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Article

Acinetobacter nematophilus sp. nov., Alcaligenes nematophilus sp. nov., Enterobacter nematophilus sp. nov., and Kaistia nematophila sp. nov., Isolated from Soil-Borne Nematodes and Proposal for the Elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the Species Level

by
Ricardo A. R. Machado
1,*,
Ameni Loulou
2,†,
Aashaq Hussain Bhat
1,†,
Maristella Mastore
3,
Céline Terrettaz
4,
Maurizio Francesco Brivio
3 and
Sadreddine Kallel
2,*
1
Experimental Biology Research Group, Institute of Biology, University of Neuchâtel, 2000 Neuchâtel, Switzerland
2
Laboratory of Bio-aggressor and Integrated Protection in Agriculture, Department of Plant Health and Environment, National Agronomic Institute of Tunisia, University of Carthage, Tunis-Mahrajène 1082, Tunisia
3
Laboratory of Comparative Immunology and Parasitology, Department of Theoretical and Applied Sciences, University of Insubria, 21100 Varese, Italy
4
Laboratory of Microbiology, Institute of Biology, University of Neuchâtel, 2000 Neuchâtel, Switzerland
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this study.
Taxonomy 2023, 3(1), 148-168; https://doi.org/10.3390/taxonomy3010012
Submission received: 19 November 2022 / Revised: 17 February 2023 / Accepted: 24 February 2023 / Published: 9 March 2023
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Abstract

:
Four bacterial strains, A-IN1T, A-TC2T, E-TC7T, and K-TC2T, isolated from soil-borne nematodes of the species Oscheius tipulae and Acrobeloides bodenheimeri, were found to represent new species of the genera Acinetobacter, Alcaligenes, Enterobacter, and Kaistia, respectively. In this study, we described these new species using a polyphasic taxonomic approach that included whole-genome and whole-proteome phylogenomic reconstructions, core genome sequence comparisons, and phenotypic characterization. Phylogenomic reconstructions using whole-genome and whole-proteome sequences show that A-IN1T is closely related to Acinetobacter guillouiae DSM 590T and to Acinetobacter bereziniae LMG 1003T. The dDDH values between A-IN1T and these latest strains are 25.1 and 39.6%, respectively, which are below the 70% divergence threshold for prokaryotic species delineation. A-TC2T is closely related to Alcaligenes faecalis subsp. faecalis DSM 30030T and to Alcaligenes faecalis subsp. phenolicus DSM 16503T. The dDDH values between A-TC2T and these latest strains are 47.0 and 66.3%, respectively. In addition, the dDDH values between Alcaligenes faecalis subsp. faecalis DSM 30030T, Alcaligenes faecalis subsp. phenolicus DSM 16503T, and Alcaligenes faecalis subsp. parafaecalis are always lower than 70%, demonstrating that the three strains represent species within the genus Alcaligenes rather than subspecies within Alcaligenes faecalis. E-TC7T is closely related to Enterobacter kobei DSM 13645T, Enterobacter chuandaensis 090028T, and to Enterobacter bugandensis STN0717-56T. The dDDH values between E-TC7T and these strains are 43.5, 42.9, and 63.7%, respectively. K-TC2T is closely related to Kaistia terrae DSM 21341T and to Kaistia defluvii JCM 18034T. The dDDH values between these strains are 29.2 and 30.7%, respectively. Several biochemical tests allow to differentiate the type strains of the newly described species from the type strains of their more closely related species. Based on the results of this polyphasic taxonomic approach, the following new species are proposed: Acinetobacter nematophilus sp. nov. with A-IN1T (=CCM 9231T =CCOS 2018T) as the type strain, Alcaligenes nematophilus sp. nov. with A-TC2T (=CCM 9230T =CCOS 2017T) as the type strain, Enterobacter nematophilus sp. nov. with E-TC7T (=CCM 9232T =CCOS 2020T) as the type strain, and Kaistia nematophila sp. nov. with K-TC2T (=CCM 9239T =CCOS 2022T) as the type strain. In addition, we propose the elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the species level. Therefore, we propose the creation of Alcaligenes parafaecalis sp. nov. with DSM 13975T as the type strain, and Alcaligenes phenolicus sp. nov. with DSM 16503T as the type strain. Our study contributes to a better understanding of the biodiversity and phylogenetic relationships of bacteria associated with soil-borne nematodes.

1. Introduction

Soil nematodes associate with several different bacterial species [1,2,3]. Certain nematodes, for instance, establish obligate mutualistic associations with entomopathogenic bacteria, which aid them to kill insects by producing toxins and digestive enzymes [4,5]. Apart from this, bacteria can also serve as food sources for free-living nematodes, aid nematodes in development, defense, reproduction and nutrient acquisition [1,2,6,7,8,9,10,11,12]. Bacteria thus play essential roles for soil-borne nematodes.
To characterize the biodiversity of bacteria associated with soil-borne nematodes, we often conduct surveys to recover soil nematodes, culture them in the laboratory, and then isolate their associated bacteria. During one of our surveys, we isolated several nematode species, which harbor four novel bacterial species of the genus Acinetobacter, Alcaligenes, Enterobacter, and Kaistia [13,14,15]. In this study, we morphologically, biochemically and molecularly characterized them to describe these new species.
The genus Acinetobacter was first described by Brisou and Prévot (1954) [16]. Members of this genus are Gram-negative coccobacilli, non-motile, non-spore-forming, aerobic, and mesophilic. Bacteria of this genus can survive under different environmental conditions and have been isolated from food, including fish, meat, cheese, milk, and vegetables [17]. Moreover, some Acinetobacter species have been recovered from activated sludge, sewage, dump sites, raw wastewater, and hydrocarbon-contaminated areas [18,19,20]. Over the past decades, some species of this genus have emerged as significant nosocomial and opportunistic pathogens causing outbreaks of colonization and infection, especially in immunosuppressed patients [21,22]. At the time of writing, this genus includes 76 species with valid published and correct names (https://lpsn.dsmz.de/genus/acinetobacter; accessed on 3 March 2023).
The genus Alcaligenes was first described by Castellani and Chalmers (1919) [23]. Members of this genus are Gram-negative, coccobacillary rods, motile, aerobic, and mesophilic. Bacteria of this genus have been isolated from plants, soil, sediment, bioprocess residues, water, and clinical samples [24,25,26]. Some species are resistant to heavy metals such as chromium, and other environmental pollutants such as phenol [24,27,28]. At the time of writing, this genus includes four species and three subspecies with valid published and correct names (https://lpsn.dsmz.de/genus/alcaligenes; accessed on 3 March 2023).
The genus Enterobacter was first described by Hormaeche and Edwards (1960) [29]. Members of this genus are Gram-negative, rod-shaped, non-spore forming, facultative anaerobic, and mesophilic [30]. Bacteria of this genus are widely distributed in nature. They are plant pathogens, colonize the human gut, and certain species can also cause urinary and respiratory infections in humans [31,32,33,34,35]. At the time of writing, this genus includes 24 species with valid published names (https://lpsn.dsmz.de/genus/enterobacter; accessed on 3 March 2023). Noteworthily, this latest list still does not reflect recent propositions to elevate the following subspecies to the species level: E. cloacae subsp. cloacae, E. cloacae subsp. dissolvens, E. hormaechei subsp. hormaechei, and E. hormaechei subsp. xiangfangensis [36]. All these species are actually independent species rather than subspecies: E. cloacae, E. dissolvens, E. hormaechei, and E. xiangfangensis [36].
The genus Kaistia was first described by Im et al. (2005) [37]. Members of this genus are Gram-negative, short rod to coccus-shaped, non-motile, predominantly aerobic although some species can be facultative anaerobes, and mesophilic [37]. They are also widely distributed in nature. Some species have been isolated from peat layers, anaerobic sludge blankets, soil sediments, freshwater, and sediment contaminated with sewage [38,39,40,41,42,43]. At the time of writing, this genus includes nine species with valid published and correct names (https://lpsn.dsmz.de/genus/kaistia; accessed on 3 March 2023).
In this study, we characterized four bacterial strains that represent four novel species for which we propose the following names: Acinetobacter nematophilus sp. nov., Alcaligenes nematophilus sp. nov., Enterobacter nematophilus sp. nov., and Kaistia nematophila sp. nov. In addition, we propose the elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the species level. Therefore, we propose the creation of the following new species: Alcaligenes parafaecalis sp. nov., and Alcaligenes phenolicus sp. nov. Our study, therefore, contributes to a better understanding of the biodiversity and phylogenetic relationships of bacteria associated with soil-borne nematodes.

2. Material and Methods

2.1. Bacteria Isolation

Bacterial strains were obtained from different soil-borne nematode strains isolated at different locations in Tunisia using the soil baiting method and different insects as baits [44] as described elsewhere [13]. This procedure was carried out to isolate entomophilic nematodes. Acinetobacter nematophilus sp. nov. A-IN1T was isolated from an unidentified soil-borne nematode, IN1, collected in the National Agronomic Institute of Tunisia (Tunis, Tunisia). These nematodes were isolated using Galleria mellonella larvae as baits. Unfortunately, we were unsuccessful in establishing long-term nematode colonies under laboratory conditions, and we could not identify these nematodes. However, insects presented typical symptoms of Heterorhabditis nematode infestation such as that the nematodes turned red within 48 h upon nematode inoculation. Alcaligenes nematophilus sp. nov. A-TC2T and Kaistia nematophila sp. nov. K-TC2T were isolated from Oscheius tipulae TC2 nematodes collected in Takilsa (Cap Bon, Tunisia). These nematodes were isolated using Ceratitis capitata pupae as baits. Enterobacter nematophilus sp. nov. E-TC7T was isolated from Acrobeloides bodenheimeri TC7 nematodes collected in Takilsa (Cap Bon, Tunisia). These nematodes were isolated using Ceratitis capitata pupae as baits. To isolate the bacteria from O. tipulae TC2 and A. bodenheimeri TC7 nematodes, approximately 200 specimens of each nematode strain were washed several times with sterile phosphate buffered saline (PBS, 1mM KH2PO4, 1mM K2HPO4, 5M NaCl, pH 7.2). Then, the nematodes were incubated in a 1% v/v sodium hypochlorite/PBS solution for 5 min under gentle orbital agitation. After this incubation period, the nematodes were recovered by decanting, washed with sterile PBS, and incubated again in a 1% v/v sodium hypochlorite/PBS solution. After this incubation period, the nematodes were again recovered by decanting, washed several times with PBS and sonicated in sterile PBS at 4 °C for 30 sec and at 100W burst using an ultrasonic processor (Labsonic-L, B-Braun Biotech Inc, Allentown, PA, USA). The nematode body fragments were pelleted by centrifugation at 650× g (10 min, 4 °C). Then, 100 µL of supernatants were cultured in lysogeny broth (LB) medium overnight at 30 °C under constant shaking (180 rpm). An aliquot of the resulting cultures was plated on LB agar medium and incubated at 30 °C for 24–48 h. Single colonies were sub-cultured and used for further experiments. To isolate the bacteria from IN1 nematodes, a few drops of hemolymph from G. mellonella larvae infested with these nematodes were cultured in LB medium. To this end, the larvae were dipped first in 70% ethanol, and then punctured with a sterile needle in the ventral part. The released hemolymph was cultured in LB medium and incubated overnight at 30 °C for 24 h under constant shaking (180 rpm). An aliquot of the resulting cultures was plated on LB agar medium and incubated at 30 °C for 24–48 h. Single colonies were sub-cultured and used for further experiments. Different procedures such as the characterization of colony and cell morphology, and 16S rRNA gene sequencing were carried out to determine culture purity. The bacteria were deposited in the Czech Collection of Microorganisms (CCM) and in the national Culture Collection of Switzerland (CCOS) under the following accession numbers: Acinetobacter nematophilus A-IN1T (=CCM 9231T =CCOS 2018T), Alcaligenes nematophilus A-TC2T (=CCM 9230T =CCOS 2017T), Enterobacter nematophilus E-TC7T (=CCM 9232T =CCOS 2020T), and Kaistia nematophila K-TC2T (=CCM 9239T =CCOS 2022T).

2.2. Bacteria Molecular Characterization

To molecularly characterize the newly isolated bacterial strains, phylogenetic and phylogenomic relationships were reconstructed using 16S rRNA gene, whole-genome and whole-proteome sequences. In addition, sequence similarity scores were calculated as described below.

2.3. 16S rRNA Gene Sequencing

To obtain 16S rRNA gene sequences, the 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the following universal primers: 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1525R (5’-AAGGAGGTGWTCCARCC-3’). The following cycling conditions were used: 1 cycle at 94 °C for 10 min followed by 40 cycles at 94 °C for 60 s, 55 °C for 60 s, 72 °C for 60 s and a final extension at 72 °C for 5 min [3,45,46]. PCR products were separated by electrophoresis in a 1% TAE-agarose gel stained with GelRed nucleic acid gel stain (Biotium), gel-purified (QIAquick Gel Purification Kit, Qiagen) and sequenced by Sanger sequencing (Microsynth AG, Balgach, Switzerland). The obtained sequences were manually curated using Bioedit 7.2.5 [47]. In addition, 16S rRNA gene sequences were obtained directly from the whole-genome sequences using the bacterial ribosomal RNA predictor Barrnap 0.7 using the following parameters: reject length threshold = 0.5; length cutoff= 0.8; and e-value= 0.00001 [48]. The obtained sequences were identical to those obtained by Sanger sequencing. Phylogenetic relationships were reconstructed using the Maximum Likelihood method based on the Kimura 2-parameter model in MEGA7 as described above [49,50,51]. Tree support was determined by the bootstrap method based on 100 replicates. Graphical representation and edition of the trees were performed with Interactive Tree of Life (v3.5.1) [52,53]. The National Center for Biotechnology Information (NCBI) accession numbers of the sequences used for these analyses are shown in Table S1.

2.4. Whole Genome Sequencing

Genome sequences were obtained as described elsewhere [54,55]. Briefly, genomic DNA was extracted and purified using the GenElute Bacterial Genomic DNA Kit (Sigma–Aldrich, Switzerland) following the manufacturer’s instructions. The resulting DNA was used for library preparation using the TruSeq DNA PCR–Free LT Library Prep (FC–121–3003) kit. Indexed libraries were then pooled at equimolar concentrations and sequenced (2 × 150 bp) on an Illumina HiSeq 3000 instrument. Genomes were assembled using the Bactopia pipeline [56]. To this end, the raw Illumina reads were quality trimmed using Trimmomatic 0.39 [57]. The resulting reads were assembled with SPAdes 3.14.1 [58]. Scaffolds with a mean read–depth smaller than 20% of the median read–depth of the longer scaffolds (≥5000 bp) as well as scaffolds that were shorter than 200 bp were removed. Minor assembly errors were corrected using Pilon 1.22 with default parameters [59]. Completeness and contamination of the assembled genomes was assessed using checkM v1.1.6 with default parameters [60].

2.5. Whole Genome and Whole Proteome-Based Phylogenies

To reconstruct whole genome-based phylogenies, genomes were first aligned using Roary 3.13.0. Genes to be considered core had to be present in 85% of the genomes with an 85% protein identity and a coverage higher than 90%. Obtained alignments were used to build phylogenomic trees using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Branch support was assessed using the Shimodaira-Hasegawa-like procedure based on 100 replicates [61]. To reconstruct whole proteome-based phylogenies, first all ORFs from all genomes were extracted using Prodigal [62]. Then, homologous genes (85% or higher similarity, and a coverage higher than 90%) were clustered using MMSEQS2 (e-value: 0.001, sensitivity: 7.5, and cover: 0.5) and MCL (Inflation = 2) [63,64,65]. Orthologous genes were then translated and aligned using MAFFT [66]. Orthologous genes had to be present in more than 85% of the genomes to be considered core. Lastly, a maximum-likelihood-based phylogenomic tree was reconstructed based on the inferred core-proteome alignment using RAxML [67,68]. Branch support was assessed using the rapid bootstrap method based on 100 replicates [69]. Graphical representation and editing of the trees were performed with the Interactive Tree of Life (v3.5.1) [52,53]. Whole-genome sequence similarities were calculated by the GBPD (Genome Blast Distance Phylogeny) method using the Genome-to-Genome Distance Calculator 2.1 and formula 2 of the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) web service (http://ggdc.dsmz.de; accessed 5 June 2022) using default parameters [70,71,72,73]. The NCBI accession numbers of the sequences used for these analyses are shown in Table S1.

2.6. Physiological, Biochemical and Morphological Characterization

To physiologically, biochemically, and morphologically characterize the newly isolated bacterial strains, bacterial cultures from single colonies were used. Cell morphology was observed under a Leica DM4 B optical microscope at 1000× magnification, with cells grown for 16 h at 28 °C on LB. Pictures were taken using a built-in camera (Leica DFC7000T). The optimum temperature for bacterial growth was evaluated on LB agar medium at 20 °C, 24 °C, 30 °C, 32 °C, 37 °C, and 42 °C. Growth on medium containing different salt concentrations and pH was evaluated in 3 mL of LB medium, using 15-mL Falcon tubes. Five NaCl concentrations were used: 1% (Regular LB medium), 2%, 3%, 5% and 7%. Five different pH were used: 3, 5, 7, 8, and 9. Each tube was inoculated with 0.1 mL of an overnight bacterial culture, then incubated for 24 h at 28 °C and 180 rpm. Three tubes per treatment were considered. Cytochrome oxidase production was tested on discs containing N,N-dimethyl-p-phenylenediamine oxalate and α-naphthol (Sigma-Aldrich, Switzerland). Catalase activity was determined by adding a drop of 10% (v/v) H2O2 into 50 µL of a 16 h-old bacterial culture. Biochemical characterization was carried out using the API20E (bioMérieux, Inc. Durham, NC, USA) according to the manufacturer’s instructions. To this end, bacteria were grown for 16h at 28 °C in LB agar Petri plates. Then, one single colony was re-suspended in 5 mL of 0.85% NaCl. The resulting bacterial solution was used to inoculate the different microtubes containing the biochemical tests. Samples were incubated at 28 °C. Results were evaluated after 24 h. Gram staining was carried out using the Gram-Color modified (phenol-free) staining kit following the manufacture’s instructions (Sigma–Aldrich, Buchs, Switzerland). Cell morphology, optimum temperature for bacterial growth, growth on medium containing different salt concentrations and pH, and Gram reaction were evaluated only in the novel species described in this study. Biochemical tests such as cytochrome oxidase production, catalase activity, and the API20E tests were evaluated, in parallel, two independent times. The reference bacterial strains were obtained from the Korean agricultural culture collection (KACC), the German Collection of Microorganisms and Cell Cultures (DSMZ), the Czechoslovak National Collection of Type Cultures (CNCTC), and the Japan Collection of Microorganisms (JCM) as described in Table S5.

3. Results and Discussion

3.1. Phylogenetic and Phylogenomic Reconstructions and Sequence Comparisons

Phylogenetic reconstructions based on 16S rRNA gene sequences show that A-IN1T is more closely related to Acinetobacter bereziniae LMG 1003T (Figure S1A). The 16S rRNA gene sequence similarity score between A-IN1T and this strain is 99.4% (Figure S1B). Based on phylogenies reconstructed from core-genome and core-proteomes, A-IN1T is more closely related to A. bereziniae LMG 1003T (Figure 1 and Figure S5A). The phylogenetic tree obtained in this study largely resembles the trees obtained in previous studies [74,75]. The digital DNA-DNA hybridization (dDDH) score between A-IN1T and this strain is 39.6% (Table 1). These values are below the 70% divergence threshold for prokaryotic species delineation [70,72,76].
Phylogenetic reconstructions based on 16S rRNA gene sequences show that A-TC2T clusters together with Alcaligenes faecalis subsp. faecalis DSM 30030T (Proposed name: Alcaligenes faecalis) and A. faecalis subsp. phenolicus DSM 16503T (Proposed name: Alcaligenes phenolicus) (Figure S2A). The 16S rRNA gene sequence similarity scores between A-TC2T and these latest strains are 99.8% in both cases (Figure S2B). Based on phylogenies reconstructed from core-genome and core-proteomes, A-TC2T is more closely related to A. faecalis subsp. phenolicus DSM 16503T (Proposed name: Alcaligenes phenolicus)(Figure 2 and Figure S5B). Previous taxonomic studies have not included core-genome phylogenies to compare [26,27]. The digital DNA–DNA hybridization (dDDH) score between A-TC2T and this strain is 66.3% (Table 2). This value is below the 70% divergence threshold for prokaryotic species delineation [70,72,76]. In addition, we observed that dDDH scores between A. faecalis subsp. faecalis DSM 30030T (Proposed name: Alcaligenes faecalis), A. faecalis subsp. parafaecalis DSM 13975T (Proposed name: Alcaligenes parafaecalis) and A. faecalis subsp. phenolicus DSM 16503T (Proposed name: Alcaligenes phenolicus) are always below 70%, demonstrating that these three strains represent species rather than subspecies (Table 2).
Phylogenetic reconstructions based on 16S rRNA gene sequences show that E-TC7T is more closely related to Enterobacter bugandensis EB-247T (Figure S3A). The 16S rRNA gene sequence similarity score between E-TC7T and this strain is 99.5% (Figure S3B). Based on phylogenies reconstructed from core-genome and core-proteomes, E-TC7T is more closely related to E. bugandensis EB-247T (Figure 3 and Figure S5C). Similar phylogenies were reconstructed in previous studies [34,36]. The digital DNA-DNA hybridization (dDDH) score between E-TC7T and this strain is 63.7% (Table 3). These values are below the 70% divergence threshold for prokaryotic species delineation [70,72,76].
Phylogenetic reconstructions based on 16S rRNA gene sequences show that K-TC2T is more closely related to Kaistia defluvii JCM 18034T (Figure S4A). The 16S rRNA gene sequence similarity score between K-TC2T and this latest strain is 99.3% (Figure S4B). Similar phylogenies were reconstructed in previous studies [38,77]. Based on phylogenies reconstructed from core-genome and core-proteomes, K-TC2T is more closely related to K. defluvii JCM 18034T (Figure 4 and Figure S5D). Recent studies have not reconstructed phylogenies using core genome sequences to compare [77]. The digital DNA-DNA hybridization (dDDH) score between K-TC2T and this strain is 30.7% (Table 4). These values are below the 70% divergence threshold for prokaryotic species delineation [70,72,76].
Based on these genomic divergence values, we propose the creation of the following new taxa: Acinetobacter nematophilus sp. nov. with A-IN1T (=CCM 9231T =CCOS 2018T) as the type strain, Alcaligenes nematophilus sp. nov. with A-TC2T (=CCM 9230T =CCOS 2017T) as the type strain, Enterobacter nematophilus sp. nov. with E-TC7T (=CCM 9232T =CCOS 2020T) as the type strain, and Kaistia nematophila sp. nov. with K-TC2T (=CCM 9239T =CCOS 2022T) as the type strain. In addition, we propose the elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. phenolicus, and Alcaligenes faecalis subsp. parafaecalis to the species level and therefore we propose the creation of Alcaligenes parafaecalis sp. nov. with DSM 13975T as the type strain, and Alcaligenes phenolicus sp. nov. with DSM 16503T as the type strain.

3.2. Genomic Features

The genomes of thirteen type strains of the genera Acinetobacter, Alcaligenes, Enterobacter, and Kaistia were sequenced in this study. Their main characteristics are summarized in Tables S2–S4. The main characteristics of the genomes of the novel species are as follows. The genome of Acinetobacter nematophilus A-IN1T contains 4360075 bp, a G+C content of 38.49, and 4046 proteins. The genome of Alcaligenes nematophilus sp. nov. A-TC2T contains 4251628 bp, a G+C content of 56.41, and 3892 proteins. The genome of Enterobacter nematophilus sp. nov. E-TC7T contains 4708215bp, a G+C content of 56.36, and 4289 proteins. The genome of Kaistia nematophila sp. nov. K-TC2T contains 5165915 bp, a G+C content of 66.75, and 4789 proteins (Tables S2 and S3). These genomes are predicted to be more than 99% complete and contain less than 2% contamination (Table S4).

3.3. Physiological and Biochemical Characteristics

Biochemical tests show that Acinetobacter nematophilus A-IN1T, Alcaligenes nematophilus A-TC2T, Enterobacter nematophilus E-TC7T, and Kaistia nematophila K-TC2T exhibit biochemical capacities that are similar to the biochemical capacities of several members of their respective genus (Table 5). However, these strains also exhibit unique biochemical capacities that differ from the biochemical capacities of their most closely related taxa (Table 5).
In particular, Acinetobacter nematophilus A-IN1T does not hydrolyze arginine, unlike Acinetobacter bereziniae DSM 25435T, its most closely related taxon. Acinetobacter bereziniae DSM 25435T has been biochemically characterized previously using different biochemical tests than the tests used in this study. However, we observed that Acinetobacter bereziniae DSM 25435T utilizes citrate, which is in good agreement with that previous study [78].
Alcaligenes nematophilus A-TC2T is negative for β-galactosidase, tryptophan deaminase, and gelatinase, unlike Alcaligenes faecalis subsp. phenolicus DSM 16503T (proposed name: Alcaligenes phenolicus), its most closely related taxon. In addition, Alcaligenes nematophilus A-TC2T does not produce hydrogen sulfide or indole, unlike Alcaligenes faecalis subsp. phenolicus DSM 16503T, which produces these molecules. Lastly, Alcaligenes nematophilus A-TC2T does not oxidize mannitol, inositol, rhamnose, or amygdalin, unlike Alcaligenes faecalis subsp. phenolicus DSM 16503T which oxidizes these compounds. In previous studies, Alcaligenes faecalis subsp. phenolicus DSM 16503T was also reported to hydrolase gelatin, while Alcaligenes endophyticus DSM 100498T, Alcaligenes faecalis subsp. faecalis DSM 30030T (Proposed name: Alcaligenes faecalis), and Alcaligenes faecalis subsp. parafaecalis DSM 13975T (Proposed name: Alcaligenes parafaecalis) were reported as gelatinase negative [26,28]. Our results are therefore in good agreement with these previous studies [26,28].
Enterobacter nematophilus E-TC7T oxidizes sucrose and melibiose, unlike Enterobacter bugandensis STN0717-56T, its most closely related taxon. In previous studies, it has been reported that most Enterobacter species do not produce indole or sulfides, but produce acetoin, and are mostly positive for citrate utilization. Our results are in good agreement with these studies [32,34,36].
Kaistia nematophila K-TC2T is positive for β-galactosidase, arginine dihydrolase, ornithine decarboxylase, and citrate utilization, unlike Kaistia defluvii JCM 18034T, its most closely related taxon. Moreover, Kaistia nematophila K-TC2T produces acetoin, unlike Kaistia defluvii JCM 18034T. In addition, Kaistia nematophila K-TC2T reduces nitrogen dioxide and oxidizes rhamnose and arabinose, unlike Kaistia defluvii JCM 18034T (Table 1). We also observe that most Kaistia species are gelatinase negative, as has been also observed in previous studies [38,39].
Noteworthily, we observed that the biochemical capacities of the newly isolated bacteria are, in some cases, very similar to the biochemical capacities of their most closely related species. For instance, Acinetobacter nematophilus A-IN1T and Acinetobacter bereziniae DSM 25435T differ only in their capacity to digest arginine, and Enterobacter nematophilus E-TC7T and Enterobacter bugandensis STN0717-56T differ only in their capacity to oxidize sucrose and melibiose (Table 5). In other cases, however, closely related species show greater phenotypic divergencies. For instance, Alcaligenes nematophilus A-TC2T and Alcaligenes faecalis subsp. phenolicus DSM 16503T differ in several traits, in a similar manner as Kaistia nematophila K-TC2T, Kaistia defluvii JCM 18034T, and Kaistia terrae DSM 21341T do. Such divergencies might reflect adaptations to their local environments and could potentially impact the outcome of interspecific interactions.

3.4. Ecology

The ecology of the type strains of the novel species described in this study and their nematode hosts have been studied previously [13,14,15]. An interesting aspect of these bacterial strains is that all of them were isolated from soil-borne nematodes. In one of our previous studies, we evaluated the insect-killing abilities of one of them, Oscheius tipulae TC2, the host of A. nematophilus A-TC2T and K. nematophila K-TC2T [13]. We observed that this nematode isolate was highly lethal against the mediterranean fly and it rapidly and effectively killed its eggs, larvae, and pupae, and also interferes with insect metamorphosis [13]. We did not extensively test the insect-killing abilities of the other two nematodes that host the other bacterial strains described in this study, Acrobeloides bodenheimeri TC7 and IN1 (unidentified), but they are also able to kill insects as we recovered them from soil samples using C. capitata pupae and G. mellonella larvae as baits, respectively [13,14,15]. In an additional study, we tested the contribution of the bacterial strains isolated in this study to the insect-killing abilities of their nematode hosts [14]. By injecting the bacteria directly in the hemocoels of G. mellonella larvae, we observed that they all were highly pathogenic. These bacterial strains therefore might be promising biocontrol agents. Further studies are required to untap their real potential under agriculturally relevant settings.

3.5. Conclusions

Considering the biochemical and genomic differences of the strains analyzed in this study, we propose the following new species: Acinetobacter nematophilus sp. nov. with A-IN1T (=CCM 9231T =CCOS 2018T) as the type strain, Alcaligenes nematophilus sp. nov. with A-TC2T (=CCM 9230T =CCOS 2017T) as the type strain, Enterobacter nematophilus sp. nov. with E-TC7T (=CCM 9232T =CCOS 2020T) as the type strain, and Kaistia nematophila sp. nov. with K-TC2T (=CCM 9239T =CCOS 2022T) as the type strain. In addition, we propose the elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the species level. Therefore, we propose the creation of Alcaligenes parafaecalis sp. nov. with DSM 13975T as the type strain, and Alcaligenes phenolicus sp. nov. with DSM 16503T as the type strain.

3.6. Protologues

  • Emended description of Alcaligenes faecalis
(fae.ca’lis. L. fem. n. faex (gen. faecis), dregs, feces; L. masc./fem. adj. suff. -alis, suffix denoting pertaining to; N.L. masc./fem. adj. faecalis, pertaining to feces, fecal). This amended description results from the proposal to elevate Alcaligenes faecalis subsp. faecalis to the species level. The description is the same as given for Alcaligenes faecalis by Castellani and Chalmers (1919), updated by Kersters and De Ley (1984) and amended by Schroll et al. (2001) and Rehfuss and Urban (2005) [23,25,28,79] with the following additions. The genome of the type strain, DSM 30030T, contains 4003996 base pairs, 3706 proteins, and a G+C content of 56.65%. The genome and the 16S rRNA gene sequences of DSM 30030T were deposited in the NCBI under the accession numbers JAPKNC01 and OP804215, respectively. The type strain is ATCC 8750T (=DSM 30030T =ATCC 8750T =CCUG 1814T =CIP 55.84T =CIP 60.80T, HAMBI 1907T =IFO 13111T =JCM 1474T =JCM 20522T =JCM 20663T =LMG 1229T =NBRC 13111T =NCAIM B.01104T =NCIB 8156T =NCIMB 8156T =NCTC 11953T =VKM B-1518T).
  • Description of Acinetobacter nematophilus sp. nov.
(ne.ma.to’phi.lus. N.L. masc. adj. suff. -philus, loving or having affinity for; N.L. masc. adj. nematophilus, nematode-loving). Cells are short rods, approx. 0.9–1.3 µm wide and 1.2–1.4 µm long (Figure S6A). Growth is observed between 20–32 °C. Poor growth occurs at 37 °C. The optimal temperature for growth is 30 °C. Bacterial growth occurs at pH between 5–9 (optimum 5–7) and not at pH 3. Bacterial growth occurs in LB medium containing between 1–3% NaCl (optimum 1–2%). Bacterial growth is inhibited in LB containing more than 3% NaCl. It is negative for β-galactosidase, arginine dihydrolase, lysine and ornithine decarboxylases, urease, tryptophan deaminase, and cytochrome oxidase. It does not produce hydrogen sulfide, indole, or nitrites. It does not digest gelatin. It does not oxidize mannitol, inositol, sorbitol, sucrose, or amygdalin. It does not reduce nitrites. It is positive for catalase, utilizes citrate, produces acetoin, and oxidizes glucose, rhamnose, melibiose, and arabinose. The genome and the 16S rRNA gene sequences of the type strain, A-IN1T, were deposited in the NCBI databank under the accession numbers JAPKMY01 and OP818110, respectively. The genome assembled contains 4360075 base pairs, 4046 proteins, and a G+C content of 38.49%. The type strain of the species is A-IN1T (=CCM 9231T =CCOS 2018T). A-IN1T was isolated from an unidentified soil-borne nematode, IN1, collected in Tunis (Tunisia).
  • Description of Alcaligenes nematophilus sp. nov.
(ne.ma.to’phi.lus. N.L. masc. adj. suff. -philus, loving or having affinity for; N.L. masc. adj. nematophilus, nematode-loving). Cells are rod-shaped, approx. 0.5–0.8 µm wide and 1.3–1.9 µm long (Figure S6B). Growth is observed between 20–42 °C. The optimal temperature for growth is 30 °C. Bacterial growth occurs at pH between 7–9 (optimum 7–8) and not at pH below 5. Bacterial growth occurs in LB medium containing between 1–7% NaCl (optimum 1–3%). It is Negative for β-galactosidase and tryptophan deaminase. It does not produce hydrogen sulfide, indole, or nitrites. It does not digest gelatin. It does not oxidase mannitol, inositol, sorbitol, sucrose, amygdalin, glucose, rhamnose, melibiose, or arabinose. It does not reduce nitrites. It is positive for arginine dihydrolase, lysine and ornithine decarboxylases, urease, cytochrome oxidase, and catalase. It utilizes citrate and produces acetoin. The genome and the 16S rRNA gene sequences of the type strain, A-TC2T, were deposited in the NCBI databank under the accession numbers JAPKMZ01 and OP804216, respectively. The genome assembled contains 4251628 base pairs, 3892 proteins, and a G+C content of 56.41%. The type strain of the species is A-TC2T (=CCM 9230T =CCOS 2017T). A-TC2T was isolated from Oscheius tipulae TC2 nematodes collected in Takilsa (Tunisia).
  • Description of Alcaligenes parafaecalis sp. nov.
(para.fae.ca’lis. (Gr. prep. para, beside, alongside of, near, like; N.L. masc./fem. adj. faecalis, specific epithet; N.L. masc./fem. adj. parafaecalis, intended to mean alongside of the species Alcaligenes faecalis). Alcaligenes parafaecalis sp. nov. results from the proposal to elevate Alcaligenes faecalis subsp. parafaecalis to the species level. The description of A. parafaecalis sp. nov. is the same as given for Alcaligenes faecalis subsp. parafaecalis by [25]. However, we found that it does not digest gelatin at 30 °C. The genome and the 16S rRNA gene sequences of the type strain, DSM 13975T, were deposited in the NCBI under the accession number JAPKNA01 and OP804213, respectively. The genome assembled contains 4004626 base pairs, 3624 proteins, and a G+C content of 56.02%. The type strain of the species is DSM 13975T (=CIP 106866T =CCUG 48316T =GT).
  • Description of Alcaligenes phenolicus sp. nov.
(phe.nol.i’cus. N. L. n. phenol common name for industrial solvent hydroxybenzene; N.L. masc. adj. phenolicus pertaining to phenol). Alcaligenes phenolicus sp. nov. results from the proposal to elevate Alcaligenes faecalis subsp. phenolicus to the species level. The description of A. phenolicus sp. nov. is the same as given for Alcaligenes faecalis subsp. phenolicus by Rehfuss and Urban (2005) [28]. The genome and the 16S rRNA gene sequences were deposited in the NCBI under the accession number JAPKNB01 and OP804214, respectively. The genome assembled contains 4238760 base pairs, 3909 proteins, and a G+C content of 56.42%. The type strain of the species is JT (=DSM 16503T =NRRL B-41076T).
  • Description of Enterobacter nematophilus sp. nov.
(ne.ma.to’phi.lus. N.L. masc. adj. suff. -philus, loving or having affinity for; N.L. masc. adj. nematophilus, nematode-loving). Cells are rod-shaped, approx. 0.9–1.2µm wide and 2.0–2.5µm long (Figure S6C). Growth is observed between 20–42 °C. The optimal temperature for growth is 37 °C. Bacterial growth occurs at pH between 5–9 (optimum 5–7) and not at pH 3. Bacterial growth occurs in LB medium containing between 1–7% NaCl (optimum 1–5%). It is negative for tryptophan deaminase. It does not produce hydrogen sulfide, indole, or nitrites. It does not oxidase sucrose or melibiose. It is positive for β-galactosidase, digests gelatin, oxidizes mannitol, inositol, sorbitol, amygdalin, glucose, rhamnose, and arabinose. It is positive for arginine, lysine, and ornithine decarboxylases, urease, cytochrome oxidase, and catalase. It utilizes citrate, produces acetoin and reduces nitrites. The genome and the 16S rRNA gene sequences of the type strain, E-TC7T, were deposited in the NCBI under the accession number JAPKNE01 and OP818089, respectively. The genome assembled contains 4708215 base pairs, 4289 proteins, and a G+C content of 56.36%. The type strain of the species is E-TC7T (=CCM 9232T =CCOS 2020T). E-TC7T was isolated from Acrobeloides bodenheimeri TC7 nematodes collected in Takilsa (Tunisia).
  • Description of Kaistia nematophila sp. nov.
(ne.ma.to’phi.la. N.L. n. nematoda nematode; N.L. fem. adj. suff. -phila, loving or having affinity for; N.L. fem. adj. nematophila, nematode-loving). Cells are short rods, approx. 0.8–1.2µm wide and 1.1–1.6 µm long (Figure S6D). Growth is observed between 20–35 °C. The optimal temperature for growth is 28–30 °C. Bacterial growth occurs at pH between 5–8 (optimum 5–7) and not at pH 3 or 9. Bacterial growth occurs in LB medium containing 1% NaCl. Bacterial growth is inhibited in LB containing more than 1% NaCl. It is negative for tryptophan deaminase and lysine decarboxylase. It does not produce hydrogen sulfide, indole, acetoin, or nitrites. It does not oxidase sucrose, melibiose, mannitol, inositol, sorbitol, amygdalin, or glucose. It does not digest gelatin. It reduces nitrites and oxidizes rhamnose and arabinose. It is positive for β-galactosidase, arginine dihydrolase, ornithine decarboxylase, urease, cytochrome oxidase, and catalase. It utilizes citrate. The genome and the 16S rRNA gene sequences of the type strain, K-TC2T, were deposited in the NCBI under the accession numbers JAPKNK01 and OP804305, respectively. The genome assembled contains 5165915 base pairs, 4789 proteins, and a G+C content of 66.75%. The type strain of the species is K-TC2T (=CCM 9239T =CCOS 2022T). K-TC2T was isolated from Oscheius tipulae TC2 nematodes collected in Takilsa (Tunisia).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/taxonomy3010012/s1, Figure S1: Phylogenetic reconstruction based on 16S rRNA gene sequences of several Acinetobacter species with validly published names; Figure S2: Phylogenetic reconstruction based on 16S rRNA gene sequences of all Alcaligenes species with validly published names; Figure S3: Phylogenetic reconstruction based on 16S rRNA gene sequences of all Enterobacter species with validly published names; Figure S4: Phylogenetic reconstruction based on 16S rRNA gene sequences of all Kastia species with validly published names; Figure S5: Phylogenetic reconstructions based on core proteome sequences; Figure S6: Photographs of the newly described bacterial species; Table S1: National Center for Biotechnology Information (NCBI) accession numbers of the bacterial sequences used in this study; Table S2: Features of the genomes generated in this study (Part 1); Table S3: Features of the bacterial genomes generated in this study (Part 2); Table S4: Completeness (%) and contamination (%) of the bacterial genomes generated in this study assessed by checkM; Table S5: Source of the bacterial strains used in this study.

Author Contributions

Conceptualization, R.A.R.M. and S.K.; Data curation, R.A.R.M.; Formal analysis, R.A.R.M.; Funding acquisition, R.A.R.M., M.F.B. and S.K. Investigation, R.A.R.M., A.L., A.H.B., M.M. and C.T. Methodology, R.A.R.M., A.L., A.H.B., M.M. and C.T.; Project administration, R.A.R.M., M.F.B. and S.K.; Resources, R.A.R.M., M.F.B. and S.K.; Supervision, R.A.R.M., M.F.B. and S.K.; Validation, R.A.R.M.; Visualization, R.A.R.M.; Writing—original draft, R.A.R.M.; Writing—review & editing, R.A.R.M., A.H.B., C.T. and S.K. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financed the Swiss National Science Foundation (Grant 186094 to RARM) and partially financed by the federated research project “Gestion intégrée contre la Cératite pour la promotion d’une agriculture durable” (PRF2019-D6P2) of Ministry of Higher Education and Scientific Research, Tunisia. The work of AHB was supported by a Postdoctoral Swiss Government Excellence Scholarship (Grant Nr. 2021.0463 to AHB). The work of C.T. is supported by the Swiss National Science Foundation (Grant 180142 to Diego Gonzalez).

Data Availability Statement

All data are provided within the manuscript. Whole genome sequences of A-IN1T, A-TC2T, E-TC7T, and K-TC2T are deposited in the National Center for Biotechnology Information (NCBI) databank under the accession numbers JAPKMY01, JAPKMZ01, JAPKNE01, and JAPKNK01, respectively; and the 16S rRNA gene sequences under the accession numbers and OP818110, OP804216, OP818089, and OP804305, respectively.

Acknowledgments

We thank the Swiss National Science Foundation and the Institute of Biology of the University of Neuchâtel (Switzerland), the University of Carthage and the National Agronomic Institute of Tunisia for their support.

Conflicts of Interest

The authors declare no conflict of interest and that no humans or animals were used for experimental purposes.

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Taxonomy 03 00012 g001 550
Figure 1. Phylogeny based on core genome sequences of several species of the genus Acinetobacter. 644754 nucleotide positions (601 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Phylogenomic trees were built using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Accession numbers of the sequences used in the analyses are shown in Table S1. Bold indicates a novel taxonomic proposal.
Figure 1. Phylogeny based on core genome sequences of several species of the genus Acinetobacter. 644754 nucleotide positions (601 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Phylogenomic trees were built using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Accession numbers of the sequences used in the analyses are shown in Table S1. Bold indicates a novel taxonomic proposal.
Taxonomy 03 00012 g001
Taxonomy 03 00012 g002 550
Figure 2. Phylogeny based on core genome sequences of all the species of the genus Alcaligenes. 408607 nucleotide positions (399 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Phylogenomic trees were built using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Accession numbers of the sequences used in the analyses are shown in Table S1. Bold indicates novel taxonomic proposals.
Figure 2. Phylogeny based on core genome sequences of all the species of the genus Alcaligenes. 408607 nucleotide positions (399 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Phylogenomic trees were built using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Accession numbers of the sequences used in the analyses are shown in Table S1. Bold indicates novel taxonomic proposals.
Taxonomy 03 00012 g002
Taxonomy 03 00012 g003 550
Figure 3. Phylogeny based on core genome sequences of all the species of the genus Enterobacter. 3055395 nucleotide positions (3071 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Phylogenomic trees were built using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Accession numbers of the sequences used in the analyses are shown in Table S1. Bold indicates a novel taxonomic proposal.
Figure 3. Phylogeny based on core genome sequences of all the species of the genus Enterobacter. 3055395 nucleotide positions (3071 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Phylogenomic trees were built using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Accession numbers of the sequences used in the analyses are shown in Table S1. Bold indicates a novel taxonomic proposal.
Taxonomy 03 00012 g003
Taxonomy 03 00012 g004 550
Figure 4. Phylogeny based on core genome sequences of all the species of the genus Kaistia. 543369 nucleotide positions (508 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Phylogenomic trees were built using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Accession numbers of the sequences used in the analyses are shown in Table S1. Bold indicates a novel taxonomic proposal.
Figure 4. Phylogeny based on core genome sequences of all the species of the genus Kaistia. 543369 nucleotide positions (508 core genes) were used in the analyses. Numbers at the nodes represent SH-like branch supports. Phylogenomic trees were built using FastTree 2.1.10 based on the Generalized Time Reversible Model (GTR) [61]. Accession numbers of the sequences used in the analyses are shown in Table S1. Bold indicates a novel taxonomic proposal.
Taxonomy 03 00012 g004
Table
Table 1. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of several species of the genus Acinetobacter. Accession numbers of the sequences used in the analyses are shown in Table S1. Colors indicate the degree of relatedness between bacterial species pairs.
Table 1. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of several species of the genus Acinetobacter. Accession numbers of the sequences used in the analyses are shown in Table S1. Colors indicate the degree of relatedness between bacterial species pairs.
A. rudis DSM 24031TA. pullicarnis S23TA. larvae BRTC-1TA. gerneri MTCC 9824TA. silvestris ANC 4999TA. sichuanensis WCHAc060041TA. defluvii WCHA30TA. wuhouensis WCHA60TA. rongchengensis WCHAc060115TA. piscicola LW15TA. guillouiae DSM 590TA. bereziniae LMG 1003TA. nematophilus sp. nov. A-IN1TA. lanii 185TA. equi 114TA. cumulans WCHAc060092TA. chengduensis WCHAc060005TA. tandoii DSM 14970TA. johnsonii DSM 69633TA. variabilis NIPH 2171TA. chinensis WCHAc010005T
A. rudis DSM 24031TID21.222.021.920.921.321.421.721.021.321.521.720.821.421.521.321.120.820.921.121.3
A. pullicarnis S23T21.2ID22.022.721.022.122.822.724.921.722.222.521.022.021.822.621.723.022.024.122.7
A. larvae BRTC-1T22.022.0ID22.220.820.922.121.821.821.622.021.921.122.021.921.420.821.321.121.721.5
A. gerneri MTCC 9824T21.922.722.2ID21.622.223.123.122.922.222.322.721.522.122.323.022.823.121.923.122.6
A. silvestris ANC 4999T20.921.020.821.6ID21.521.622.022.022.122.122.021.521.721.721.221.021.020.720.320.8
A. sichuanensis WCHAc060041T21.322.120.922.221.5ID27.024.227.122.923.126.622.522.022.123.222.522.021.421.623.2
A. defluvii WCHA30T21.422.822.123.121.627.0ID25.428.023.223.428.422.722.422.723.722.422.322.022.424.7
A. wuhouensis WCHA60T21.722.721.823.122.024.225.4ID24.324.224.524.423.322.122.724.622.922.022.322.627.5
A. rongchengensis WCHAc060115T21.024.921.822.922.027.128.024.3ID36.424.527.123.221.821.922.521.823.221.722.422.9
A. piscicola LW15T21.321.721.622.222.122.923.224.236.4ID25.224.223.621.322.322.021.621.821.321.322.2
A. guillouiae DSM 590T21.522.222.022.322.123.123.424.524.525.2ID26.225.122.022.422.221.922.421.922.922.1
A. bereziniae LMG 1003T21.722.521.922.722.026.628.424.427.124.226.2ID39.622.022.422.421.521.621.822.923.0
A. nematophilus sp. nov. A-IN1T20.821.021.121.521.522.522.723.323.223.625.139.6ID21.221.621.320.920.720.820.821.6
A. lanii 185T21.422.022.022.121.722.022.422.121.821.322.022.021.2ID22.522.922.221.821.722.421.7
A. equi 114T21.521.821.922.321.722.122.722.721.922.322.422.421.622.5ID22.622.522.021.621.521.8
A. cumulans WCHAc060092T21.322.621.423.021.223.223.724.622.522.022.222.421.322.922.6ID33.821.622.322.423.5
A. chengduensis WCHAc060005T21.121.720.822.821.022.522.422.921.821.621.921.520.922.222.533.8ID21.221.321.922.2
A. tandoii DSM 14970T20.823.021.323.121.022.022.322.023.221.822.421.620.721.822.021.621.2ID22.422.520.6
A. johnsonii DSM 69633T20.922.021.121.920.721.422.022.321.721.321.921.820.821.721.622.321.322.4ID22.021.1
A. variabilis NIPH 2171T21.124.121.723.120.321.622.422.622.421.322.922.920.822.421.522.421.922.522.0ID22.2
A. chinensis WCHAc010005T21.322.721.522.620.823.224.727.522.922.222.123.021.621.721.823.522.220.621.122.2ID
Table
Table 2. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of all the species of the genus Alcaligenes. Accession numbers of the sequences used in the analyses are shown in Table S1. Colors indicate the degree of relatedness between bacterial species pairs.
Table 2. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of all the species of the genus Alcaligenes. Accession numbers of the sequences used in the analyses are shown in Table S1. Colors indicate the degree of relatedness between bacterial species pairs.
A. endophyticus DSM 100498TA. pakistanensis KCTC 42083TA. parafaecalis sp. nov. DSM 13975TA. aquatilis BU33NA. faecalis DSM 30030TA. phenolicus sp. nov. DSM 16503TA. nematophilus sp. nov. A-TC9T
A. endophyticus DSM 100498TID18.218.118.218.018.018.2
A. pakistanensis KCTC 42083T18.2ID32.729.530.830.530.4
A. parafaecalis sp. nov. DSM 13975T18.132.7ID34.136.835.735.5
A. aquatilis BU33N18.229.534.1ID41.639.038.4
A. faecalis DSM 30030T18.030.836.841.6ID49.147.0
A. phenolicus sp. nov. DSM 16503T18.030.535.739.049.1ID66.3
A. nematophilus sp. nov. A-TC9T18.230.435.538.447.066.3ID
Table
Table 3. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of all the species of the genus Enterobacter. Accession numbers of the sequences used in the analyses are shown in Table S1. Colors indicate the degree of relatedness between bacterial species pairs.
Table 3. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of all the species of the genus Enterobacter. Accession numbers of the sequences used in the analyses are shown in Table S1. Colors indicate the degree of relatedness between bacterial species pairs.
E. cancerogenus ATCC33241TE. quasihormaechei WCHEQ120003TE. hormaechei ATCC 49162TE. xiangfangensis LMG27195TE. hoffmannii DSM 14563TE. oligotrophicus CCA6TE. soli LMG 25861TE. wuhouensis WCHEW120002TE. huaxiensis WCHEHu090008TE. quasimori 090044TE. mori LMG 25706TE. ludwigii EN-119TE. dissolvens ATCC 23373TE. cloacae DSM 30054TE. kobei DSM 13645TE. chuandaensis 090028TE. bugandensis EB-247TE. nematophilus sp. nov. E-TC7TE. sichuanensis WCHECL1597TE. chengduensis WCHECl-C4TE. asburiae ATCC 35953TE. dykesii E1TE. vonholyi E13TE. roggenkampii DSM 16690TE. quasiroggenkampii WCHECL1060T
E. cancerogenus ATCC33241TID31.731.631.531.532.129.532.032.132.331.930.430.730.831.231.731.831.831.232.032.431.831.532.031.5
E. quasihormaechei WCHEQ120003T31.7ID51.652.853.533.529.833.933.734.934.031.432.132.333.435.336.735.332.433.934.233.733.334.333.6
E. hormaechei ATCC 49162T31.651.6ID60.058.033.830.233.933.835.034.231.632.432.933.735.435.735.332.634.534.633.833.334.333.7
E. xiangfangensis LMG27195T31.552.860.0ID66.633.830.233.833.835.034.231.532.532.633.435.235.735.232.834.134.833.933.734.734.0
E. hoffmannii DSM 14563T31.553.558.066.6ID33.830.033.733.834.834.131.432.132.533.435.035.534.932.534.034.433.533.134.233.5
E. oligotrophicus CCA6T32.133.533.833.833.8ID31.634.034.534.934.732.733.333.233.334.134.133.932.933.734.833.933.734.433.9
E. soli LMG 25861T29.529.830.230.230.031.6ID30.730.831.030.830.330.430.330.230.630.530.730.330.731.331.030.530.830.8
E. wuhouensis WCHEW120002T32.033.933.933.833.734.030.7ID39.638.037.932.833.333.233.936.235.836.334.135.937.736.635.636.035.6
E. huaxiensis WCHEHu090008T32.133.733.833.833.834.530.839.6ID37.637.432.132.832.733.535.235.035.133.635.236.435.334.535.134.6
E. quasimori 090044T32.334.935.035.034.834.931.038.037.6ID66.833.034.634.436.138.538.138.536.438.940.940.039.339.840.2
E. mori LMG 25706T31.934.034.234.234.134.730.837.937.466.8ID32.634.034.035.437.737.037.535.237.339.938.937.637.537.3
E. ludwigii EN-119T30.431.431.631.531.432.730.332.832.133.032.6ID34.434.234.634.734.834.734.635.036.135.535.035.534.9
E. dissolvens ATCC 23373T30.732.132.432.532.133.330.433.332.834.634.034.4ID62.134.935.635.535.837.135.536.736.135.636.136.0
E. cloacae DSM 30054T30.832.332.932.632.533.230.333.232.734.434.034.262.1ID35.735.635.635.536.936.237.235.835.536.335.8
E. kobei DSM 13645T31.233.433.733.433.433.330.233.933.536.135.434.634.935.7ID42.443.143.539.243.342.741.540.941.340.6
E. chuandaensis 090028T31.735.335.435.235.034.130.636.235.238.537.734.735.635.642.4ID53.956.139.742.745.644.543.143.442.7
E. bugandensis EB-247T31.836.735.735.735.534.130.535.835.038.137.034.835.535.643.153.9ID63.739.942.845.144.143.344.243.2
E. nematophilus sp. nov. E-TC7T31.835.335.335.234.933.930.736.335.138.537.534.735.835.543.556.163.7ID40.242.945.744.943.643.842.7
E. sichuanensis WCHECL1597T31.232.432.632.832.532.930.334.133.636.435.234.637.136.939.239.739.940.2ID42.645.244.244.245.544.6
E. chengduensis WCHECl-C4T32.033.934.534.134.033.730.735.935.238.937.335.035.536.243.342.742.842.942.6ID52.750.949.748.949.5
E. asburiae ATCC 35953T32.434.234.634.834.434.831.337.736.440.939.936.136.737.242.745.645.145.745.252.7ID69.357.251.751.7
E. dykesii E1T31.833.733.833.933.533.931.036.635.340.038.935.536.135.841.544.544.144.944.250.969.3ID57.451.151.5
E. vonholyi E13T31.533.333.333.733.133.730.535.634.539.337.635.035.635.540.943.143.343.644.249.757.257.4ID56.056.4
E. roggenkampii DSM 16690T32.034.334.334.734.234.430.836.035.139.837.535.536.136.341.343.444.243.845.548.951.751.156.0ID65.1
E. quasiroggenkampii WCHECL1060T31.533.633.734.033.533.930.835.634.640.237.334.936.035.840.642.743.242.744.649.551.751.556.465.1ID
Table
Table 4. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of all the species of the genus Kaistia. Accession numbers of the sequences used in the analyses are shown in Table S1. Colors indicate the degree of relatedness between bacterial species.
Table 4. Pairwise comparison of digital DNA-DNA Hybridization (dDDH) scores (%) of all the species of the genus Kaistia. Accession numbers of the sequences used in the analyses are shown in Table S1. Colors indicate the degree of relatedness between bacterial species.
K. soli DSM 19436TK. geumhonensis DSM 18799TK. hirudinis DSM 25966TK. algarum JCM 31803TK. dalseonensis DSM 18800TK. granuli DSM 23481TK. adipata DSM 17808TK. nematophila sp. nov. K-TC2TK. terrae DSM 21341TK. defluvii JCM 18034T
K. soli DSM 19436TID22.221.021.021.320.820.921.220.721.0
K. geumhonensis DSM 18799T22.2ID21.520.821.321.021.321.520.721.3
K. hirudinis DSM 25966T21.021.5ID24.121.821.221.421.520.821.4
K. algarum JCM 31803T21.020.824.1ID21.421.020.921.220.921.1
K. dalseonensis DSM 18800T21.321.321.821.4ID21.821.922.221.822.1
K. granuli DSM 23481T20.821.021.221.021.8ID30.027.827.127.4
K. adipata DSM 17808T20.921.321.420.921.930.0ID28.025.727.1
K. nematophila sp. nov. K-TC2T21.221.521.521.222.227.828.0ID29.230.9
K. terrae DSM 21341T20.720.720.820.921.827.125.729.2ID30.7
K. defluvii JCM 18034T21.021.321.421.122.127.427.130.930.7ID
Table
Table 5. Phenotypic characters of the different strains described in this study and of the type strains of closely related species. 1: Acinetobacter bereziniae DSM 25435T; 2: Acinetobacter guillouiae DSM 590T; 3: Acinetobacter nematophilus sp. nov. A-IN1T; 4: Alcaligenes endophyticus DSM 100498T; 5: Alcaligenes faecalis subsp. faecalis DSM 30030T (Proposed name: Alcaligenes faecalis); 6: Alcaligenes nematophilus sp. nov. A-TC2T; 7: Alcaligenes faecalis subsp. parafaecalis DSM 13975T (Proposed name: Alcaligenes parafaecalis sp. nov.); 8: Alcaligenes faecalis subsp. phenolicus DSM 16503T (Proposed name: Alcaligenes phenolicus sp. nov.); 9: Enterobacter bugandensis STN0717-56T; 10: Enterobacter chuandaensis 090028T; 11: Enterobacter kobei DSM 13645T; 12: Enterobacter nematophilus sp. nov. E-TC7T; 13: Kaistia algarum JCM 31803T; 14: Kaistia dalseonensis DSM 18800T; 15: Kaistia defluvii JCM 18034T; 16: Kaistia geumhonensis DSM 18799T; 17: Kaistia nematophila sp. nov. K-TC2T; and 18: Kaistia terrae DSM 21341T. +: positive reaction. −: negative reaction.
Table 5. Phenotypic characters of the different strains described in this study and of the type strains of closely related species. 1: Acinetobacter bereziniae DSM 25435T; 2: Acinetobacter guillouiae DSM 590T; 3: Acinetobacter nematophilus sp. nov. A-IN1T; 4: Alcaligenes endophyticus DSM 100498T; 5: Alcaligenes faecalis subsp. faecalis DSM 30030T (Proposed name: Alcaligenes faecalis); 6: Alcaligenes nematophilus sp. nov. A-TC2T; 7: Alcaligenes faecalis subsp. parafaecalis DSM 13975T (Proposed name: Alcaligenes parafaecalis sp. nov.); 8: Alcaligenes faecalis subsp. phenolicus DSM 16503T (Proposed name: Alcaligenes phenolicus sp. nov.); 9: Enterobacter bugandensis STN0717-56T; 10: Enterobacter chuandaensis 090028T; 11: Enterobacter kobei DSM 13645T; 12: Enterobacter nematophilus sp. nov. E-TC7T; 13: Kaistia algarum JCM 31803T; 14: Kaistia dalseonensis DSM 18800T; 15: Kaistia defluvii JCM 18034T; 16: Kaistia geumhonensis DSM 18799T; 17: Kaistia nematophila sp. nov. K-TC2T; and 18: Kaistia terrae DSM 21341T. +: positive reaction. −: negative reaction.
Bacterial Strain
AcinetobacterAlcaligenesEnterobacterKaistia
Biochemical test123456789101112131415161718
β-Galactosidase−−−−−−−+++++++−+++
Arginine dihydrolase++−+++++++++−+−++−
Lysine decarboxylase−−−−+++++−++−+−+−−
Ornithine decarboxylase−−−+−+++++++−+−++−
Citrate utilization++++++++++++++−+++
H2S production−−−−−−−+−−−−−−−−−−
Urease−−−++++++−++++++++
Tryptophan deaminase−−−−+−−+−−−−−−−−−−
Indole production−−−−−−−+−−−−−−−−−−
Acetoin production++++++++++++−++−−−
Gelatinase−−−−−−−++−++−−−−−−
Glucose oxidation+−+−−−−−++++−−−−−−
Mannitol oxidation−−−−−−−+++++−−−−−−
Inositol oxidation−−−−−−−++−−+−−−−−−
Sorbitol oxidation−−−−−−−−+−++−−−−−−
Rhamnose oxidation+−+−−−−+++++−−−−+−
Sucrose oxidation−−−−−−−−+++−−−−−−−
Melibiose oxidation+−+−−−−−+−+−−−−−−−
Amygdalin oxidation−−−−−−−+++++−−−−−−
Arabinose oxidation+−+−−−−−++++−−−−+−
(Cytochrome) oxidase−−−+++++++++++++++
Catalase++++++++++++++++++
NO2 production−−−−−−−−−−+−−+−−−−
NO2 reduction to N2 gas−−−−−−−+++−+−−−++−
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Machado, R.A.R.; Loulou, A.; Bhat, A.H.; Mastore, M.; Terrettaz, C.; Brivio, M.F.; Kallel, S. Acinetobacter nematophilus sp. nov., Alcaligenes nematophilus sp. nov., Enterobacter nematophilus sp. nov., and Kaistia nematophila sp. nov., Isolated from Soil-Borne Nematodes and Proposal for the Elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the Species Level. Taxonomy 2023, 3, 148-168. https://doi.org/10.3390/taxonomy3010012

AMA Style

Machado RAR, Loulou A, Bhat AH, Mastore M, Terrettaz C, Brivio MF, Kallel S. Acinetobacter nematophilus sp. nov., Alcaligenes nematophilus sp. nov., Enterobacter nematophilus sp. nov., and Kaistia nematophila sp. nov., Isolated from Soil-Borne Nematodes and Proposal for the Elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the Species Level. Taxonomy. 2023; 3(1):148-168. https://doi.org/10.3390/taxonomy3010012

Chicago/Turabian Style

Machado, Ricardo A. R., Ameni Loulou, Aashaq Hussain Bhat, Maristella Mastore, Céline Terrettaz, Maurizio Francesco Brivio, and Sadreddine Kallel. 2023. "Acinetobacter nematophilus sp. nov., Alcaligenes nematophilus sp. nov., Enterobacter nematophilus sp. nov., and Kaistia nematophila sp. nov., Isolated from Soil-Borne Nematodes and Proposal for the Elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the Species Level" Taxonomy 3, no. 1: 148-168. https://doi.org/10.3390/taxonomy3010012

APA Style

Machado, R. A. R., Loulou, A., Bhat, A. H., Mastore, M., Terrettaz, C., Brivio, M. F., & Kallel, S. (2023). Acinetobacter nematophilus sp. nov., Alcaligenes nematophilus sp. nov., Enterobacter nematophilus sp. nov., and Kaistia nematophila sp. nov., Isolated from Soil-Borne Nematodes and Proposal for the Elevation of Alcaligenes faecalis subsp. faecalis, Alcaligenes faecalis subsp. parafaecalis, and Alcaligenes faecalis subsp. phenolicus to the Species Level. Taxonomy, 3(1), 148-168. https://doi.org/10.3390/taxonomy3010012

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