Proposal for Acinetobacter higginsii sp. nov. to accommodate organisms of human clinical origin previously classified as Acinetobacter genomic species 16.

In 1989, Bouvet and Jeanjean delineated five proteolytic genomic species (GS) of Acinetobacter, each with two to four human isolates. Three were later validly named, whereas the remaining two (GS15 and GS16) have been awaiting nomenclatural clarification. Here we present the results of the genus-wide taxonomic study of 13 human strains classified as GS16 (n=10) or GS15 (n=3). Based on core genome phylogenetic analysis, the strains formed two respective but closely related phylogroups within the Acinetobacter haemolytic clade. The intraspecies genomic average nucleotide identity based on blast (ANIb) values for GS16 and GS15 reached ≥94.9 % and ≥98.7, respectively, whereas ANIb values between them were 92.5-93.5% and those between them and the known species were ≤91.5 %. GS16 and GS15 could be differentiated from the other Acinetobacter species by their ability to lyse gelatin and sheep blood and to assimilate d,l-lactate, along with their inability to acidify d-glucose and assimilate glutarate. In contrast, GS16 and GS15 were indistinguishable from one another by metabolic/physiological features or whole-cell MALDI-TOF mass spectra. All the GS15/GS16 genomes contained genes encoding a class D ß-lactamase, Acinetobacter-derived cephalosporinase and aminoglycoside 6'-N-acetyltransferase. Searching NCBI databases revealed genome sequences of three additional isolates of GS16, but none of GS15. We conclude that our data support GS16 as representing a novel species, but leave the question of the taxonomic status of GS15 open, given its close relatedness to GS16 and the small number of available strains. We propose the name Acinetobacter higginsii sp. nov. for GS16, with the type strain NIPH 1872T (CCM 9243T=CIP 70.18T=ATCC 17988T).

Delineation of a novel environmental phylogroup of the genus Acinetobacter encompassing Acinetobacter terrae sp. nov., Acinetobacter terrestris sp. nov. and three other tentative species.

This study aimed to define the taxonomic position and structure of a novel, taxonomically unique group of 26 Acinetobacter strains, provisionally designated Taxon 24 (T24). The strains were recovered from soil and freshwater ecosystems (n = 21) or animals (n = 5) in Czechia, Scotland, Germany, the Netherlands and Turkey between 1993 and 2015. They were non-glucose-acidifying, nonhemolytic, nonproteolytic, growing at 32 °C and on acetate and ethanol as single carbon sources, but not on 4-hydroxybenzoate and mostly not at 37 °C. Their whole-genome sequences were 3.0-3.7 Mb in size, with GC contents of 39.8-41.3%. Based on core genome phylogenetic analysis, the 26 strains formed a distinct clade within the genus Acinetobacter, with strongly supported subclades termed T24A (n = 11), T24B (n = 8), T24C (n = 2), T24D (n = 3) and T24E (n = 2). The internal genomic ANIb values for these subclades were >94.8%, while the ANIb values between them were <92.5%. The results of MALDI-TOF MS-based analyses agreed with this classification. The five subclades differed from each other in the results of one to six carbon source assimilation tests. Given the genomic and phenotypic distinctness, internal coherence, numbers of available strains and geographically diverse origin of T24A and T24B, we propose the names Acinetobacter terrae sp. nov. and Acinetobacter terrestris sp. nov. for these two taxa, respectively. The type strains are ANC 4282v (= CCM 8986T = CCUG 73811T = CNCTC 8082T) and ANC 4471T (= CCM 8985T = CCUG 73812T = CNCTC 8093T), respectively. We conclude that these two species together with the other T24 strains represent a widely dispersed Acinetobacter clade primarily associated with terrestrial ecosystems.

Revising the taxonomy of the Acinetobacter lwoffii group: The description of Acinetobacter pseudolwoffii sp. nov. and emended description of Acinetobacter lwoffii.

In 1986, Bouvet and Grimont delineated two related taxa of the genus Acinetobacter termed genospecies (GS) 8 and 9. They proposed the name Acinetobacter lwoffii for GS8, which included the supposed type strain (CIP 64.10). As the authenticity of CIP 64.10 was later questioned, this study aimed at reassessing the taxonomy of these genospecies. We investigated 52 strains of GS8 or GS9, including CIP 64.10 and the genuine type strain of A. lwoffii (NCTC 5866T). All strains were subjected to the genus-wide comparative analyses of MALDI-TOF whole-cell mass spectra, rpoB gene sequences and metabolic traits while whole-genome sequences were analysed for 16 strains. The strains were classified into two distinct groups corresponding to GS8 (n=15) and GS9 (n=37). CIP 64.10 fell within GS8 whereas NCTC 5866T belonged to GS9. Intraspecies ANIb values for the genomes of GS8 (n=6) and GS9 (n=10) were ≥96.1% and ≥95.4%, respectively, whereas the ANIb values between them were 86.8-88.6%. Based on core genome phylogeny, GS8 and GS9 formed a distinct clade within the genus, with two respective, strongly supported subclades. GS8 and GS9 were similar in physiological and catabolic properties but were separable by MALDI-TOF MS. We conclude that the name A. lwoffii pertains to GS9 and not to GS8 as originally assumed and that these groups represent two species. We propose the name Acinetobacter pseudolwoffii sp. nov. for GS8, with ANC 5044T (=CCM 8638T=CCUG 67963T=CIP 111642T) as the type strain, and provide the emended description of A. lwoffii.

Limitations of routine MALDI-TOF mass spectrometric identification of Acinetobacter species and remedial actions.

A set of 204 taxonomically well-defined strains belonging to 17 Acinetobacter spp., including 11 recently described species (A. albensis, A. bohemicus, A. colistiniresistens, A. courvalinii. A. dispersus, A. gandensis, A. modestus, A. proteolyticus, A. seifertii, A. variabilis, and A. vivianii) and six species of the so-called haemolytic clade (A. beijerinckii, A. gyllenbergii, A. haemolyticus, A. junii, A. parvus, and A. venetianus), were subjected to MALDI-TOF mass spectrometric profiling. The identification outputs were evaluated using the current version (8.0.0.0) of the commercially available Bruker Daltonics, Biotyper database, which does not contain reference entries for six of the species tested. Up to 29% of the strains were falsely identified as different Acinetobacter spp. present in the Biotyper database, resulting mostly from the close phylogenetic relationship of species of the haemolytic clade. To obtain more reliable identification, extending the commercial database showed only partial improvement, while the use of an alternative MALDI matrix solution (strongly acidified ferulic acid) allowed correct identification of nearly all problematic strains.

Acinetobacter wuhouensis sp. nov., isolated from hospital sewage.

We recovered eight strains of the genus Acinetobacter from hospital sewage at West China Hospital in Chengdu, China. Based on the comparative analysis of the rpoB sequence, these strains formed a strongly supported and internally coherent cluster (intra-cluster identity of ≥98.0 %), which was clearly separated from all known Acinetobacter species (≤91.1 %). The eight strains also formed a tight and distinct cluster based on the genus-wide comparison of whole-cell mass fingerprints generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In addition, the combination of their ability to assimilate 2,3-butanediol and phenylacetate, but not 4-hydroxybenzoate, and the inability to grow at 37 °C could distinguish these eight strains from all known Acinetobacter species. Whole-genomic sequencing has been performed for two selected strains, WCHA60T and WCHA62. There were 96.65 % average nucleotide identity (ANI) and 72 % in silico DNA-DNA hybridization (isDDH) values between WCHA60T and WCHA62, suggesting that the two strains indeed belonged to the same species. In contrast, the ANI and isDDH values between the two strains and the known Acinetobacter species were <83 and <30 %, respectively; both of which were far below the cut-off to define a bacterial species. Therefore, the eight strains should be considered to represent a novel species of the genus Acinetobacter, for which the name Acinetobacterwuhouensis sp. nov. is proposed. The type strain is WCHA60T (=CCTCC AB 2016204T=GDMCC 1.1100T=KCTC 52505T).

Acinetobacter guangdongensis Feng et al. 2014 is a junior heterotypic synonym of Acinetobacter indicus Malhotra et al. 2012.

A draft whole-genome sequence was obtained for Acinetobacter guangdongensis strain KCTC 42012T and compared against those of the type strains of all Acinetobacter species with validly published names. High similarity was found to Acinetobacter indicus CCM 7832T (average nucleotide identity based on blast and digital DNA-DNA hybridization values of 96.3 and 70.4 %, respectively). In addition, the metabolic, physiological and chemotaxonomic features of KCTC 42012T were shown to be congruent with those of A. indicus. We conclude that Acinetobacter guangdongensisFeng et al. 2014 is a later heterotypic synonym of Acinetobacter indicus Malhotra et al. 2012.

Acinetobacter colistiniresistens sp. nov. (formerly genomic species 13 sensu Bouvet and Jeanjean and genomic species 14 sensu Tjernberg and Ursing), isolated from human infections and characterized by intrinsic resistance to polymyxins.

Strains of the genusAcinetobacter, classified as genomic species 13BJ/14TU have been previously associated with human infections and resistance to colistin. To clarify the taxonomy of this provisional group, we investigated 24 strains that have been isolated from humans since the 1960s in 10 countries. The genus-wide analysis of the rpoB and gyrB sequences of all strains and whole-genome sequences of strains representing different rpoB/gyrB genotypes showed that the 24 strains formed a distinct monophyletic group within the so-called haemolytic clade of the genus Acinetobacter. The distinctness of the group at the species level was supported by the results of the cluster analysis of the whole-cell protein fingerprints generated by matrix-assisted laser desorption ionization-time-of-flight MS. The 24 strains had very similar metabolic features and could be distinguished from other members of the genus by the combination of strong haemolytic and proteolytic activities and the ability to oxidize d-glucose and grow on phenylacetate and/or l-phenylalanine. The minimum inhibitory concentrations of the 24 strains to colistin and polymyxin B ranged from 16 to 64 mgl-1 and from 4 to 32 mgl-1, respectively, so uniformly reaching the current clinical resistance breakpoint (4 mg l-1) for these drugs. Genus-wide comparison revealed that such a consistently high level of resistance to polymyxins is a unique feature among species of the genus Acinetobacter,which occur in humans. We conclude that genomic species 13BJ/14TU represents a biologically meaningful and medically relevant species, for which the name Acinetobacter colistiniresistens sp. nov. is proposed. The type strain is NIPH 2036T (=CCM 8641T=CIP 110478T=CCUG 67966T=CNCTC 7573T).

Acinetobacter pakistanensis Abbas et al. 2014 is a later heterotypic synonym of Acinetobacter bohemicus Krizova et al. 2014.

Two novel species names, Acinetobacter bohemicus and Acinetobacter pakistanensis, appeared on validation list no. 161 (January 2015) under priority numbers 26 and 28, respectively. As the published data suggested a high similarity of the organisms associated with these names, we aimed to define their taxonomic relationship. The study set included all strains used in the original nomenclatural proposals, i.e. 25 strains of A. bohemicus and one strain of A. pakistanensis. The average nucleotide identity values (95.9 and 96.1 % based on blast and MUMmer, respectively) between the whole-genome sequences of A. bohemicus ANC 3994T and A. pakistanensis KCTC 42081T supported the identity of these type strains at the species level. Based on the genus-wide comparative analyses of the rpoB sequences and whole-cell fingerprints generated by matrix-assisted laser desorption/ionization-time-of-flight MS, A. pakistanensis KCTC 42081T fell within the respective clusters formed by the 25 A. bohemicus strains. The same picture was obtained on the basis of comparative analysis of 16S rRNA gene sequences of KCTC 42081T and three A. bohemicus strains. Finally, the metabolic and physiological features of KCTC 42081T were found to be congruent with those of A. bohemicus. Based on these results, we conclude that Acinetobacter pakistanensis is a later heterotypic synonym of Acinetobacter bohemicus.

Acinetobactercelticus sp. nov., a psychrotolerant species widespread in natural soil and water ecosystems.

A novel, taxonomically unique group of six strains of the genus Acinetobacter was discovered during an exploratory study on strains culturable from soil and water natural ecosystems in the Bohemian part of the Czech Republic. Based on the comparative analyses of the 16S rRNA gene, gyrB and rpoB sequences, these strains formed strongly supported and internally coherent clusters (intracluster identities of ≥99.9, ≥96.1 and ≥97.3 %, respectively), which were clearly separated from all known species of the genus Acinetobacter (≤98.7, ≤83.2 and ≤88.9 %, respectively). The distinctness of the group at the species level was evidenced also by the results of the genus-wide analyses of the whole-cell mass fingerprints of the six strains generated by matrix-assisted laser desorption/ionization-time-of-flight MS and the whole-genome sequence of a group member, ANC 4603T. Compared with the known species of the genus Acinetobacter, all six strains exhibited a unique phenotype, characterized by psychrotolerance (growth at 1 °C through 28 °C), the inability to grow at 32 °C and the ability to assimilate l-aspartate and malonate but not 2,3-butanediol or citrate. Based on these results, the name Acinetobacter celticus sp. nov. is proposed for the taxon represented by the six strains. The type strain is ANC 4603T (=CCM 8700T=CCUG 69239T=CNCTC 7549T).

Acinetobacter pragensis sp. nov., found in soil and water ecosystems.

This study aimed to define the taxonomic status of a novel, phenetically distinct group of seven strains belonging to the genus Acinetobacter, which were isolated from environmental soil and water samples collected in Central Bohemia, Czech Republic. Comparative sequence analyses of the 16S rRNA, gyrB and rpoB genes showed that all these strains formed respective tight clusters (intracluster sequence similarities of ≥99.8, ≥98.1 and ≥98.3 %, respectively), which were distant from all known Acinetobacter species (≤98.2, ≤84.0 and ≤88.9 %, respectively). The average nucleotide identity and digital DNA-DNA hybridization values (≤83.5 and ≤27.4 %, respectively) between the whole-genome sequence of a group representative (strain ANC 4149T) and those of known taxa were far below the thresholds used to discriminate between bacterial species. The seven strains also formed a tight and distinct cluster based on the genus-wide comparison of whole-cell mass fingerprints generated by matrix-assisted laser desorption/ionization time-of-flight MS and could be distinguished from all other members of the genus Acinetobacter by the combination of their ability to assimilate glutarate and l-tartrate and inability to grow at 37 °C and on l-aspartate. It is concluded that the seven strains represent a novel species for which the name Acinetobacter pragensis sp. nov. is proposed. The type strain is ANC 4149T (=CCM 8637T=CCUG 67962T=CNCTC 7530T).

Taxonomy of haemolytic and/or proteolytic strains of the genus Acinetobacter with the proposal of Acinetobacter courvalinii sp. nov. (genomic species 14 sensu Bouvet & Jeanjean), Acinetobacter dispersus sp. nov. (genomic species 17), Acinetobacter modestus sp. nov., Acinetobacter proteolyticus sp. nov. and Acinetobacter vivianii sp. nov.

We aimed to define the taxonomic status of 40 haemolytic and/or proteolytic strains of the genus Acinetobacter which were previously classified into five putative species termed as genomic species 14BJ (n=9), genomic species 17 (n=9), taxon 18 (n=7), taxon 19 (n=6) and taxon 20 (n=9). The strains were recovered mostly from human clinical specimens or soil and water ecosystems and were highly diverse in geographical origin and time of isolation. Comparative analysis of the rpoB and gyrB gene sequences of all strains, and the whole-genome sequences of selected strains, showed that these putative species formed five respective, well-supported clusters within a distinct clade of the genus Acinetobacter which typically, although not exclusively, encompasses strains with strong haemolytic activity. The whole-genome-based average nucleotide identity (ANIb) values supported the species status of each of these clusters. Moreover, the distinctness and coherence of the clusters were supported by whole-cell profiling based on MALDI-TOF MS. Congruent with these findings were the results of metabolic and physiological testing. We conclude that the five putative taxa represent respective novel species, for which the names Acinetobacter courvalinii sp. nov. (type strain ANC 3623T=CCUG 67960T=CIP 110480T=CCM 8635T), Acinetobacter dispersus sp. nov. (type strain ANC 4105T=CCUG 67961T=CIP 110500T=CCM 8636T), Acinetobacter modestus sp. nov. (type strain NIPH 236T=CCUG 67964T=CIP 110444T=CCM 8639T), Acinetobacter proteolyticus sp. nov. (type strain NIPH 809T=CCUG 67965T=CIP 110482T = CCM 8640T) and Acinetobacter vivianii sp. nov. (type strain NIPH 2168T=CCUG 67967T=CIP 110483T=CCM 8642T) are proposed.

Acinetobacter albensis sp. nov., isolated from natural soil and water ecosystems.

We have studied the taxonomic position of a phenetically unique group of eight strains of the genus Acinetobacter which were isolated from soil and water samples collected in protected landscape areas in the Czech Republic. Each of the comparative sequence analyses of the 16S rRNA, gyrB and rpoB genes showed that the eight strains formed a cohesive and tight cluster (intracluster sequence identities of ≥ 99.9 %, ≥ 98.5 % and ≥ 97.7 %, respectively), which was clearly separated from all hitherto known species of the genus Acinetobacter ( ≤ 98.6 %, ≤ 84.5 % and ≤ 89.3 %, respectively). Congruent with these findings were the results of comparative sequence analysis of three additional housekeeping genes (gltA, pyrG and recA). This genotypic distinctness was mirrored by the uniqueness of the combination of a number of independent phenotypic markers including the whole-cell spectra produced by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) MS and physiological and metabolic features. The most useful phenotypic features to differentiate the eight strains from all known species of the genus Acinetobacter were the ability to assimilate tricarballylate and the inability to grow at 35 °C or to assimilate ethanol or l-histidine. We conclude that the eight strains represent a novel environmental species for which the name Acinetobacter albensis sp. nov. is proposed. The type strain is ANC 4874T ( = CCUG 67281T = CCM 8611T).

Acinetobacter seifertii sp. nov., a member of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex isolated from human clinical specimens.

This study aimed to define the taxonomic status of a phenetically distinct group of 16 strains that corresponds to Acinetobacter genomic species 'close to 13TU', a provisional genomic species of the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex recognized by Gerner-Smidt and Tjernberg in 1993. These strains have been isolated in different countries since the early 1990s and were mostly recovered from human clinical specimens. They were compared with 45 reference strains representing the known taxa of the ACB complex using taxonomic methods relevant to the genus Acinetobacter. Based on sequence analysis of the concatenated partial sequences (2976 bp) of seven housekeeping genes, the 16 strains formed a tight and well-supported cluster (intracluster sequence identity of ≥98.4 %) that was clearly separated from the other members of the ACB complex (≤94.7 %). The species status of the group was supported by average nucleotide identity values of ≤91.7 % between the whole genome sequence of representative strain NIPH 973(T) (NCBI accession no. APOO00000000) and those of the other species. In addition, whole-cell matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS analyses indicated the distinctness of the group at the protein level. Metabolic and physiological tests revealed several typical features of the group, although they did not allow its reliable differentiation from the other members of the ACB complex. We conclude that the 16 strains represent a distinct novel species, for which we propose the name Acinetobacter seifertii sp. nov. The type strain is NIPH 973(T) ( = CIP 110471(T) = CCUG 34785(T) = CCM 8535(T)).

A taxonomically unique Acinetobacter strain with proteolytic and hemolytic activities recovered from a patient with a soft tissue injury.

A taxonomically unique bacterial strain, Acinetobacter sp. A47, has been recovered from several soft tissue samples from a patient undergoing reconstructive surgery owing to a traumatic amputation. The results of 16S rRNA, rpoB, and gyrB gene comparative sequence analyses showed that A47 does not belong to any of the hitherto-known taxa and may represent an as-yet-unknown Acinetobacter species. The recognition of this novel organism contributes to our knowledge of the taxonomic complexity underlying infections caused by Acinetobacter.

Acinetobacter variabilis sp. nov. (formerly DNA group 15 sensu Tjernberg & Ursing), isolated from humans and animals.

We aimed to define the taxonomic status of 16 strains which were phenetically congruent with Acinetobacter DNA group 15 described by Tjernberg & Ursing in 1989. The strains were isolated from a variety of human and animal specimens in geographically distant places over the last three decades. Taxonomic analysis was based on an Acinetobacter-targeted, genus-wide approach that included the comparative sequence analysis of housekeeping, protein-coding genes, whole-cell profiling based on matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), an array of in-house physiological and metabolic tests, and whole-genome comparative analysis. Based on analyses of the rpoB and gyrB genes, the 16 strains formed respective, strongly supported clusters clearly separated from the other species of the genus Acinetobacter. The distinctness of the group at the species level was indicated by average nucleotide identity values of ≤82 % between the whole genome sequences of two of the 16 strains (NIPH 2171(T) and NIPH 899) and those of the known species. In addition, the coherence of the group was also supported by MALDI-TOF MS. All 16 strains were non-haemolytic and non-gelatinase-producing, grown at 41 °C and utilized a rather limited number of carbon sources. Virtually every strain displayed a unique combination of metabolic and physiological features. We conclude that the 16 strains represent a distinct species of the genus Acinetobacter, for which the name Acinetobacter variabilis sp. nov. is proposed to reflect its marked phenotypic heterogeneity. The type strain is NIPH 2171(T) ( = CIP 110486(T) = CCUG 26390(T) = CCM 8555(T)).