Genome-based taxonomic reclassication of Acinetobacter species using type and reference strains

Acinetobacter species are widely distributed in the environment and clinical settings worldwide and serve as natural reservoirs of antimicrobial resistance genes and occasional human pathogens responsible for nosocomial infections. In this study, we performed genomic analysis of Acinetobacter seohaensis DSM 16313, a type strain of the proposed Acinetobacter species. This species was estimated to be evolutionary close to Acinetobacter towneri but the genome sequence of A. seohaensis was not publicly available. Pangenome analysis of the genome sequence of A. seohaensis along with those of genome-available type and reference strains of 82 Acinetobacter species including A. towneri suggested that three groups of Acinetobacter species, A. seohaensis and A. towneri; Acinetobacter pullorum and Acinetobacter portensis; and Acinetobacter idrijaensis, Acinetobacter mesopotamicus, and Acinetobacter lwoi, were phylogenetically very similar to each other. Genome comparisons based on in silico DNA-DNA hybridization and the average nucleotide identity conrmed that these three groups of Acinetobacter species are conspecic. Based on the rules of priority, A. seohaensis, A. pullorum, and A. idrijaensis/A. mesopotamicus should be reclassied as later heterotypic synonyms of A. towneri, A. portensis, and A. lwoi, respectively.


Main Text
Acinetobacter towneri, belonging to the genus Acinetobacter whose members are gram-negative aerobic coccobacilli, is often isolated from water environments worldwide (1). This species has become increasingly important in recent years as a natural reservoir of antimicrobial resistance (AMR) genes (2)(3)(4)(5)(6). Acinetobacter baumannii, a cause of opportunistic infections in humans, has acquired resistance mechanisms to various antimicrobials, including clinically important carbapenems, making antimicrobial therapy di cult (7). AMR genes, such as those for carbapenem-hydrolyzing enzymes (carbapenemases), have spread among environmental and clinical Acinetobacter species via mobile gene elements such as plasmids (2)(3)(4). Tigecycline is a last-resort antimicrobial with promising activity against carbapenemaseproducing gram-negative bacteria, including Acinetobacter species; however, mobile genes for tigecyclineinactivating enzymes, tet(X), have also emerged in A. towneri (5, 6) and A. baumannii (8)(9)(10). Accumulation of such clinically relevant AMR genes in environmental bacteria such as A. towneri and their transmission to human pathogenic bacteria such as A. baumannii poses a global public health threat.
As of July 1, 2021, the List of Prokaryotic names with Standing in Nomenclature (LSPN) listed 92 species of the genus Acinetobacter (https://lpsn.dsmz.de/genus/acinetobacter). Of these, Acinetobacter venetianus and Acinetobacter refrigeratoris (formerly Acinetobacter refrigeratorensis) were listed in duplicate. Additionally, Acinetobacter grimontii, Acinetobacter guangdongensis, Acinetobacter pakistanensis, and Acinetobacter dijkshoorniae were later identi ed as different species (Acinetobacter junii, Acinetobacter indicus, Acinetobacter bohemicus, and Acinetobacter lactucae, respectively) (11)(12)(13)(14); therefore, the LSPN lists 86 unique species of Acinetobacter (Table S1). Of these, 68 species were validly published in the International Journal of Systematic and Evolutionary Microbiology (IJSEM), whereas the remaining 18 species were not validly published (Table S1). To date, the genome sequences of type and reference strains of 79 species are available in the NCBI database, with all 68 species published in the IJSEM (68/68, 100%) and 11 species not validly published (11/18, 61.1%) (Table S1). Although not listed in the LSPN, one novel species of Acinetobacter, Acinetobacter kanungonis, has been validly published in the IJSEM (15), and two novel species of Acinetobacter, Acinetobacter rongchengensis and Acinetobacter tianfuensis, have been proposed from large-scale reanalysis on 3,956 genomes of Acinetobacter species in public databases and published in another journal (16) (Table S1).
During molecular epidemiological analysis of carbapenem-resistant A. towneri isolates from hospital sewage in Japan, we performed genomic analysis of Acinetobacter seohaensis DSM 16313, a proposed type strain whose genome sequence was estimated to be similar to that of A. towneri but the genome sequence of A. seohaensis was not publicly available (17). The 16S rRNA gene sequence of A. seohaensis DSM 16313 (accession no. AY633608) (17) was shown to be nearly identical (99.8%) to that of A. towneri DSM 14962 T (type strain, accession no. EF611416). The Illumina sequencing library (pairedend, insert size 500-900 bp) was prepared using the Nextera XT DNA Library Prep Kit (Illumina). Wholegenome sequencing using the HiSeq X system (Illumina) was performed, followed by de novo assembly of Illumina reads using Shovill v1.1.0 (https://github.com/tseemann/shovill) with default parameters.
The resulting draft genome sequence of A. seohaensis DSM 16313 (accession no. BPEQ00000000) consisted of 298 contigs with a genome size of 2,99 Mbp and GC content of 41.3%.
The priority of prokaryotic names is governed by the International Code of Nomenclature of Prokaryotes (25). Rule 23a of the code states that, "In a given position, a species can bear only one correct epithet, that is, the earliest that is in accordance with the Rules of this Code". Rules 23b, 24a, and 24b establish the priority of names based on their dates of valid publication in the IJSEM. In our case, A. towneri was validly published in the IJSEM in July 2003 (1) and A. seohaensis was published in another journal in November 2007 (17); A. portensis was validly published in the IJSEM in August 2020 (21) and A. pullorum was published in another journal in April 2020 (20); A. lwo i was validly published in the International Journal of Systematic and Evolutionary Bacteriology (predecessor journal of the IJSEM) in April 1986 (24), and A. idrijaensis and A. mesopotamicus were published in other journals in November 2014 and October 2020, respectively (22,23). Based on the rules of priority, A. seohaensis, A. pullorum, and A. idrijaensis/A. mesopotamicus are later heterotypic synonyms of A. towneri, A. portensis, and A. lwo i, respectively. Core genome phylogeny of publicly available genomes of type and reference strains of 83 species of Acinetobacter. Bar lengths represent the number of substitutions per site in the core genome. Names of type and reference strains of Acinetobacter species, accession nos. of the genome sequences, years when each strain was validly published in the International Journal of Systematic and Evolutionary Microbiology (or years when each strain was published in another journal), and average nucleotide