The best of both worlds: a proposal for further integration of Candidatus names into the International Code of Nomenclature of Prokaryotes.

The naming of prokaryotes is governed by the International Code of Nomenclature of Prokaryotes (ICNP) and partially by the International Code of Nomenclature for Algae, Fungi and Plants (ICN). Such codes must be able to determine names of taxa in a universal and unambiguous manner, thus serving as a common language across different fields and activities. This unity is undermined when a new code of nomenclature emerges that overlaps in scope with an established, time-tested code and uses the same format of names but assigns different nomenclatural status values to the names. The resulting nomenclatural confusion is not beneficial to the wider scientific community. Such ambiguity is expected to result from the establishment of the 'Code of Nomenclature of Prokaryotes Described from DNA Sequence Data' ('SeqCode'), which is in general and specific conflict with the ICNP and the ICN. Shortcomings in the interpretation of the ICNP may have exacerbated the incompatibility between the codes. It is reiterated as to why proposals to accept sequences as nomenclatural types of species and subspecies with validly published names, now implemented in the SeqCode, have not been implemented by the International Committee on Systematics of Prokaryotes (ICSP), which oversees the ICNP. The absence of certain regulations from the ICNP for the naming of as yet uncultivated prokaryotes is an acceptable scientific argument, although it does not justify the establishment of a separate code. Moreover, the proposals rejected by the ICSP are unnecessary to adequately regulate the naming of uncultivated prokaryotes. To provide a better service to the wider scientific community, an alternative proposal to emend the ICNP is presented, which would result in Candidatus names being regulated analogously to validly published names. This proposal is fully consistent with previous ICSP decisions, preserves the essential unity of nomenclature and avoids the expected nomenclatural confusion.

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).

Novel lipophosphonoxin-loaded polycaprolactone electrospun nanofiber dressing reduces Staphylococcus aureus induced wound infection in mice.

Active wound dressings are attracting extensive attention in soft tissue repair and regeneration, including bacteria-infected skin wound healing. As the wide use of antibiotics leads to drug resistance we present here a new concept of wound dressings based on the polycaprolactone nanofiber scaffold (NANO) releasing second generation lipophosphonoxin (LPPO) as antibacterial agent. Firstly, we demonstrated in vitro that LPPO released from NANO exerted antibacterial activity while not impairing proliferation/differentiation of fibroblasts and keratinocytes. Secondly, using a mouse model we showed that NANO loaded with LPPO significantly reduced the Staphylococcus aureus counts in infected wounds as evaluated 7 days post-surgery. Furthermore, the rate of degradation and subsequent LPPO release in infected wounds was also facilitated by lytic enzymes secreted by inoculated bacteria. Finally, LPPO displayed negligible to no systemic absorption. In conclusion, the composite antibacterial NANO-LPPO-based dressing reduces the bacterial load and promotes skin repair, with the potential to treat wounds in clinical settings.

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.

Strain "Acinetobacter mesopotamicus" GC2 Does Not Represent a Novel Species, but Belongs to the Species Acinetobacter lwoffii as Revealed by Whole-Genome Sequence-Based Analysis.

The formal scientific name "Acinetobacter mesopotamicus" has recently been proposed for bacterial strain GC2 isolated from soil (Curr Microbiol 77:3192-3200). However, the recalculation of the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) parameters revealed that their values for the genome sequences of GC2 and Acinetobacter lwoffii NCTC 5866T were as high as 95.7% and 66.1%, respectively. Moreover, the ANI and dDDH values for the genome of GC2 tested against those of 10 reference strains of A. lwoffii were 95.5-96.7% and 65.5-74.2%, respectively, and fall within the intraspecies range of these values known for A. lwoffii. These data indicate that strain GC2 belongs to A. lwoffii and contradicts the results of the authors of the proposal, who found the ANI and dDDH values for the GC2 and A. lwoffii DSM 2403T genomes to be 88.87% and 36.1%, respectively. The misclassification of strain GC2 is likely to result from the use of an incorrect reference genome sequence for in silico comparative analyses.

Pathogenic Acinetobacter species including the novel Acinetobacter dijkshoorniae recovered from market meat in Peru.

Species of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex are important human pathogens which can be recovered from animals and food, potential sources for their dissemination. The aim of the present study was to characterise the Acinetobacter isolates recovered from market meat samples in Peru. From July through August 2012, 138 meat samples from six traditional markets in Lima were cultured in Lysogeny and Selenite broths followed by screening of Gram-negative bacteria in selective media. Bacterial isolates were identified by MALDI-TOF MS and DNA-based methods and assessed for their clonal relatedness and antimicrobial susceptibility. Twelve Acinetobacter isolates were recovered from calf samples. All but one strain were identified as members of the clinically-relevant Acinetobacter calcoaceticus-Acinetobacter baumannii complex: 9 strains as Acinetobacter pittii, 1 strain as A. baumannii, and 1 strain as the recently described novel species A. dijkshoorniae. The remaining strain could not be identified at the species level unambiguously but all studies suggested close relatedness to A. bereziniae. All isolates were well susceptible to antibiotics. Based on macrorestriction analysis, six isolates were further selected and some of them were associated with novel MLST profiles. The presence of pathogenic Acinetobacter species in human consumption meat might pose a risk to public health as potential reservoirs for their further spread into the human population. Nevertheless, the Acinetobacter isolates from meat found in this study were not multidrug resistant and their prevalence was low. To our knowledge, this is also the first time that the A. dijkshoorniae species is reported in Peru.

Acinetobacter cumulans sp. nov., isolated from hospital sewage and capable of acquisition of multiple antibiotic resistance genes.

We studied the taxonomic position of six phenetically related strains of the genus Acinetobacter, which were recovered from hospital sewage in China and showed different patterns of resistance to clinically important antibiotics. Whole-genome sequencing of these strains and genus-wide phylogeny reconstruction based on a set of 107 Acinetobacter core genes indicated that they formed a separate and internally cohesive clade within the genus. The average nucleotide identity based on BLAST and digital DNA-DNA hybridization values between the six new genomes were 97.25-98.67% and 79.2-89.3%, respectively, whereas those between them and the genomes of the known species were ≤78.57% and ≤28.5%, respectively. The distinctness of the strains at the species level was also supported by the results of the cluster analysis of the whole-cell protein fingerprints generated by MALDI-TOF MS. Moreover, the strains displayed a catabolically unique profile and could be differentiated from the phylogenetically closest species at least by their inability to grow on d,l-lactate. A total of 18 different genes were found in the six genome sequences which encode resistance to seven classes of antimicrobial agents, including clinically important carbapenems, oxyimino-cephalosporins, or aminoglycosides. These genes occurred in five different combinations, with three to 10 different genes per strain. We conclude that the six strains represent a novel Acinetobacter species, for which we propose the name Acinetobacter cumulans sp. nov. to reflect its ability to acquire and cumulate diverse resistance determinants. The type strain is WCHAc060092T (ANC 5797T=CCTCC AB 2018119T=GDMCC 1.1380T=KCTC 62576T).

Differentiation of Taxonomically Closely Related Species of the Genus Acinetobacter Using Raman Spectroscopy and Chemometrics.

In recent years, several efforts have been made to develop quick and low cost bacterial identification methods. Genotypic methods, despite their accuracy, are laborious and time consuming, leaving spectroscopic methods as a potential alternative. Mass and infrared spectroscopy are among the most reconnoitered techniques for this purpose, with Raman having been practically unexplored. Some species of the bacterial genus Acinetobacter are recognized as etiological agents of nosocomial infections associated with high rates of mortality and morbidity, which makes their accurate identification important. The goal of this study was to assess the ability of Raman spectroscopy to discriminate between 16 Acinetobacter species belonging to two phylogroups containing taxonomically closely related species, that is, the Acinetobacter baumannii-Acinetobacter calcoaceticus complex (six species) and haemolytic clade (10 species). Bacterial spectra were acquired without the need for any sample pre-treatment and were further analyzed with multivariate data analysis, namely partial least squares discriminant analysis (PLSDA). Species discrimination was achieved through a series of sequential PLSDA models, with the percentage of correct species assignments ranging from 72.1% to 98.7%. The obtained results suggest that Raman spectroscopy is a promising alternative for identification of Acinetobacter species.

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).

Identification of Acinetobacter seifertii isolated from Bolivian hospitals.

Acinetobacter seifertii is a recently described species that belongs to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex. It has been recovered from clinical samples and is sometimes associated with antimicrobial resistance determinants. We present here the case of three A. seifertii clinical isolates which were initially identified as Acinetobacter sp. by phenotypic methods but no identification at the species level was achieved using semi-automated identification methods. The isolates were further analysed by whole genome sequencing and identified as A. seifertii. Due to the fact that A. seifertii has been isolated from serious infections such as respiratory tract and bloodstream infections, we emphasize the importance of correctly identifying isolates of the genus Acinetobacter at the species level to gain a deeper knowledge of their prevalence and clinical impact.

Ability of phages to infect Acinetobacter calcoaceticus-Acinetobacter baumannii complex species through acquisition of different pectate lyase depolymerase domains.

Bacteriophages are ubiquitous in nature and represent a vast repository of genetic diversity, which is driven by the endless coevolution cycle with a diversified group of bacterial hosts. Studying phage-host interactions is important to gain novel insights into their dynamic adaptation. In this study, we isolated 12 phages infecting species of the Acinetobacter baumannii-Acinetobacter calcoaceticus complex which exhibited a narrow host range and similar morphological features (podoviruses with short tails of 9-12 nm and isometric heads of 50-60 nm). Notably, the alignment of the newly sequenced phage genomes (40-41 kb of DNA length) and all Acinetobacter podoviruses deposited in Genbank has shown high synteny, regardless of the date and source of isolation that spans from America to Europe and Asia. Interestingly, the C-terminal pectate lyase domain of these phage tail fibres is often the only difference found among these viral genomes, demonstrating a very specific genomic variation during the course of their evolution. We proved that the pectate lyase domain is responsible for phage depolymerase activity and binding to specific Acinetobacter bacterial capsules. We discuss how this mechanism of phage-host co-evolution impacts the tail specificity apparatus of Acinetobacter podoviruses.

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).