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Acinetobacter baumannii has successfully spread during the last decades as one of the main critically important pathogens. However, many aspects including plasmids, are still under-investigated. Here, we report the complete sequence of an Acinetobacter baumannii strain, belonging to the ST25 IP (Institut Pasteur) sequence type recovered in 2012 in Lebanon, using a combination of Illumina MiSeq and Oxford Nanopore sequencing and a hybrid assembly approach. This strain (Cl107) carries a 198 kb plasmid called pCl107 that encodes the MPF I conjugative transfer system. The plasmid carries the aacA1, aacC2, sul2 , strAB , and tetA (B) antibiotic resistance genes. pCl107 region encompassing the sul2 , strAB , tetA (B) is closely related to AbGRI1 chromosomal resistance islands, which are widespread in A. baumannii strains belonging to Global Clone 2. The resistance region found in pCl107 is one of the missing links in the evolutionary history of the AbGRI1 islands. pCl107 also contains a BREX Type 1 region and represents one of the two main evolution patterns observed in BREX clusters found in plasmids related to pCl107. pCl107 also harbours a ptx phosphonate metabolism module, which plays an ancestral structure compared to other large plasmids in ST25 strains. While the uric acid metabolic module found in pCl107 is incomplete, we identified possible ancestors from plasmids and chromosomes of Acinetobacter spp. Our analyses indicate a complex evolutionary history of plasmids related to pCl107 with many links to multiple antibiotic resistance and metabolic pathways., Competing Interests: None declared, (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Objectives: To identify the origins of resistance in a sporadic extensively resistant Acinetobacter baumannii isolate., Methods: The complete genome of RCH52 was determined by combining available Illumina short reads with MinION (Oxford Nanopore) long reads using Unicycler. Bioinformatic searches were used to identify features of interest., Results: The complete genome of RCH52 revealed an unusual chromosomal region containing all of the antibiotic resistance genes, except tet39, which is in a plasmid. A 129 585 bp segment was bounded by inversely oriented copies of ISAba1 and included two groups of resistance genes separated by the large segment of the backbone of type 1 IncC plasmids that lies between the ARI-A and ARI-B resistance islands but does not include the replication region. The ISAba1-bounded segment was located in a novel integrative element that had integrated into the chromosomal thyA gene but provided a replacement thyA gene. Several resistance genes are derived from either the ARI-A or the ARI-B resistance islands found in IncC plasmids that have been brought together by an IS26-mediated deletion of the original plasmid. This non-replicating circular molecule (or translocatable unit) has been incorporated into a smaller ISAba1-bounded unit that includes oxa23 in Tn2008B via homologous recombination between sul2-CR2-floR segments found in both., Conclusions: The plasmids shared by most Gram-negative pathogens, including the broad host range IncC plasmids, have not been detected in Acinetobacter species. However, it seems likely that they can conjugate into members of this genus and contribute pre-existing clusters of antibiotic resistance genes., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To determine the genetic context of genes conferring antibiotic resistance on the carbapenem-resistant Acinetobacter baumannii Cl415, recovered in 2017 at El Youssef Hospital Centre in Akkar Governorate, North Lebanon., Methods: Antibiotic resistance phenotype for 22 antibiotics was determined using disc diffusion or MIC determination. The whole-genome sequence of Cl415 was determined using a combination of the Illumina MiSeq and Oxford Nanopore (MinION) platforms. Complete genome was assembled using Unicycler and antibiotic resistance determinants and ISs were identified using ResFinder and ISFinder, respectively., Results: Cl415 is a global clone 2 (GC2) strain and belongs to the most common STs of this clone, ST2IP and ST218OX. Cl415 is resistant to several antibiotics, including aminoglycosides and carbapenems to a high level. Genomic analysis of Cl415 revealed that it carries four chromosomal AbaR4 copies. One copy was found in the comM gene replacing the AbGRI1 island. Cl415 also contains a novel variant of AbGRI2, herein called AbGRI2-15, carrying only the blaTEM and aphA1 resistance genes. Cl415 belongs to a subclade of GC2 strains that appear to have diverged recently with a wide geographical distribution., Conclusions: The resistance gene complement of Cl415 was found in the chromosome with four oxa23 located in AbaR4 copies and the remaining genes in a novel variant of the AbGRI2 resistance island. Cl415 was isolated in Lebanon, but phylogenetic analysis suggests that Cl415 represents a new lineage with global distribution within GC2., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: To analyse the context of genes conferring antibiotic resistance in two carbapenem-resistant Acinetobacter baumannii isolates recovered in Tehran, Iran., Methods: The antibiotic resistance phenotype for 28 antibiotics was determined using disc diffusion. The whole genome sequences of ABH008 and ABS200 were determined using the Illumina HiSeq X Ten platform. Resistance genes were identified using ResFinder and multilocus sequence types were determined using the Oxford and Institut Pasteur schemes., Results: Isolates ABH008 and ABS200, recovered in 2012 and 2013, respectively, in two different Tehran hospitals, belong to the common global clone 1 lineage, ST1 IP and ST231 OX . They are resistant to sulfamethoxazole, tetracycline, gentamicin, amikacin, third-generation cephalosporins and carbapenems. Despite being isolated in different hospitals, phylogenetic analysis indicated they are closely related. Consistent with this, both isolates carry catA1, sul1, aacC1 and aadA1 in a novel variant of the AbaR3-type resistance island, named AbaR31. Both isolates are resistant to amikacin and carbapenems owing to aphA6 and oxa23 , respectively. The oxa23 gene is located in the AbaR4 resistance island, and aphA6 in Tn aphA6 , and both mobile elements are in an ∼90 kbp plasmid encoding the putative RepAci6 replication initiation protein. Resistance to third-generation cephalosporins is due to the acquisition by homologous recombination of a 5 kb DNA segment that contains ISAba1- ampC from a ST623 strain., Conclusions: The resistance gene complements of ABH008 and ABS200 were found in AbaR31 and a plasmid that encodes RepAci6. The close genetic relationship of ABH008 and ABS200, despite each being recovered from different hospitals, indicates transmission between the two hospitals., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: To understand the acquisition of resistance genes by a non-GC1, non-GC2 Acinetobacter baumannii strain responsible for a 4 year outbreak at a Sydney hospital., Methods: Representative isolates were screened for resistance to antibiotics. Three were subjected to WGS using Illumina HiSeq. One genome was completed with MinION long reads. Resistance regions were compared with known sequences using bioinformatics., Results: Isolates were resistant to third-generation cephalosporins, gentamicin and tobramycin, sulfamethoxazole and erythromycin. Sequenced isolates were ST49 (Institut Pasteur scheme) and ST128 (Oxford scheme) and carried KL11 at the capsule locus and OCL8 at the lipooligosaccharide outer core locus. The complete genome of isolate J9 revealed that the resistance genes were all in plasmids; pRAY* contained aadB, and a large plasmid, pJ9-3, contained sul2 and floR genes and a dif module containing the mph(E)-msr(E) macrolide resistance genes. Transposon Tn6168, consisting of a second copy of the chromosomal ampC gene region flanked by ISAba1s, confers resistance to third-generation cephalosporins. Tn6168 is located inside the mph(E)-msr(E) dif module. pJ9-3 includes a set of four dif modules and the orientation of the pdif sites, XerC-XerD or XerD-XerC, alternates. A large transposon, Tn6175, containing tniCABDE transposition genes and genes annotated as being involved in heavy metal metabolism, uptake or export was found in the comM gene. Other ST49:ST128:KL11:OCL8 genomes found in the GenBank WGS database carried Tn6175 but neither of the plasmids carrying the resistance genes., Conclusions: An early carbapenem-susceptible A. baumannii outbreak recorded in Australia was caused by an unusual clone that had acquired plasmids carrying antibiotic resistance genes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: Recent reports indicate the emergence of a new carbapenemase-producing Klebsiella pneumoniae clone, ST307. We sought to better understand the global epidemiology and evolution of this clone and evaluate its association with antimicrobial resistance (AMR) genes., Methods: We collated information from the literature and public databases and performed a comparative analysis of 95 ST307 genomes (including 37 that were newly sequenced)., Results: We show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the blaCTX-M-15 ESBL gene and several other AMR determinants., Conclusions: Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance has occasionally emerged., (© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: To determine the identity and context of genes conferring antibiotic resistance in a sporadic multiply antibiotic-resistant Acinetobacter baumannii recovered at Royal Children's Hospital, Brisbane., Methods: The antibiotic resistance phenotype for 23 antibiotics was determined using disc diffusion or MIC determination. The whole-genome sequence of RCH51 was determined using the Illumina HiSeq platform. Antibiotic resistance determinants were identified using ResFinder. Plasmids were recovered by transformation., Results: Isolate RCH51 belongs to the uncommon STs ST103 IP (7-3-2-1-7-1-4) and ST514 OX (1-52-29-28-18-114-7). It was found to be resistant to sulfamethoxazole, tetracycline, gentamicin, tobramycin and kanamycin and also exhibited reduced susceptibility to imipenem (MIC 2 mg/L) and meropenem (MIC 6 mg/L). RCH51 carries the oxa235 , sul2 , floR , aadB and tet39 resistance genes, all located on plasmids. The largest of the three plasmids, pRCH51-3, is 52 789 bp and carries oxa235 in the ISAba1-bounded transposon Tn 6252 , as well as sul2 and floR . pRCH51-3 represents a new A. baumannii plasmid family that is potentially conjugative as it contains several genes predicted to encode transfer functions. However, conjugation of pRCH51-3 was not detected. The aadB and tet39 resistance genes were each found in small plasmids identical to the known plasmids pRAY*-v1 and pRCH52-1, respectively., Conclusions: The resistance gene complement of RCH51 was found in three plasmids. pRCH51-3, which carries the oxa235 , sul2 and floR resistance genes, represents a new, potentially conjugative A. baumannii plasmid type., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: The objective of this study was to locate the antibiotic resistance determinants in the multiply antibiotic-resistant Acinetobacter baumannii isolate D4., Methods: The genome was sequenced using Illumina HiSeq and assembled de novo using Velvet. PCR was used to link the relevant contigs and fill the gaps. Sequences were compared with ones in GenBank and annotated., Results: A sporadic A. baumannii isolate D4, recovered in Sydney in 2006 from a wound, was multiply antibiotic resistant. D4 is ST25 (Institut Pasteur scheme) and exhibited resistance to third-generation cephalosporins and reduced susceptibility to ciprofloxacin, as well as resistance to aminoglycosides (gentamicin, kanamycin, neomycin and tobramycin) and further older antibiotics, nalidixic acid, sulfamethoxazole, streptomycin, spectinomycin and trimethoprim. The gyrA gene has a mutation consistent with nalidixic acid resistance. The bla PER conferring cephalosporin resistance, together with the aadB, aadA13/2, aadA2, strAB and sul1 resistance genes, are located within a 29 173 bp complex class 1 integron that includes three copies of intI1, three cassette arrays and two copies of the 3'-conserved segment. This integron is adjacent to the resG gene of an integrative genomic resistance island, AGI1 (Acinetobacter genomic island 1), with a backbone related to that of islands in the SGI1, SGI2 and PGI1 families. AGI1 is located at the 3'-end of the chromosomal trmE (formerly thdF) gene., Conclusions: AGI1 represents a new lineage of genomic resistance islands that belongs in the same broad group as members of the SGI1, SGI2 and PGI1 families. Genes conferring resistance to cephalosporins, aminoglycosides and sulphonamides are located in a complex class 1 integron within AGI1., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate., Methods: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted., Results: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus., Conclusions: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: To locate the acquired antibiotic resistance genes, including the amikacin resistance transposon TnaphA6, in the genome of an Australian isolate belonging to Acinetobacter baumannii global clone 1 (GC1)., Methods: A multiply antibiotic-resistant GC1 isolate harbouring TnaphA6 was sequenced using Illumina HiSeq, and reads were used to generate a de novo assembly and determine multilocus sequence types (STs). PCR was used to assemble the AbaR chromosomal resistance island and a large plasmid carrying TnaphA6. Plasmid DNA sequences were compared with ones available in GenBank. Conjugation experiments were conducted., Results: The A. baumannii GC1 isolate G7 was shown to include the AbaR3 antibiotic resistance island. It also contains an 8.7 kb cryptic plasmid, pAb-G7-1, and a 70,100 bp plasmid, pAb-G7-2, carrying TnaphA6. pAb-G7-2 belongs to the Aci6 Acinetobacter plasmid family. It encodes transfer functions and was shown to conjugate. Plasmids related to pAb-G7-2 were detected in further amikacin-resistant GC1 isolates using PCR. From the genome sequence, isolate G7 was ST1 (Institut Pasteur scheme) and ST231 (Oxford scheme). Using Oxford scheme PCR-based methods, the isolate was ST109 and this difference was traced to a single base difference resulting from the inclusion of the original primers in the gpi segment analysed., Conclusions: The multiply antibiotic-resistant GC1 isolate G7 carries most of its resistance genes in AbaR3 located in the chromosome. However, TnaphA6 is on a conjugative plasmid, pAb-G7-2. Primers developed to locate TnaphA6 in pAb-G7-2 will simplify the detection of plasmids related to pAb-G7-2 in A. baumannii isolates.

Objectives: To explore the cause of third-generation cephalosporin resistance in Australian Acinetobacter baumannii isolates belonging to global clone 1 (GC1)., Methods: GC1 isolates from Australia were tested for resistance to ceftazidime and cefotaxime using disc diffusion and MICs. PCR was used to determine the context of ISAba1-ampC configurations and amplicons were sequenced. The level of transcripts was measured using quantitative real-time PCR. Multilocus sequence typing was performed., Results: All ceftazidime- and cefotaxime-resistant isolates carried an appropriately oriented ISAba1 adjacent to the ampC gene and ISAba1 increased ampC transcripts 8-12-fold. In three isolates, the ampC gene next to ISAba1 was not in the normal chromosomal position. Instead, ISAba1 was 7 bp upstream of an additional copy of ampC located in a 3155 bp duplicated segment of the chromosome that differs from the resident GC1 segment by 2.3% but is almost identical to the corresponding region in several non-GC1 draft genomes. The duplicated segment is bounded by directly oriented copies of ISAba1 and flanked by a 9 bp direct duplication. This 5.5 kb transposon, named Tn6168, is in the same position in the chromosome of the three Australian isolates and the GC1 isolate AB0057. Tn6168 was also detected in an unrelated A. baumannii strain, where it was in a different location. The central part of Tn6168 was probably acquired from a sequence type ST32 (Institut Pasteur scheme) A. baumannii strain., Conclusions: The ISAba1-ampC configuration, which increases ampC expression, can be part of a composite transposon Tn6168.

Objectives: To determine the cause of resistance to the aminoglycosides gentamicin and tobramycin in Acinetobacter isolates and the location of the resistance genes., Methods: Australian Acinetobacter baumannii isolates were screened for resistance to aminoglycosides. PCR followed by restriction digestion of amplicons was used to detect genes and plasmids. Plasmids were isolated and examined by restriction digestion. Plasmid DNA sequences were determined and bioinformatic analysis was used to identify features. The sequence of the bla(OXA-Ab) gene and multilocus sequence typing were used to determine strain types., Results: Isolates that exhibited resistance to gentamicin, kanamycin and tobramycin were of diverse strain types. These isolates all carried the aadB gene cassette, and in all but one the cassette was in a 6 kb plasmid similar to pRAY. The three plasmid sequences determined revealed multiple frame-shift differences in the available pRAY sequence that altered the reading frames. In pRAY*, mobA and mobC mobilization genes were identified, but no potential replication initiation protein was found. pRAY*-v1 differed from pRAY* by 66 single-base differences, and pRAY*-v2 included two insertion sequences, ISAba22, located upstream of the aadB gene cassette, and IS18-like, within ISAba22., Conclusions: The plasmid pRAY* and variants are widely distributed in Acinetobacter spp. and are the most common cause of resistance to gentamicin and tobramycin. Mobilization genes should assist in the dissemination of pRAY* and its variants.

Objectives: To explore the diversity of genomic resistance islands in multiply antibiotic-resistant Acinetobacter baumannii isolates in global clone 1 (GC1) from Australian hospitals., Methods: PCR was used to characterize isolates, detect antibiotic resistance genes and insertion sequences and screen for genomic resistance islands. Structures of genomic islands were determined by PCR mapping and sequencing. Multilocus sequence typing was performed using the Oxford scheme., Results: Eleven isolates that belong to GC1 were found among 90 A. baumannii isolated between 2001 and 2010 at Australian hospitals, and 5 were carbapenem resistant. Ten isolates had the features characteristic of AbaR3 and related islands, but one carbapenem-resistant isolate did not. Instead, D36 carried the bla(OXA-23) gene in transposon Tn2006, with Tn2006 in AbaR4, and AbaR4 in the chromosomal comM gene, replacing the AbaR3-type island usually associated with multiply antibiotic-resistant GC1 isolates. D36 was resistant to gentamicin, kanamycin and tobramycin due to the aadB gene cassette in the context found in plasmid pRAY and to sulfamethoxazole due to the sul2 gene. D36 was of a rare sequence type (ST), ST247. Bioinformatic analysis identified five potential transposition genes in the AbaR backbone transposons., Conclusions: Substantial diversity was observed among the GC1 isolates. This is the first report of AbaR4 replacing the AbaR3-type island seen in most GC1 isolates, and D36 represents a distinct new GC1 lineage. The AbaRs are members of a large, previously undocumented family of transposons that target comM.