GBE The Environmental Acinetobacter baumannii Isolate DSM30011 Reveals Clues into the Preantibiotic Era Genome Diversity, Virulence Potential, and Niche Range of a Predominant Nosocomial Pathogen Guillermo D. Repizo1,2,*, Alejandro M. Viale2,Vıtor Borges3,Marıa M. Cameranesi2, Najwa Taib4, Martın Espariz2,Ce´lineBrochier-Armanet4,Joao~ Paulo Gomes3, and Suzana P. Salcedo1 1Laboratory of Molecular Microbiology and Structural Biochemistry, CNRS UMR5086, University of Lyon, France 2Departamento de Microbiologia, Instituto de Biologia Molecular y Celular de Rosario (IBR, CONICET), Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Argentina 3Bioinformatics Unit, Department of Infectious Diseases, National Institute of Health, Lisbon, Portugal 4Laboratoire de Biome´ trie et Biologie Evolutive, Univ. Lyon, Universite´ Lyon 1, CNRS, UMR5558, Villeurbanne, France *Corresponding author: E-mail: [email protected]. Accepted: August 24, 2017 Data deposition: The whole genome shotgun project for A. baumannii DSM30011 has been deposited at DDBJ/ENA/GenBank under the accession JJOC00000000. The version described in this paper is version JJOC02000000. Abstract Acinetobacter baumannii represents nowadays an important nosocomial opportunistic pathogen whose reservoirs outside the clinical setting are obscure. Here, we traced the origins of the collection strain A. baumannii DSM30011 to an isolate first reported in 1944, obtained from the enriched microbiota responsible of the aerobic decomposition of the resinous desert shrub guayule. Whole-genome sequencing and phylogenetic analysis based on core genes confirmed DSM30011 affiliation to A. baumannii. Comparative studies with 32 complete A. baumannii genomes revealed the presence of 12 unique accessory chromosomal regions in DSM30011 including five encompassing phage-related genes, five containing toxin genes of the type-6 secretion system, and one with an atypical CRISPRs/cas cluster. No antimicrobial resistance islands were identified in DSM30011 agreeing with a general antimicrobial susceptibility phenotype including folate synthesis inhibitors. The marginal ampicillin resistance of DSM30011 most likely derived from chromosomal ADC-type ampC and blaOXA-51-type genes. Searching for catabolic pathways genes revealed several clusters involved in the degradation of plant defenses including woody tissues and a previously unreported atu locus responsible of aliphatic terpenes degradation, thus suggesting that resinous plants may provide an effective niche for this organism. DSM30011 also harbored most genes and regulatory mechanisms linked to persistence and virulence in pathogenic Acinetobacter species. This strain thus revealed important clues into the genomic diversity, virulence potential, and niche ranges of the preantibiotic era A. baumannii population, and may provide an useful tool for our understanding of the processes that led to the recent evolution of this species toward an opportunistic pathogen of humans. Key words: comparative genomics, preantibiotic era bacterium, virulence factors, CRISPRs/cas. Introduction Among the genus, the phylogenetically closely-related species The genus Acinetobacter,familyMoraxellaceae,class composing the Acinetobacter calcoaceticus–Acinetobacter Gammaproteobacteria, is characterized by Gram-negative baumannii (Acb) complex represent nowadays important op- aerobic coccobacilli of ubiquitous environmental distribution portunistic pathogens (Antunes et al. 2014). Infections due to and large metabolic capabilities (Bouvet and Grimont 1986). A. baumannii in particular, rarely reported in healthcare ß The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2292 Genome Biol. Evol. 9(9):2292–2307. doi:10.1093/gbe/evx162 Advance Access publication August 26, 2017 Acinetobacter baumannii DSM30011 Genomic Analysis GBE settings before the 1970s, rapidly increased in importance contamination remains debated (Eveillard et al. 2013; with the global spread of a limited number of epidemic clonal Antunes et al. 2014). complexes (CC) possessing multidrug-resistance (MDR) phe- Acinetobacter baumannii DSM30011 is a collection strain notypes (Diancourt et al. 2010; Antunes et al. 2014). Strains amenable for genetic manipulation (Wilharm et al. 2013). This composing the CCs generally contain plasmids and organism is capable of killing Galleria mellonella larvae, out- chromosomally-located resistance islands (RI) and genomic competing other clinically-relevant bacterial species in a type- islands (GI) encompassing different transposons and integrons 6 secretion system (T6SS)-dependent manner, and forming which play pivotal roles in both antimicrobial and heavy metal biofilms and biopellicles (Repizo et al. 2015), that is, traits resistance (Di Nocera et al. 2011; Nigro et al. 2011; Antunes generally associated to bacterial persistence in different envi- et al. 2014; Touchon et al. 2014). Also, CC strains carry a large ronments including the clinical setting (Roca et al. 2012; repertoire of insertion sequences (ISs) capable of mediating Longo et al. 2014; Nait Chabane et al. 2014). Here, we traced genome rearrangements, deletions, insertions, inversions, and DSM30011 origins to a strain originally deposited at the NRRL gene overexpression with strong adaptive significances (Roca in 1943 as Achromobacter lacticum 4-h (NRRL B-551, et al. 2012; Antunes et al. 2014; Touchon et al. 2014). The Agricultural Research Service Culture Collection, ARS, U.S. combination of the above factors, added to the intrinsic resis- Department of Agriculture; see supplementary table S1, tance of A. baumannii to desiccation and fever-associated Supplementary Material online for details). This strain was temperatures, are considered main factors of persistence of isolated after enrichment of the resin-degrading natural pathogenic strains in the nosocomial environment (Roca et al. microbiota responsible of the aerobic decomposition of gua- 2012; Antunes et al. 2014). yule (Parthenium argentatum Gray), a resin-producing angio- Whole-genome sequence (WGS) comparisons have be- sperm of the Asteraceae family common to arid and semiarid come commonplace in examining strain-to-strain variability areas of the south-western United States and north-central and in comparing pathogenic strains with environmental, Mexico (Allen et al. 1944; Naghski et al. 1944). DSM30011 less aggressive relatives in efforts to identify underlying ge- thus provided us with an environmental A. baumannii strain netic determinants and mechanisms responsible for pheno- isolated by the middle of last century just before the onset of typic dissimilarities. When applied to A. baumannii,these the antibiotic era (Antunes et al. 2014), and therefore well- approaches provided valuable information on both the pre- differentiated both temporally and epidemiologically from the sent A. baumannii population structure and the mechanisms clinical strains predominant nowadays. Here, we conducted a of acquisition and evolution of antimicrobial resistance (Di comparative genomic analysis of DSM30011 with other Nocera et al. 2011, Roca et al. 2012; Antunes et al. 2014; Acinetobacter strains to obtain clues into the genomic diver- Touchon et al. 2014). It is now generally accepted that A. sity and virulence potential of the preantibiotic era A. bau- baumannii arose from other members of the Acb complex mannii population. as the result of an ancient population bottleneck, followed by a recent population expansion from a few clinically- Materials and Methods relevant clones endowed with a diverse arsenal of resistance genes (Diancourtetal.2010; Antunes et al. 2014; Touchon Strain Sources et al. 2014). Still, the identification of A. baumannii virulence The DSM30011 strain used for genome sequencing in this traits has remained elusive, and in fact large genetic variations work was obtained from the DSMZ-German Collection of have been found both between and within CC members Microorganisms and Cell Cultures (Braunschweig, suggesting a complex and even a multifactorial nature of Germany), and was kindly provided to us by Dr X. mechanisms involved (Diancourt et al. 2010; Roca et al. Charpentier (CIRI, Lyon, France). The original isolation and 2012; Antunes et al. 2014). In this context it has recently different denominations assigned to this strain by various col- been emphasized the importance of a deeper genomic study lections are detailed below, under “Tracing DSM30011 ori- of non-clinical (“environmental”) isolates to clarify both the gins” and in supplementary table S1, Supplementary Material virulence potential of the aboriginal A. baumannii population online. Acinetobacter sp. strains NCIMB8208 and and the evolutionary paths that led toward an opportunistic NCIMB8209 were obtained from the National Collection of pathogen lifestyle (Antunes et al. 2014). However, and con- Industrial Food and Marine Bacteria (NCIMB, Aberdeen, trasting with the environmental ubiquity of most other mem- Scotland). The A. baumannii type strains ATCC19606 and bers of the genus, the natural habitats and infection reservoirs