bioRxiv preprint doi: https://doi.org/10.1101/710913; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 The plasmid diversity of Acinetobacter bereziniae HPC229 provides clues on the 2 ability of the species to thrive on both clinical and environmental habitats 3 4 Marco Brovedan, Guillermo D. Repizo, Patricia Marchiaro, Alejandro M. Viale*, and 5 Adriana Limansky*. 6 7 Instituto de Biología Molecular y Celular de Rosario (IBR), Departamento de 8 Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, 9 Universidad Nacional de Rosario (UNR), 2000 Rosario, Argentina. 10 11 *Corresponding authors. Alejandro M. Viale and Adriana Limansky. viale@ibr- 12 conicet.gov.ar; [email protected] 13 14 Running title: Acinetobacter bereziniae plasmid diversity 15 16 Abstract 17 Acinetobacter bereziniae is an environmental microorganism with increasing 18 clinical incidence, and may thus provide a model for a bacterial species bridging the gap 19 between the environment and the clinical setting. A. bereziniae plasmids have been 20 poorly studied, and their characterization could offer clues on the causes underlying the 21 leap between these two radically different habitats. Here we characterized the whole 22 plasmid content of A. bereziniae HPC229, a clinical strain previously reported to harbor 23 a 44-kbp plasmid, pNDM229, conferring carbapenem and aminoglycoside resistance. 24 We identified five extra plasmids in HPC229 ranging from 114 to 1.3 kbp, including 25 pAbe229-114 (114 kbp) encoding a MOBP111 relaxase and carrying heavy metal bioRxiv preprint doi: https://doi.org/10.1101/710913; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 26 resistance, a bacteriophage defense BREX system and four different toxin-antitoxin 27 (TA) systems. Two other replicons, pAbe229-15 (15.4 kbp) and pAbe229-9 (9.1 kbp), 28 both encoding MOBQ1 relaxases and also carrying TA systems, were found. The three 29 latter plasmids contained Acinetobacter Rep_3 superfamily replication initiator protein 30 genes. HPC229 also harbors two smaller plasmids, pAbe229-4 (4.4 kbp) and pAbe229- 31 1 (1.3 kbp), the former bearing a ColE1-type replicon and a TA system, and the latter 32 lacking known replication functions. Comparative sequence analyses against deposited 33 Acinetobacter genomes indicated that the above five HPC229 plasmids were unique, 34 although some regions were also present in other of these genomes. The transfer, 35 replication, and adaptive modules in pAbe229-15, and the stability module in pAbe229- 36 9, were bordered by sites potentially recognized by XerC/XerD site-specific tyrosine 37 recombinases, thus suggesting a potential mechanism for their acquisition. The presence 38 of Rep_3 and ColE1-based replication modules, different mob genes, distinct adaptive 39 functions including resistance to heavy metal and other environmental stressors, as well 40 as antimicrobial resistance genes, and a high content of XerC/XerD sites among 41 HPC229 plasmids provide evidence of substantial links with bacterial species derived 42 from both environmental and clinical habitats. 43 44 Introduction 45 Plasmids are extra-chromosomal self-replicating DNA molecules that act as 46 efficient vectors of horizontal gene transfer (HGT) across bacterial populations, thus 47 facilitating adaptation of particular individuals and derived clonal lineages to novel 48 and/or fluctuating environmental conditions [1,2]. Bacterial plasmids are assemblies of 49 different modules encompassing replication, mobilization and stability functions in 50 what is defined as the plasmid backbone [3]. This conserved structure is generally bioRxiv preprint doi: https://doi.org/10.1101/710913; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 51 accompanied by accessory genes that provide adaptive functions, including 52 pathogenicity/virulence determinants, antimicrobial resistance genes, and/or other traits 53 depending on the selective context [1,3]. Plasmids are also carriers of mobile genetic 54 elements such as insertion sequences (IS), transposons, and different genomic islands, 55 thus becoming vessels for the transport of information from a communal gene pool [1]. 56 They additionally provide scaffolds where different genetic rearrangements can occur 57 via different homologous and non-homologous recombination processes, events that 58 contribute not only to their own evolution but also the survival of their hosts under 59 different evolutionary pressures [4]. 60 The identification of plasmid types provides relevant information about their impact 61 on the physiology of their hosts and their modes of transmission [3]. Current plasmid 62 typing schemes exploit the more conserved backbone modules associated with 63 replication (Rep typing) and/or mobility (MOB typing) [3,5]. Still, a caveat of these 64 schemes is that plasmids lacking Rep or MOB genes escape classification schemes [6]. 65 In this context, the rapid advance of whole genome sequence methodologies (WGS), by 66 providing complete (or near-complete) plasmid sequences, has helped to overcome in 67 part these limitations by providing valuable insights into the evolution of the accesory 68 regions of the plasmids present in particular bacterial groups [7]. Thus, the 69 characterization not only of the plasmid backbone but also of accesory genes including 70 the complete repertoire of mobile genetic elements, and the identification of potential 71 recombinatorial hot spots in the plasmid sequence, may provide many clues on the 72 evolutionary processes that shaped their structures and drove their persistence in a given 73 bacterial group, as well as their potentiality of dissemination to novel hosts [8-12]. 74 We have recently demonstrated [13] that A. baumannii plasmids can exploit the 75 XerC/XerD site-specific recombination system [14] to mediate fusions and resolutions bioRxiv preprint doi: https://doi.org/10.1101/710913; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 76 of different replicons, some of them carrying blaOXA-58 and aphA6 genes conferring 77 carbapenem and aminoglycoside resistance, respectively. This may represent a powerful 78 mechanism not only to drive plasmid structural rearrangements but also host range 79 expansions [13]. Several authors have proposed in fact that the Acinetobacter 80 XerC/XerD recombination system may mediate the mobilization of different DNA 81 segments carrying adaptive or stability traits, a situation that includes antimicrobial 82 resistance, heavy metal resistance, and toxin-antitoxin (TA) genes among others 83 [6,13,15-18]. 84 We have recently characterized by WGS a carbapenem-resistant Acinetobacter 85 bereziniae strain isolated in Argentina designated HPC229 [19]. HPC229 carries a 86 blaNDM-1-bearing carbapenem-resistance plasmid of 44 kbp designated pNDM229 [20], 87 which displays a similar structure than plasmid pNDM-BJ01 carried by an A. lwoffii 88 strain isolated in China [21]. This strongly suggested that pNDM229 was acquired by 89 HPC229 as the result of HGT from other member of the genus, and selected due to the 90 antimicrobial pressure common to the clinical setting [20]. As a species, A. bereziniae 91 has a wide distribution and has been isolated from various sources including soils, 92 vegetables, animals, and human-associated environments such as wastewaters [22-25]. 93 More recently, A. bereziniae has been also increasingly associated to healthcare- 94 associated infections in humans, and is currently considered an emerging Acinetobacter 95 opportunistic pathogen [24,26]. In fact, although A. bereziniae isolates were in the past 96 generally susceptible to most antimicrobials of clinical use [23], since 2010 a number of 97 carbapenem-resistant clinical strains bearing IMP-, SIM-, VIM- or NDM-type metallo- 98 β-lactamases (MβL) have been reported [20,24,27,28]. MβL genes are generally carried 99 in these strains by plasmids, suggesting both the mechanism of their acquisition and 100 potential spread to other Gram-negative species [20,24]. A. bereziniae might easily bioRxiv preprint doi: https://doi.org/10.1101/710913; this version posted July 22, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 101 acquire plasmids, and has been proposed recently as a reservoir of clinically-relevant 102 antimicrobial resistance genes [24]. Contrary to the acquired resistance plasmids 103 mentioned above, the available information on the natural plasmids circulating among 104 the population