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Antimicrobial resistance (AMR) is a global threat to human, animal, and environmental health. Studying AMR in environmental bacteria is crucial to understand the emergence and dissemination of resistance genes and pathogens, and to identify potential reservoirs and transmission routes. This study provides novel insights into the genomic diversity and AMR potential of environmental Acinetobacter species. By comparing the genomes of aquatic Acinetobacter isolates with clinical and non-clinical strains, we revealed that they are highly divergent yet carry pdif modules that encode resistance to antibiotics commonly used in clinical settings. We also demonstrated that an environmental A. baumannii isolate can acquire clinically relevant plasmids and carries virulence factors similar to those of hospital-associated strains. These findings suggest that environmental Acinetobacter species may serve as reservoirs and vectors of clinically important genes. Consequently, further research is warranted to comprehensively understand the ecology and evolution of this genus.

This study examines the genetic contexts and evolutionary steps responsible for the formation of the widely spread transposon Tn6925 carrying blaTEM and aacC2e, which confers resistance to beta-lactam and aminoglycoside antibiotics in Gram-negative bacteria. The blaTEM-1 and aacC2e genes were found in several transposons. They were first observed within an IS26 bounded 3.7 kb transposon (Tn6925) on several Acinetobacter baumannii plasmids located within a 4.7 kb dif module. Truncated and expanded variations of Tn6925 were found across other A. baumannii plasmids, as well as in other Gram-negative bacteria (including Vibrio cholerae). Moreover, blaTEM-1 and aacC2e were in much larger resistance-heavy transposons including the ISAba1-bounded 24.6 kb (here called Tn6927), found in an A. baumannii chromosome. A novel ISKpn12-bounded transposon was also observed to contain blaTEM and aacC2e which was found interrupting Tn5393 along with an IS26 pseudo-compound transposon to form a 24.9 kb resistance island in an Acinetobacter pittii plasmid. Multiple mobile genetic elements are involved in the formation of transposon structures that circulate blaTEM and aacC2e. Among these, IS26 and ISAba1 appear to have played a major role in the formation and spread of these elements in the Acinetobacter species.

Antibiotic resistance is a growing global concern in the field of medicine as it renders bacterial infections difficult to treat and often more severe. Acinetobacter baumannii is a gram-negative bacterial pathogen causing a wide range of infections, including pneumonia, sepsis, urinary tract infections, and wound infections. A. baumannii has emerged as a significant healthcare-associated pathogen due to its high level of antibiotic resistance. The global spread of antibiotic-resistant strains of A. baumannii has resulted in limited treatment options, leading to increased morbidity and mortality rates, especially in vulnerable populations such as the elderly and immunocompromised individuals, as well as longer hospital stays and higher healthcare costs. Further complicating the situation, multi- and pan-drug-resistant strains of A. baumannii are becoming increasingly common, and these deadly strains are resistant to all or almost all available antibiotics. A. baumannii employs various clever strategies to develop antibiotic resistance, including horizontal transfer of resistance genes, overexpression of inherent efflux pumps that remove drugs from the cell, intrinsic mutations, combined with natural selection under antibiotic selective pressure leading to emergence of successful resistance clones. The typical multidrug resistance phenotype of A. baumannii is, therefore, an orchestrated collimation of all these mechanisms combined with the worldwide spread of "global clones," rendering infections caused by this pathogen challenging to control and treat. To address the escalating problem of antibiotic resistance in A. baumannii, there is a need for increased surveillance, strict infection control measures, and the development of new treatment strategies, requiring a concerted effort by healthcare professionals, researchers, and policymakers.

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.

Acinetobacter baumannii is an important opportunistic pathogen known for its high levels of resistance to many antibiotics, particularly those considered last resorts such as colistin and carbapenems. Plasmids of this organism are increasingly associated with the spread of clinically important antibiotic resistance genes. Although A. baumannii is a ubiquitous organism, to date, most of the focus has been on studying strains recovered from clinical samples ignoring those isolated in the environment (soil, water, food, etc.). Here, we analysed the genetic structures of eight novel plasmids carried by an environmental colistin-resistant A. baumannii (strain E-072658) recovered in a recycled fibre pulp in a paper mill in Finland. It was shown that E-072658 carries a new variant of the mcr-4 colistin resistance gene (mcr-4.7) in a novel Tn3-family transposon (called Tn6926) carried by a novel plasmid p8E072658. E-072658 is also resistant to sulphonamide compounds; consistent with this, the sul2 sulphonamide resistance gene was found in a pdif module. E-072658 also carries six additional plasmids with no antibiotic resistance genes, but they contained several pdif modules shared with plasmids carried by clinical strains. Detailed analysis of the genetic structure of all eight plasmids carried by E-072658 showed a complex evolutionary history revealing genetic exchange events within the genus Acinetobacter beyond the clinical or environmental origin of the strains. This work provides evidence that environmental strains might act as a source for some of the clinically significant antibiotic resistance genes.

The present work describes the evolution of a resistance phenotype to a multitargeting antimicrobial agent, namely, silver nanoparticles (nanosilver; NAg), in the globally prevalent bacterial pathogen Acinetobacter baumannii. The Gram-negative bacterium has recently been listed as a critical priority pathogen requiring novel treatment options by the World Health Organization. Through prolonged exposure to the important antimicrobial nanoparticle, the bacterium developed mutations in genes that encode the protein subunits of organelle structures that are involved in cell-to-surface attachment as well as in a cell envelope capsular polysaccharide synthesis-related gene. These mutations are potentially correlated with stable physiological changes in the biofilm growth behavior and with an evident protective effect against oxidative stress, most likely as a feature of toxicity defense. We further report a different adaptation response of A. baumannii to the cationic form of silver (Ag+). The bacterium developed a tolerance phenotype to Ag+, which was correlated with an indicative surge in respiratory activity and changes in cell morphology, of which these are reported characteristics of tolerant bacterial populations. The findings regarding adaptation phenomena to NAg highlight the risks of the long-term use of the nanoparticle on a priority pathogen. The findings urge the implementation of strategies to overcome bacterial NAg adaptation, to better elucidate the toxicity mechanisms of the nanoparticle, and preserve the efficacy of the potent alternative antimicrobial agent in this era of antimicrobial resistance. IMPORTANCE Several recent studies have reported on the development of bacterial resistance to broad-spectrum antimicrobial silver nanoparticles (nanosilver; NAg). NAg is currently one of the most important alternative antimicrobial agents. However, no studies have yet established whether Acinetobacter baumannii, a globally prevalent nosocomial pathogen, can dev

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.

Abstract 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 ST25IP (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 MPFI 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.

Acinetobacter baumannii is a critically important pathogen known for its widespread antibiotic resistance and ability to persist in hospital-associated environments. Whilst the majority of A. baumannii infections are hospital-acquired, infections from outside the hospital have been reported with high mortality. Despite this, little is known about the natural environmental reservoir(s) of A. baumannii and the virulence potential underlying non-clinical strains. Here, we report the complete genome sequences of six diverse strains isolated from environments such as river, soil, and industrial sites around the world. Phylogenetic analyses showed that four of these strains were unrelated to representative nosocomial strains and do not share a monophyletic origin, whereas two had sequence types belonging to the global clone lineages GC1 and GC2. Further, the majority of these strains harboured genes linked to virulence and stress protection in nosocomial strains. These genotypic properties correlated well with in vitro virulence phenotypic assays testing resistance to abiotic stresses, serum survival, and capsule formation. Virulence potential was confirmed in vivo, with most environmental strains able to effectively kill Galleria mellonella greater wax moth larvae. Using phenomic arrays and antibiotic resistance profiling, environmental and nosocomial strains were shown to have similar substrate utilisation patterns although environmental strains were distinctly more sensitive to antibiotics. Taken together, these features of environmental A. baumannii strains suggest the existence of a strain-specific distinct gene pools for niche specific adaptation. Furthermore, environmental strains appear to be equally virulent as contemporary nosocomial strains but remain largely antibiotic sensitive.

Acinetobacter baumannii is an opportunistic pathogen that has become difficult to eradicate mainly because of its high level of antibiotic resistance. Other features that contribute to this organism's success are the ability to compete for nutrients and iron. Recently, several novel Tn7-family transposons that encode synthesis and transport of siderophore and iron uptake systems were characterised. Here, another Tn7-type transposon (named Tn6553) is described. Tn6553 contains a set of iron utilisation genes with a transposition module related to Tn7. Tn7-family transposons that carry iron uptake systems facilitate the spread of these functions in Acinetobacter strains. Given that Tn7 is known to transpose efficiently into its preferred target site, finding siderophore functions on Tn7 family transposons is important in the context of dissemination of virulence genes amongst Acinetobacter strains.

Carbapenem-resistant Acinetobacter baumannii (CRAb) is a public health threat accounting for a significant number of hospital-acquired infections. Despite the importance of this pathogen, there is scarce literature on A. baumannii molecular epidemiology and evolutionary pathways relevant to resistance emergence in South American strains. We analyzed the genomic context of 34 CRAb isolates recovered from clinical samples between 2010 and 2013 from two hospitals in Santiago, Chile, using whole-genome sequencing. Several Institut Pasteur scheme sequence types (STs) were identified among the 34 genomes studied here, including ST1, ST15, ST79, ST162, and ST109. No ST2 (the most widespread sequence type) strain was detected. Chilean isolates were phylogenetically closely related, forming lineages specific to South America (e.g., ST1, ST79, and ST15). The genomic contexts of the resistance genes were diverse: while genes were present in a plasmid in ST15 strains, all genes were chromosomal in ST79 strains. Different variants of a small Rep_3 plasmid played a central role in the acquisition of the oxa58 carbapenem and aacC2 aminoglycoside resistance genes in ST1, ST15, and ST79 strains. The aacC2 gene along with blaTEM were found in a novel transposon named Tn6925 here. Variants of Tn7 were also found to play an important role in the acquisition of the aadA1 and dfrA1 genes. This work draws a detailed picture of the genetic context of antibiotic resistance genes in a set of carbapenem-resistant A. baumannii strains recovered from two Chilean hospitals and reveals a complex evolutionary picture of antibiotic resistance gene acquisition events via multiple routes involving several mobile genetic elements. IMPORTANCE Treating infections caused by carbapenem-resistant A. baumannii (CRAb) has become a global challenge given that CRAb strains are also often resistant to a wide range of antibiotics. Analysis of whole-genome sequence data is now a standard approach for studying the

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.

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Acinetobacter baumannii is a successful opportunistic pathogen that can compete for iron under iron-limiting conditions. Here, large novel transposons that carry genes for synthesis and transport of the fimsbactin siderophores present in some A. baumannii strains were examined. Tn6171, originally found in the A. baumannii global clone 1 (GC1) lineage 2 isolate D36, includes tns genes encoding proteins related to the TnsA, TnsB, TnsC transposition proteins (50-59 % identity), TnsD targeting protein (43 % identity) and TnsE (31 % identity) of Tn7, and is found in the chromosome downstream of the glmS gene, the preferred location for Tn7, flanked by a 5 bp target site duplication. Tn6171 is bounded by 29 bp inverted repeats and, like Tn7, includes additional TnsB binding sites at each end. Tn6171 or minor variants were detected in the equivalent location in complete or draft genomes of several further A. baumannii isolates belonging to GC1 [sequence type (ST) 1, ST81, ST94, ST328, ST623, ST717], GC2 (ST2) and ST10. However, in some of these isolates the surrounding glmS region was clearly derived from a different A. baumannii lineage, indicating that the transposon may have been acquired by replacement of a segment of the chromosome. A recombination-free phylogeny revealed that there were several transposon acquisition events in GC1. The GC1 isolates were mainly lineage 2, but a potential third lineage was also detected. A related transposon, designated Tn6552, was detected in ATCC 17978 (ST437) and other ST437 isolates. However, the Tn6552 tnsD targeting gene was interrupted by an ISAba12, and Tn6552 is not downstream of glmS.

The worldwide distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) has become a global concern, particularly in countries where antibiotic prescription is not tightly regulated. However, knowledge of the genomic aspects of CRAB from many parts of the world is still limited. Here, 50 carbapenem-resistant A. baumannii isolates recovered at a single hospital in Tehran, Iran, during several outbreaks in 2012 and 2013 were found to be resistant to multiple antibiotics. They were examined using PCR mapping and multilocus sequence typing (MLST). All Iranian strains belonged to sequence type 328 in the Institut Pasteur MLST scheme (ST328IP), a single-locus variant of ST81IP, and all Iranian strains contained two carbapenem resistance genes, oxa23 and oxa24. The oxa23 gene is in the transposon Tn2006 in AbaR4, which interrupts the chromosomal comM gene. Phylogenetic analysis using whole-genome sequence (WGS) data for 9 isolates showed that they belonged to the same clade, designated the ST81/ST328 clade, within lineage 2 of global clone 1 (GC1). However, there were two groups that included either KL13 or KL18 at the K locus (KL) for capsular polysaccharide synthesis and either a tet39 or an aadB resistance gene, respectively. The genetic context of the resistance genes was determined, and the oxa24 (OXA-72 variant) and tet39 (tetracycline resistance) genes were each in a pdif module in different plasmids. The aadB gene cassette (which encodes gentamicin, kanamycin, and tobramycin resistance) was harbored by pRAY*, and the aphA6 gene (which encodes amikacin resistance) and sul2 gene (which encodes sulfamethoxazole resistance) were each harbored by a different plasmid. The sequences obtained here will underpin future studies of GC1 CRAB strains from the Middle East region.IMPORTANCE Carbapenem-resistant Acinetobacter baumannii strains are among the most critical antibiotic-resistant bacteria causing hospital-acquired infections and treatment failures. The global

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Salmonella genomic island 1 (SGI1) is an integrative mobilisable element that plays an important role in the capture and spread of multiple drug resistance. To date, SGI1 has been found in clinical isolates of Salmonella enterica serovars, Proteus mirabilis, Morganella morganii, Acinetobacter baumannii, Providencia stuartii, Enterobacter spp, and recently in Escherichia coli. SGI1 preferentially targets the 3´-end of trmE, a conserved gene found in the Enterobacteriaceae and among members of the Gammaproteobacteria. It is, therefore, hypothesised that SGI1 and SGI1‐related elements (SGI1-REs) may have been acquired by diverse bacterial genera. Here, Bitsliced Genomic Signature Indexes (BIGSI) was used to screen the NCBI Sequence Read Archive (SRA) for putative SGI1-REs in Gammaproteobacteria. Novel SGI‐REs were identified in diverse genera including Cronobacter spp, Klebsiella spp, and Vibrio spp and in two additional isolates of Escherichia coli. An extensively drug‐resistant human clonal lineage of Klebsiella pneumoniae carrying an SGI1‐RE in the United Kingdom and an SGI1-RE that lacks a class 1 integron were also identified. These findings provide insight into the origins of this diverse family of clinically important genomic islands and expand the knowledge of the potential host range of SGI1-REs within the Gammaproteobacteria.

Stenotrophomonas maltophilia isolate CF13 is a multidrug-resistant isolate that was recovered in Sydney, Australia, in 2011, from a sputum sample from an individual with cystic fibrosis. The genome sequence of CF13 was completed using long- and short-read technologies.