describe plasmids belonging to both of these two groups. Granted that the supporting data was not included in the original reports and that the consensus iteron sequences have since been shown to be essentially identical, we have addressed the question again. The original IncA plasmid, RA1, and the IncC plasmid pRMH760, were introduced into the same cell by transformation, and were found to be maintained stably for over 100 generations in the absence of selection for either plasmid, i.e. they were compatible. We conclude that use of the term IncA/C for this important plasmid group is indeed incorrect and it causes unnecessary confusion. Granted the importance of IncC plasmids in the spread of antibiotic resistance genes, we recommend that use of the misleading terms IncA/C, IncA/C 1 and IncA/C 2 should cease. In addition, RA1 and pRMH760 were shown to each completely prevent entry of the other via conjugative transfer into the cell they reside in., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The IncC plasmid pB2-1, from a Klebsiella pneumoniae isolate recovered in Brisbane prior to 1995, belongs to a subtype of type 1 IncC plasmids, here designated type 1a, that includes those carrying carbapenem resistance genes such as bla NDM and bla KPC . pB2-1 carries a 2358bp deletion in the rhs1 gene found in four other type 1a IncC plasmids. pB2-1 confers resistance to ampicillin, gentamicin, kanamycin, neomycin, tobramycin, sulfamethoxazole, tetracycline and trimethoprim. It transferred at a frequency of 4.7×10 -3 transconjugants per donor, similar to that of another type 1a plasmid pDGO100 but ten-fold lower than for its closest relative pRMH760. This difference may be due to a single amino acid substitution in TraL. pB2-1 has an ISEc52 insertion in the dsbC gene, demonstrating that dsbC is not essential for transfer. pB2-1 lacks the ARI-B insertion and hence the sul2 gene. The resistance genes sul1, dfrA10, aphA1a, bla TEM , aadB, and tetA(B) are all in the ARI-A island, in a configuration that has evolved from ARI-A of pRMH760 in two steps. A 10.3kb segment extending from the catA1 gene to the end of pDUmer module was lost via homologous recombination between two copies of IS4321. In addition, a 5.3kb segment extending from IS1326 to the left end of Tn4352B was replaced with an 18.7kb tet(B)-containing segment bounded on one end by IS1 and on the other by IS26. The IS26-bounded transposon Tn4352B was shown to be stable in K. pneumoniae in contrast to the high instability observed in E. coli., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The 167.5kb sequence of the conjugative IncC plasmid pIP40a, isolated from a Pseudomonas aeruginosa in 1969, was analysed. pIP40a confers resistance to kanamycin, neomycin, ampicillin, sulphonamides and mercuric ions, and several insertions in a type 1 IncC backbone were found, including copies of IS3, Tn1000 and a novel mercury resistance transposon, Tn6182. The antibiotic resistance genes were in two locations. Tn6023, containing the aphA1 kanamycin and neomycin resistance gene, is in a partial copy of Tn1/Tn2/Tn3 (bla TEM , ampicillin resistance) in the kfrA gene, and the sul2 sulphonamide resistance gene is in the integrative element GIsul2 in the position of ARI-B islands. The 11.5kb class II transposon Tn6182 is only distantly related to other class II transposons, with at most 33% identity between the TnpA of Tn6182 and TnpA of other group members. In addition, the inverted repeats are 37bp rather than 38bp, and the likely resolution enzyme is a tyrosine recombinase (TnpI). Re-annotation of GIsul2 revealed genes predicted to confer resistance to arsenate and arsenite, but resistance was not detected. The location of GIsul2 confirms it as the progenitor of the ARI-B configurations seen in many IncC plasmids isolated more recently. However, GIsul2 has integrated at the same site in type 1 and type 2 IncC plasmids, indicating that it targets this site. Analysis of the distribution of GIsul2 revealed that it in addition to its chromosomal integration site at the 3'-end of the guaA gene, it has also integrated into other plasmids, increasing its mobility., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The ampicillin resistance plasmid pCERC7, carrying transposon Tn2 with an IS4 insertion, was detected in the draft genome of a commensal Escherichia coli isolate. The genome data also revealed that this isolate belongs to ST131, clade B. pCERC7 is 9712bp comprised of a 3319bp backbone, Tn2::IS4 (6388bp) and 5bp of target site duplication, and was present at a copy number of 40. pCERC7 is related to several plasmids composed of only the backbone, or the backbone with the Tn2 insertion in the same position. These plasmids have been found previously in Escherichia coli or Salmonella enterica recovered in several different countries from as early as the 1970s. This group was named the NTP16 group after the best studied example. pCERC7 was annotated using available information about plasmids in this group and additional analyses. The backbone includes genes for RNA I and RNA II to initiate replication and the Tn2 interrupts a gene found here to encode a protein 66% identical to the Rom regulatory protein of ColE1. NTP16 family plasmids include a gene, previously designated mobA, that was found to encode a homologue (53% identical) of the NikA relaxase accessory protein of the conjugative IncI1 plasmid R64, which is known to bind to the R64 oriT. However, a nikB relaxase gene is not present, indicating that a relaxase must be supplied in trans for mobilisation by R64 to occur, as demonstrated previously for NTP16. Hence, MobA of NTP16 and relatives was renamed NikA. Upstream of nikA, we found a region closely related to the oriT of R64. pCERC7 and all members of the NTP16 family also include a multimer resolution site, nmr, similar to the cer site of ColE1. The backbone of the NTP16 family also includes genes for a demonstrated toxin-antitoxin system, LsoAB. Several more distantly related groups of plasmids that include a very closely related nmr-nikA-oriT segment (99.4-93.7% DNA identity) were identified in the GenBank non-redundant DNA database. All use an RNA I/RNA II-Rom system for replication initiation, but each contains a unique fragment adjacent to the nmr site. The segment of the NTP16/pCERC7 group that encodes the LsoAB toxin-antitoxin system is replaced by a different segment in other family groups. The point at which the sequences diverge is between the XerC and XerD sites of the dif site at one end of nmr, suggesting that the evolution of this broad group of plasmids involves XerC/XerD recombination., (Copyright © 2016 Elsevier Inc. All rights reserved.)

IncC (A/C 2 ) plasmids are known to play an important role in the spread of multiple antibiotic resistance determinants, including extended-spectrum β-lactamases and carbapenamases, amongst Gram negative bacterial populations. The ability to identify and track these plasmids is valuable in epidemiological and clinical studies. A recent comparative analysis of the backbones of sequenced IncC plasmids identified two distinct lineages, type 1 and type 2, with different evolutionary histories. Here, a simple PCR method to rapidly assign plasmids to one of these lineages by detecting variable regions in the backbone was developed. This PCR scheme uses two primer pairs to assign the plasmid to a lineage, and an additional two PCRs can be used to detect the i1 and i2 insertions, which are only found in type 2. PCRs were also developed to detect the presence or absence of the sul2-containing ARI-B island, which is found in some plasmids belonging to both type 1 and type 2, and the ARI-A island found in most type 1 plasmids. The PCR strategy was validated using sequenced type 1 plasmids pRMH760 and pDGO100, and the type 2 plasmid pSRC119-A/C, and a collection of non-IncC plasmids in Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae backgrounds. An IncC plasmid detected in an antibiotic susceptible commensal E. coli isolate was examined and found to be a type 1, lacking any antibiotic resistance islands and missing a large backbone segment. Examination of pIP40a, an IncC plasmid isolated in Paris in 1969, by PCR revealed that it belongs to type 1 but lacks ARI-A. However, it includes both ends of the integrative element GIsul2, whereas only remnants of one end of this element are found in more recently isolated IncC plasmids. The sequence of pIP40a was determined and confirmed the assignment to type 1 and revealed the presence of a complete copy of GIsul2., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Acinetobacter baumannii is an important nosocomial pathogen that often complicates treatment because of its high level of resistance to antibiotics. Though plasmids can potentially introduce various genes into bacterial strains, compared to other Gram-negative bacteria, information about the unique A. baumannii plasmid repertoire is limited. Here, whole genome sequence data was used to determine the plasmid content of strain A297 (RUH875), the reference strain for the globally disseminated multiply resistant A. baumannii clone, global clone 1(GC1). A297 contains three plasmids. Two known plasmids were present; one, pA297-1 (pRAY*), carries the aadB gentamicin, kanamycin and tobramycin resistance gene and another is an 8.7kb cryptic plasmid often found in GC1 isolates. The third plasmid, pA297-3, is 200kb and carries the sul2 sulphonamide resistance gene and strAB streptomycin resistance gene within Tn6172 and a mer mercuric ion resistance module elsewhere. pA297-3 transferred sulphonamide, streptomycin and mercuric ion resistance at high frequency to a susceptible A. baumannii recipient, and contains several genes potentially involved in conjugative transfer. However, a relaxase gene was not found. It also includes several genes encoding proteins involved in DNA metabolism such as partitioning. However, a gene encoding a replication initiation protein could not be found. pA297-3 includes two copies of a Miniature Inverted-Repeat Transposable Element (MITE), named MITE-297, bracketing a 77.5kb fragment, which contains several IS and the mer module. Several plasmids related to but smaller than pA297-3 were found in the GenBank nucleotide database. They were found in different A. baumannii clones and are wide spread. They all contain either Tn6172 or a variant in the same position in the backbone as Tn6172 in pA297-3. Some related plasmids have lost the segment between the MITE-297 copies and retain only one MITE-297. Others have segments of various lengths between two MITE-297 copies, and these can be derived from the region in pA297-3 via a deletion adjacent to IS related to IS26 such as IS1007 or IS1007-like. pA297-3 and its relatives represent a third type of conjugative Acinetobacter plasmid that contributes to the dissemination of antibiotic resistance in this species., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Both the Salmonella genomic islands (SGI) and the conjugative IncC plasmids are known to contribute substantially to the acquisition of resistance to multiple antibiotics, and plasmids in the A/C group are known to mobilize the Salmonella genomic island SGI1, which also carries multiple antibiotic resistance genes. Plasmid pRMH760 (IncC; A/C 2 ) was shown to mobilize SGI1 variants SGI1-I, SGI1-F, SGI1-K and SGI2 from Salmonella enterica to Escherichia coli where it was integrated at the preferred location, at the end of the trmE (thdF) gene. The plasmid was transferred at a similar frequency. However, we observed that co-transfer of the SGI and the plasmid was rarer. In E. coli to E. coli transfer, the frequency of transfer of the IncC plasmid pRMH760 was at least 1000-fold lower when the donor carried SGI1-I or SGI1-K, indicating that the SGI suppresses transfer of the plasmid. In addition, pRMH760 was rapidly lost from both E. coli and S. enterica strains that also carried SGI1-I, SGI1-F or SGI2. However, plasmid loss was not seen when the SGI1 variant was SGI1-K, which lacks two segments of the SGI1 backbone. The complete sequence of the SGI1-I and SGI1-F were determined and SGI1-K also carries two single base substitutions relative to SGI1-I. The IncA (A/C 1 ) plasmid RA1 was also shown to mobilize SGI2-A and though there are significant differences between the backbones of IncA and IncC plasmids, RA1 was also rapidly lost when SGI2-A was present in the same cell. We conclude that there are multiple interactions, both cooperative and antagonistic, between an IncA or IncC plasmid and the SGI1 and SGI2 family genomic islands., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Most A/C plasmids sequenced to date were recovered in the last two decades. To gain insight into the evolution of this group, the IncC plasmid pDGO100, found in a multiply antibiotic-resistant Escherichia coli strain isolated in 1981, was sequenced. pDGO100 belongs to the type 1 lineage and carries an ARI-A antibiotic resistance island but not an ARI-B island. The A/C2 backbone of pDGO100 has a deletion in the rhs1 gene previously found in pRMH760 and differs by only six single base pair substitutions from pRMH760, recovered at the same hospital 16years later. This confirms that the separation of type 1 and type 2 IncC plasmids is long standing. The ARI-A islands are also closely related, but pRMH760 contains Tn4352B in tniA of Tn402, while in pDGO100, Tn4352 has inserted into merA of pDUmer. pDGO100 also carries an additional 46kb insertion that includes a Tn1696-like transposon with the dfrB3 gene cassette. This insertion was identified as a novel integrating element, with an int gene at one end, and also includes the fec iron uptake operon that has been acquired from the E. coli chromosome. Related integrating elements carrying the same int gene were found in A/C2, IncHI1, and IncHI2 plasmids, and in the chromosomes of Enterobacter cloacae, Klebsiella oxytoca, and Cronobacter sakazakii isolates. In the Enterobacteriaceae chromosomes, these integrating elements appear to target a gene encoding a radical SAM superfamily protein. In the A/C2, IncHI1, and IncHI2 plasmids, genes encoding a phosphoadenosine phosphosulfate reductase were interrupted. The extremities of the integrating element are highly conserved, whilst the internal gene content varies. The detection of integrative elements in plasmids demonstrates an increased range of locations into which this type of mobile element can integrate and insertion in plasmids is likely to assist their spread., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The rare sulphonamide resistance gene sul3 was found in the commensal Escherichia coli ST95 strain 22.1-R1 that was isolated in 2010 from the faeces of a healthy Australian adult. The genome of 22.1-R1 was sequenced and a 144,344bp RepFII/FIB plasmid, pCERC3, carrying sul3 was assembled. The sul3 gene is part of a class 1 integron featuring a sul3-containing conserved segment (sul3-CS) that replaced the classic sul1-containing 3'-conserved segment (3'-CS) usually seen in class 1 integrons. The integron contained the cassette array dfrA12-orfF-aadA2-cmlA1-aadA1-qacH, conferring resistance to trimethoprim, streptomycin, spectinomycin, chloramphenicol and quaternary ammonium compound. Two additional antibiotic resistance genes, blaTEM (ampicillin resistance) and tetA(B) (tetracycline) were adjacent to the integron, forming a single resistance region. In pCERC3, the sul3-type class 1 integron was flanked by sequence derived from the tnp and mer modules of Tn21 and was in the same location as In2, the sul1-containing In5-type class 1 integron of Tn21. At one end the sequence extends into Tn2670-derived sequence and then into sequence derived from the plasmid NR1 (R100). Examination of the sequences of eleven more complete sul3-containing plasmids in GenBank confirmed the relationship between sul3-associated integrons and Tn21/Tn2670/NR1. This suggests that the events that formed sul3-associated class 1 integrons occurred within the Tn21/Tn2670 context, most likely in NR1 or a related plasmid. The backbone of pCERC3 is most closely related to the backbones of ColV virulence plasmids and contains a complete ColV operon as well as several virulence associated genes and gene clusters. Hence, pCERC3 is both an antibiotic resistance and virulence plasmid., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A collection of 111 commensal Escherichia coli isolated from 84 faecal samples from healthy Australian adults were screened using PCR-based replicon typing. Each isolate represented a distinct strain found in a particular faecal sample. Fifty-one isolates were resistant to one or more of 12 antibiotics tested. FII and FIB replicons were most common and usually found together. The FII replicon was detected in 63 isolates (35 susceptible, 28 resistant), the FIB replicon was present in 65 (32 susceptible, 33 resistant) and 54 (30 susceptible, 24 resistant) included both. Other replicon types were found infrequently (A/C, I1, K, L/M, P, R, Y, FIA and FIC) or not at all (HI1, HI2, N, T, U, W, X). Only the B/O amplicon, found in 21 resistant but only 4 susceptible isolates, was associated with antibiotic resistance. Detailed analysis of this group revealed that the B/O PCR also detected Z plasmids of several distinguishable types. PCR assays were developed to detect the two repA genes (repABKI and repAZ) found in members of the I-complex (I, B/O, K and Z plasmids). These assays distinguished the B/O and Z plasmids detected by the original "B/O" PCR. One isolate carried repABKI and the remainder carried repAZ. These genes were also detected in further isolates in the collection. Conjugative transfer of resistance genes was detected for the B/O plasmid and two Z groups. Evidence for transfer of repAZ plasmids in the human colon in the absence of antibiotic selection was also obtained., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Plasmids belonging to incompatibility groups A and C (now A/C) were among the earliest to be associated with antibiotic resistance in Gram-negative bacteria. A/C plasmids are large, conjugative plasmids with a broad host range. The prevalence of A/C plasmids in collections of clinical isolates has revealed their importance in the dissemination of extended-spectrum β-lactamases and carbapenemases. They also mobilize SGI1-type resistance islands. Revived interest in the family has yielded many complete A/C plasmid sequences, revealing that RA1, designated A/C1, is different from the remainder, designated A/C2. There are two distinct A/C2 lineages. Backbones of 128-130 kb include over 120 genes or ORFs encoding proteins of at least 100 amino acids, but very few have been characterized. Genes potentially required for replication, stability and transfer have been identified, but only the replication system of RA1 and the regulation of transfer have been studied. There is enormous variety in the antibiotic resistance genes carried by A/C2 plasmids but they are usually clustered in larger regions at various locations in the backbone. The ARI-A and ARI-B resistance islands are always at a specific location but have variable content. ARI-A is only found in type 1 A/C2 plasmids, which disseminate blaCMY-2 and blaNDM-1 genes, whereas ARI-B, carrying the sul2 gene, is found in both type 1 and type 2. This review summarizes current knowledge of A/C plasmids, and highlights areas of research to be considered in the future., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The IncHI1 plasmid pSRC27-H from Salmonella enterica serovar Typhimurium carries a region containing several genes that confer resistance to different antibiotics, and this resistance region is in the same position as related resistance regions in a group of sequenced IncHI1 plasmids from various sources that includes pHCM1. Four further additional segments are found in pHCM1 relative to another IncHI1 plasmid, R27. Using PCR or DNA sequencing to detect the presence or absence of each of these additional segments in the same position in the IncHI1 backbone, plasmid pSRC27-H was found to include them. However, in one case the additional segment was smaller in pSRC27-H, lacking a transposon carrying a second resistance region in pHCM1. The sequences of IncHI1 plasmids, pO111&#95;1 and pMAK1, were also examined and found to share the same or closely related additional segments. The structure of the additional material in pHCM1, pO111&#95;1 and pMAK1 was examined, and potential novel transposons were identified. These additional segments define an IncHI1 lineage (pHCM1, pO111&#95;1, pMAK1, pSRC27-H) which we designated type 2 to distinguish it from type 1 (R27, pAKU&#95;1, pP-stx-12). A segment from the Escherichia coli genome and an adjacent copy of IS1 in pHCM1 was defined by comparison to pO111&#95;1 and pMAK1, which lack it. pSRC27-H also lacks it. This structure is present in the same position in R27 and type 1 plasmids, but in the opposite orientation, and appears to have been incorporated via IS1-mediated transposition. The PCRs developed provide a simple means of distinguishing type 1 and type 2 IncHI1 plasmids based on the presence or absence of variable regions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Integrons are genetic elements which are capable of acquiring genes by site-specific recombination. The most common integron structure consists of two conserved segments flanking a variable region where many different antibiotic resistance genes have been found. The integrons In6 and In7, present in the plasmids pSa and pDGO100, respectively, are unusual in that they include a duplication of the sulI gene which is located within the integron 3'-conserved segment. To further investigate the structure of these integrons, the DNA sequence of the segment located between the two sulI genes was determined. In In7 this segment is 2822 bases long and includes a trimethoprim resistance gene, dhfrX, at one end. The corresponding region in In6 is 4.5 kb and is nearly identical to the In7 segment over the first 2105 bases. In the region unique to In6, a cat gene, conferring chloramphenicol resistance, has replaced the dhfrX gene of In7. This location thus represents a second variable region where different antibiotic resistance genes are found, but the way in which genes become associated with this second variable region is not known. The overall similarity of the structures of In6 and In7 suggests that the additional DNA segments found in these integrons have a common origin, and a possible mechanism for the origin of integrons with partial 3'-conserved segment duplications is presented.

The sequence of the insert region of the integron In1 found in the IncN plasmid R46 was completed. The insert region is 2929 bases long and includes four gene cassettes, two of which are identical copies of the oxa2 gene cassette flanking an aadA1 cassette. The fourth cassette encodes an open reading frame orfD. From comparison of these data with published maps and sequences it is argued that the integrons found in the IncN plasmids pCU1 and R1767 and in the transposon Tn2410 are closely related to In1 from R46. Both site-specific gene insertion and recA-dependent recombination are likely to have contributed to the evolution of these integrons.

The sequence of the Tn1696 determinant for inducible nonenzymatic chloramphenicol resistance has been determined. The cml region, the fourth insert of the Tn1696 integron, is 1547 bases and includes a 59-base element at the 3' end, as is typical of integron inserts. One gene, designated cmlA and predicting a polypeptide of 44.2 kDa, is encoded in the insert. However, the cmlA region shows one feature not previously found in an integron insert. A promoter is located within the cmlA insert, and translational attenuation signals related to those of the inducible cat and ermC genes found in gram-positive organisms are also present. The regulatory region includes a leader peptide of nine amino acids, a ribosome stall sequence related to those preceding cat genes, and two alternative pairs of stem-loop structures which either sequester or disclose the ribosome binding site and start codon preceding the cmlA gene.

A family of novel potentially mobile DNA elements called integrons, has recently been described (H. W. Stokes and R. M. Hall, 1989, Mol. Microbiol. 3, 1669-1683). The integrons present in the plasmids pDGO100 and pSa are unusual in that they include a duplication of the sulI gene which is located in one of the two conserved segments that make up these elements. In order to define the nature of the duplication in pDGO100, we have sequenced the sulI gene region located between the aadB and the dhfr genes of pDGO100. This region includes the first 1355 bases of the 2026-base 3'-conserved segment present in the integrons of Tn21, R46 and R388, and the sequence identity in pDGO100 ceases 24 bases beyond the end of the sulI gene. This position corresponds to the center of a 59-base element, a remnant of which is located at the end of sulI. This finding suggests that the 59-base element may have been involved in the event which gave rise to the partial duplication.

Tn5 conferred resistance to the related antibiotics, phleomycins, bleomycins, and tallysomycins in Escherichia coli and Salmonella typhimurium. For pure phleomycins the level of resistance was influenced by the structure of the terminal basic group. Deletion derivatives of a pBR322::Tn5 plasmid were used to show that the phleomycin resistance determinant is located between the previously identified neomycin and streptomycin resistance determinants. The pattern of expression of phleomycin and neomycin resistance in the deletion derivatives suggests that the phleomycin resistance gene is transcribed from the same promoter, PL, which is essential for expression of neomycin and streptomycin resistance. The location of the phleomycin resistance determinant correlates with the location of an open reading frame in the Tn5 sequence, which codes for a polypeptide of 126 amino acids.