Revisiting the taxonomy of the genus Elizabethkingia using whole-genome sequencing, optical mapping, and MALDI-TOF, along with proposal of three novel Elizabethkingia species: Elizabethkingia bruuniana sp. nov., Elizabethkingia ursingii sp. nov., and Elizabethkingia occulta sp. nov.
The genus Elizabethkingia is genetically heterogeneous, and the phenotypic similarities between recognized species pose challenges in correct identification of clinically derived isolates. In addition to the type species Elizabethkingia meningoseptica, and more recently proposed Elizabethkingia miricola, Elizabethkingia anophelis and Elizabethkingia endophytica, four genomospecies have long been recognized. By comparing historic DNA–DNA hybridization results with whole genome sequences, optical maps, and MALDI-TOF mass spectra on a large and diverse set of strains, we propose a comprehensive taxonomic revision of this genus. Genomospecies 1 and 2 contain the type strains E. anophelis and E. miricola, respectively. Genomospecies 3 and 4 are herein proposed as novel species named as Elizabethkingia bruuniana sp. nov. (type strain, G0146T = DSM 2975T = CCUG 69503T = CIP 111191T) and Elizabethkingia ursingii sp. nov. (type strain, G4122T = DSM 2974T = CCUG 69496T = CIP 111192T), respectively. Finally, the new species Elizabethkingia occulta sp. nov. (type strain G4070T = DSM 2976T = CCUG 69505T = CIP 111193T), is proposed.
KeywordsAAI ANI Elizabethkingia MALDI-TOF SNPs Taxonomy
First observed as a causative agent of neonatal meningitis by King (1959), Elizabethkingia infections can cause a variety of conditions including necrotizing fasciitis (Lee et al. 2006), endophthalmitis (Young et al. 2014), pneumonia (da Silva and Pereira 2013), and sepsis (Green et al. 2008; Ramanan and Razonable 2013). Elizabethkingia infections are most commonly observed in immunocompromised patients, mechanically ventilated patients, and neonates, but have been reported to cause meningitis in an immunocompetent adult (Hayek et al. 2013). Once Elizabethkingia infections occur, they have a high mortality rate, with reports of 25% for patients undergoing dialysis (Ratnamani and Rao 2013) and up to 57% for neonates with meningitis (Bloch et al. 1997). A review of 118 patients with Elizabethkingia bacteremia found an overall 14-day mortality rate of 23.4%, and approximately a five-fold increase in incidence per 100,000 admissions over an eight-year period (Hsu et al. 2011).
While hospital outbreaks have usually been attributed to Elizabethkingia meningoseptica, there have been recent reports of Elizabethkingia anophelis causing outbreaks in Intensive Care Units (Teo et al. 2014). A review of cases of bacteremia in Hong Kong hospitals that were caused by Elizabethkingia found that E. anophelis was frequently the causative agent, with an associated high degree of morbidity and mortality (Lau et al. 2016). The largest recognized outbreak to date of E. anophelis occurred in the spring of 2016, sickening 64 people in Wisconsin and nearby states of the United States (Perrin et al. 2017).
DNA–DNA hybridization was initially used to describe five distinct groups of Elizabethkingia strains (E. meningoseptica and genomospecies 1 through 4) (Ursing and Bruun 1987), but there are no known consistent phenotypic characteristics that define the various genomospecies of Elizabethkingia (Bruun and Ursing 1987). Additional Elizabethkingia species were later described with no comparison to the genomospecies reference strains, and each species was defined based on the description of a single strain: Elizabethkingia miricola was described in 2003 as Chryseobacterium miricola, and moved to the newly-formed Elizabethkingia genus in 2005, followed by E. anophelis in 2011 and Elizabethkingia endophytica in 2015 (Kim et al. 2005; Kampfer et al. 2011, 2015; Li et al. 2003). E. endophytica was subsequently recognized as a later subjective synonym of E. anophelis (Doijad et al. 2016), based on whole genome sequence analysis. Taxonomic correspondence of these recently named species with the genomospecies previously defined by DNA–DNA hybridization has not been formally addressed. In this paper, we have analyzed historical strains that had originally been assigned to genomospecies 1–4, modern type strains, and isolates recently obtained from clinical sources using whole genome sequencing (WGS) and optical mapping, and explored the use of MALDI-TOF mass spectrometry and targeted gene sequencing as identification methods.
Materials and methods
Strain selection and phenotypic testing
Strains used in this study
Taxon originally described as
Acc # contigs
Acc # complete
ENA sample id
Elizabethkingia genomosp. 1
Elizabethkingia genomosp. 1
Elizabethkingia genomosp. 1
Elizabethkingia genomosp. 1
Elizabethkingia genomosp. 1
G0146 (H) (T)
Elizabethkingia genomosp. 3
Elizabethkingia genomosp. 3
Elizabethkingia genomosp. 3
KC1913 (H) (T)
NBRC 12535 (T)
Elizabethkingia genomosp. 2
Elizabethkingia genomosp. 2
Elizabethkingia genomosp. 2
G4070 (H) (T)
Elizabethkingia genomosp. 4
G4122 (H) (T)
Elizabethkingia genomosp. 4
Elizabethkingia genomosp. 4
Genome sequencing and assembly
At CDC, strains were grown on heart infusion agar according to manufacturer instructions (Difco) and supplemented with 5% rabbit blood (Hemostat Laboratories) at 35 °C. DNA extraction for WGS was performed using the CTAB protocol provided by the Department of Energy’s Joint Genome Institute (JGI Bacterial DNA Isolation CTAB Protocol), libraries were prepared using the Illumina TruSeq DNA sample prep kit, and genomes were sequenced on an Illumina MiSeq using a 2 × 250 paired-end protocol as described previously (Nicholson et al. 2016). Using CLC Genomics Workbench version 7.51. (CLCbio, Aarhus, Denmark) adapters were removed and reads were trimmed based on quality (limit = 0.02), then the resulting reads were assembled using the de Bruijn graph method of de novo assembly. Contigs ≥500 bp that had an average mapping coverage ≥50× were selected for further analysis. Contigs were split at the positions of any ambiguous (“N”) nucleotides in the assembly. Selected genomes were closed based on orientation of the contigs as determined by optical mapping (see below), with the exact sequence of contig joins informed by read mapping.
At the Collection of Institut Pasteur (CIP), strains were cultivated on trypticase soy agar (Bio-Rad) at 30 °C and DNA was extracted using the MagNA Pure 96 robotic System with the MagNA Pure 96 DNA and Viral Nucleic Acid small volume kit (Roche Diagnostics). Libraries were constructed using the Nextera XT DNA Library Preparation kit (Illumina, Inc., San Diego, CA) and sequencing done on a NextSeq-500 instrument using a 2 × 150 paired-end protocol. Read trimming and clipping was performed with AlienTrimmer v.0.4.0 (Criscuolo and Brisse 2013), followed by sequencing error correction with Musket v.1.1 (Liu et al. 2013), and next by coverage homogenization with khmer v.1.3 (Crusoe et al. 2015). Processed reads were finally used to perform de novo assembly with SPAdes v3.6.2 (Bankevich et al. 2012).
Positions 1939-3629 in the 3825 bp rpoB gene sequence were sequenced as described previously (Shewmaker et al. 2011), with minor modifications: Elizabethkingia-specific PCR primers were designed (EK_rpoB_fwd: 5′-ATGGGATCTAACATGAT-3′ and EK_rpoB_rev: 5′-GCCCAAACCTCCATCTC-3′), and the amplicon was sequenced using these primers plus two additional primers (EKrpoB1154F: 5′-GGGGATAAAATGGCRGG-3′ and EKrpoB1154R 5′-CCYGCCATTTTATCCCC-3′). To compare rpoB for all genomes used in this analysis, the sequence of each predicted rpoB PCR product was located using BLAST, and aligned within CLC genomics workbench. Maximum likelihood (ML) trees were generated using MEGA v6 (Tamura et al. 2013).
In silico genome comparisons
The average nucleotide identity BLASTN (ANIb) method was described by Goris et al. and has been implemented in the Jspecies software package (Goris et al. 2007; Richter and Rossello-Mora 2009). Two-way average amino acid identity (AAI) scores were calculated, and percentage of conserved proteins (POCP) scores were calculated as described by Qin et al. (2014). Proteomes from each genome were generated by Prodigal v2.6.2 (Hyatt et al. 2010). For each pairwise comparison, an all-versus-all search of all proteins was carried out using BLASTp v2.4.0+ (Altschul et al. 1997) in both directions. If both directions of BLASTp searches resulted in the same protein match (pair) and exceeded 40% in amino acid identity and 50% in coverage length, we included the protein sequences for computing the arithmetic mean sequence identity. In silico genome comparisons based on calculating genome-to-genome distances as described by (Auch et al. 2010a, b; Meier-Kolthoff et al. 2013), were determined using their Genome-to-Genome Distance Calculator tool (GGDC) (http://ggdc.dsmz.de/distcalc2.php), and rounded to the nearest integer. Single nucleotide polymorphism (SNP) trees were generated using the HarvestTools (Treangen et al. 2014), and exported Newick files were edited with MEGA v6 (Tamura et al. 2013). Additional data visualizations were produced using JMP v11 (SAS Institute Inc., Cary, NC).
Core genome phylogeny
The core orthologous genome of Elizabethkingia was calculated from Prodigal-generated (v2.6.2) (Hyatt et al. 2010) sequences of each Elizabethkingia (n = 63) isolate used as input for Roary v3.6.8 (Page et al. 2015). Highly related homologs were initially identified with CD-HIT v4.6 (Fu et al. 2012) by clustering sequences with five iterations beginning at 100% and going as low as 98% identity (0.5% decrement steps). Subsequent sequences were aligned to each other in an all-against-all fashion with BLASTp v2.4.0+ and a minimum identity of 40% was required. Sequence clusters were then identified with the mcl v14-137 algorithm (Enright et al. 2002) and paralogous sequences were discarded. The set of core orthologous genes were individually aligned with the codon-aware PRANK v.140603 software (Loytynoja and Goldman 2005). Concatenated alignments of these 2259 genes were filtered for invariant sites and the resulting 10,49,915 sites per isolate were analyzed to determine the most appropriate evolutionary model, using jModelTest v2.1.10 (Guindon and Gascuel 2003; Darriba et al. 2012). The JC69 model was then used in RAxML v8.2.9 (Stamatakis 2014) to generate 100 ML pseudoreplicate topologies, and 100 bootstraps provided convergence according to the extended majority—rule consensus tree criterion (Pattengale et al. 2010). The resulting tree was edited with MEGA v6 (Tamura et al. 2013).
Whole genome optical mapping
22 strains of Elizabethkingia, including at least two representatives for each proposed species, were compared by application of the OpGen optical mapping platform (OpGen, Inc., Gaithersburg, Maryland). High molecular weight genomic DNA from overnight grown bacterial cells was purified with Argus HMW DNA Isolation Kit (OpGen, Inc.) and examined for quality and concentration using the ARGUS QCards. The Enzyme Chooser function of MapManager version 1.3 (OpGen, Inc.) identified NcoI restriction endonuclease to be optimal for optical map production because its cleavage of reference genomes would result in fragments that average 6–12 kilobase pairs (kbp) in size, with no fragments larger than 80 kbp. Individual genomic DNA fragments were loaded onto a glass surface of a MapCard (OpGen, Inc.) using the microfluidic device, washed and then digested with NcoI, stained with JOJO-1 through the ARGUS MapCard Processor (OpGen, Inc.). Map cards afterward were scanned and analyzed by automated fluorescent microscopy using the ARGUS Whole Genome Mapper v3.2.4 (OpGen, Inc.). The single molecule restriction map collections were then tiled according to overlapping fragment patterns to produce a consensus whole genome map. This map was imported into MapSolver v3.2 (OpGen, Inc.) along with predicted in silico maps of contigs derived from WGS, using the same restriction enzyme for ordering and orientation of contigs during genome circularization. In silico predicted optical maps of complete genomes were scaled according to the size of sequenced genomes. Final alignments were clustered in MapSolver v3.2 using a nearest neighbor algorithm to evaluate a similarity among Elizabethkingia strains.
Matrix-Assisted Laser Desorption/Ionization-Time Of Flight (MALDI-TOF) mass spectrometry was performed using the BioTyper (Bruker, Germany). Main Spectrum Profiles (MSPs) were created to represent each genomospecies, using the historic strains and type strains of each species. For each MSP, cells were extracted using Bruker’s Formic Acid/Acetonitrile Procedure and overlaid with HCCA Matrix. Spectra were obtained using Bruker’s Flex Control and MALDI Biotyper 3 Software. The reproducibility of these spectra was confirmed using a whole-cell direct transfer, overlaid by HCCA matrix, on the same strains as well as additional strains with sequenced genomes. MSP’s (spectral profiles) are publically available using CDC’s MicrobeNet—a free, curated reference tool. (https://microbenet.cdc.gov/; see supplemental text). Real-Time Classification was performed using Bruker RTC Software.
Results and discussion
Criteria for determination of species among Elizabethkingia strains
Traditional DNA–DNA hybridization (tDDH) results from representatives of all Elizabethkingia species (Holmes et al. 2013) correlate with in silico methods
tDDH at 70 °C
GGDC: formula 1
GGDC: formula 2
GGDC: formula 3
GGDC: formula 1
GGDC: formula 2
GGDC: formula 3
WGS contigs have been shown for other species to produce ANIb and GGDC formula 2 predicted DDH (henceforth referred to simply as “predicted DDH”) results indistinguishable from those produced using complete circularized genomes (Richter and Rossello-Mora 2009; Auch et al. 2010a), and we confirmed that this was also the case for Elizabethkingia strains (see Supplemental text); WGS contig sets were used for all subsequent analyses.
Elizabethkingia meningoseptica is phylogenetically distinct from other Elizabethkingia species
The relatively large phylogenetic distance between E. meningoseptica strains and strains from other Elizabethkingia species raised the question of whether they really do belong to the same genus.
Percentage of Conserved Proteins (POCP) between type strains of Elizabethkingia anophelis (DSM 23781), Elizabethkingia bruuniana (G0146), Elizabethkingia meningoseptica (KC1913), Elizabethkingia miricola (DSM 14571), Elizabethkingia occulta (G4070), Elizabethkingia ursingii (G4122), Chryseobacterium gleum (ATCC 35910), and Sphingobacterium spiritivorum (ATCC 33861)
E. anophelis strains constitute genomospecies 1 and Elizabethkingia miricola strains constitute genomospecies 2
Our earlier report describing WGS data for each genomospecies noted that the genome sequence of the type strain of Elizabethkingia anophelis was consistent with it belonging to genomospecies 1, and that the 16S rRNA gene of JM-87 (the type strain of “E. endophytica”) was identical to that of the historic strain F3201, which tDDH also classified as member of genomospecies 1 (Holmes et al. 2013). This similarity was borne out by comparisons of the whole genome sequences of both strains, which had an ANIb >98.70%, and a predicted DDH of 92%. Strain JM-87 had an ANIb ≥97%, and a predicted DDH ≥88%, compared to all of the genomospecies 1 strains, including the type strain of E. anophelis (DSM 23781), consistent with the recognition (Doijad et al. 2016; Perrin et al. 2017) that strain JM-87 is an Elizabethkingia anophelis strain.
The type strain of E. miricola (DSM 14571T) is most similar to the historic genomospecies 2 strains, with a predicted DDH of 70%, and an ANIb value slightly above 96%. We identified two additional strains that had marked similarity with the E. miricola type strain, and several others that were more closely related to the historic genomospecies 2 strains, as evidenced both by their phylogenetic proximity and their predicted DDH values. Strain EM-CHUV in the public domain (Opota et al. 2016), and strains CSID_3000516464 and CSID_3000516998 from this work, were similarly predicted to be E. miricola by ANIb. Several of the strains that were considered to be E. miricola (based on ANIb of ≥95%) had a predicted DDH of slightly less than 70%. A predicted DDH of at least 65% was determined to be sufficient for inclusion of the strains in the E. miricola species since the 9%.
Proposed nomenclature for the historically recognized genomospecies 3 and 4
Both ANIb and predicted DDH provide quantitative confirmation of the earlier DNA–DNA hybridization results that genomospecies 3 and 4 strains are species that are distinct from E. miricola and from each other. Core genome phylogenetic analysis and a SNP analysis of all strains that were not E. meningoseptica or E. anophelis (Supplemental Fig. 4) produced results consistent with ANIb and predicted DDH. Strain ATCC 33958 from the public domain was found to belong to genomospecies 3, as was strain BM10, by these methods. Modern strains were identified that belong to either genomospecies 3 or 4, as were strains retrieved from the CDC and CIP collections. This large set of well-characterized and historically recognized strains, which could not be provided with validly published names using pre-genomic technology, can now be named based on their complete genome sequences. In recognition of the foundational work done by Jan Ursing and Brita Bruun investigating the genus Elizabethkingia, we propose to name genomospecies 3 as Elizabethkingia bruuniana sp. nov. (bruun.i.a’na. N.L. fem. adj. bruuniana, named in honour of Brita Bruun), and genomospecies 4 as Elizabethkingia ursingii sp. nov. (ur.sing’i.i. N.L. gen. n. ursingii, of Ursing, named in honour of Jan Ursing).
A third novel Elizabethkingia species is proposed as Elizabethkingia occulta
The strain G4070 had been originally identified as belonging to genomospecies 4, but its optical map showed that it was unlike the other genomospecies 4 strains, and both ANIb and GGDC put it outside of that genomospecies. This suggested that strain G4070 was not actually a member of genomospecies 4, but instead a representative of its own novel genomospecies. MALDI-TOF mass spectra of strains from the CDC strain collection were reviewed to locate strains with spectra similar to G4070, and a subset of these had their rpoB sequenced. Strain F8124 was thereby identified as potentially belonging to the same genomospecies as G4070, and this similarity was confirmed by whole genome sequencing with an ANIb of 99.4% and a predicted DDH of 96. Strain F8124 (CL50/86 = CCUG 15909 = GIFU 2120) had been originally published as a founding member of the combination Sphingobacterium mizutae (Yabuuchi et al. 1983) due to its phenotypic similarities. Later studies based on tDDH showed that it was distinct from S. mizutae strains and it was instead assigned to the F. meningosepticum species (Holmes et al. 1988), but not included in the experiments that delineated the Elizabethkingia genomospecies. Here we propose that both strains belong to a novel species that we name Elizabethkingia occulta sp. nov. (oc.cul’ta. L. fem. adj. occulta hidden), to reflect that it was hiding in plain sight.
Elizabethkingia species identification by MALDI-TOF mass spectrometry and target gene sequencing
Using an expanded spectrum database (https://microbenet.cdc.gov/), analysis by MALDI-TOF mass spectrometry can reliably identify E. anophelis and E. meningoseptica, but cannot distinguish between the remaining species. 274 Elizabethkingia strains from the CDC collection of mostly clinical isolates were analyzed using MALDI-TOF mass spectrometry, and only 23 (8%) were found to be E. meningoseptica. 210 (71%) were E. anophelis, and 41 (14%) were one of the other Elizabethkingia species.
Both the 16S rRNA and rpoB genes were evaluated as target genes for Elizabethkingia species identification. The five copies of the 16S rRNA gene present in all Elizabethkingia genomes can be quite different from each other. The most extreme example of this among the strains described here was E. ursingii strain G4123, which contained three distinct variants of the 16S rRNA gene, one being most similar to all five from the E. ursingii type strain, two being most similar to E. bruuniana strains, and two matching each other but otherwise unique. This confounds the use of 16S rRNA sequence comparisons for Elizabethkingia species identification.
Phenotypic testing cannot reliably distinguish Elizabethkingia strains
As Ursing and Bruun reported (Bruun and Ursing 1987), there is a great deal of phenotypic variability among Elizabethkingia strains, even those belonging to the same genomospecies. Results of our testing of the historic strains are shown in Supplemental Table 2. When the Elizabethkingia genus was first published, urease was the only biochemical test that was consistently different between E. meningoseptica strains (negative) and the type strain of E. miricola (positive), but our tests show that all of the genomospecies 1 strains and some of the genomospecies 3 and 4 strains were also positive for urease, while genomospecies 2 strains G4071 and G4121 were negative. Similarly, “E. endophytica” was described as being negative for acid production from cellobiose as compared to E. anophelis, which was positive. While we confirmed these results for the strains described, three of the four genomospecies 1 strains most closely related to E. anophelis strain R26 were negative in the cellobiose assay, while the strain that was most closely related to the “E. endophytica” strain JM-87 (F3201) was positive. We did not perform cellular fatty acid testing on these strains, as all Elizabethkingia have been previously described as having polar lipid profiles that are very similar to each other, and to species of the genus Chryseobacterium (Kampfer et al. 2015).
For many decades, CDC’s Special Bacteriology Reference Laboratory performed a series of standard phenotypic tests on all strains that were added to the collection, and these results were reviewed in hope of discovering any consistent phenotypic difference between the three groups (E. meningoseptica, E. anophelis, and all others) that could be distinguished by MALDI-TOF mass spectrometry. Supplemental Table 3 summarizes this review, showing that they cannot be reliably distinguished by phenotype alone. Certain characteristics were found to be almost entirely strain-dependent (variable within a species), and not at all useful in species determination; these were urease, acid production from lactose, and growth on Simmons’ citrate (citrate as a sole carbon source).
Although is it likely that labs will rely on DNA sequence analysis and/or MALDI-TOF mass spectrometry when classifying Elizabethkingia strains, phenotypic descriptions have been published for Elizabethkingia species previously. We have combined all available information to update the existing descriptions, and provide the traditional phenotypic descriptions of the newly named species.
Emended description of the genus Elizabethkingia
Elizabethkingia (E.liz.a.beth.kin′gi.a. N.L. fem. n. Elizabethkingia in honour of Elizabeth O. King, who first described the bacteria associated with infant meningitis as [Flavobacterium] meningosepticum in 1959).
Cells are Gram-negative, non-motile, non-spore-forming rods (0.5 × 1.0–2.5 μm). Good growth is observed on TSA and nutrient agar at 28–37 °C, but no growth is observed at 5 or 42 °C. Colonies are white or yellow, semi-translucent, circular and shiny with entire edges. Catalase, phosphatase and β-galactosidase activities are positive. Indole is produced. Casein is hydrolysed, but starch is not. Malonate is not utilized. Acid is not produced from galactose, melezitose, raffinose, adonitol, dulcitol, sorbitol, or inositol, or salicin. The fatty acid profile consists largely of 15 : 0 iso, 17 : 0 iso 3-OH and summed feature 4 (15 : 0 iso 2-OH and/or 16 : 1 ω7 c/t). Menaquinone MK-6 is the predominant quinone. The G+C content of the DNA is 35.0–38.2 mol%.
The type species is E. meningoseptica. This emended genus description is represented in the Digital Protologue by taxon number GA00018.
Emended description of Elizabethkingia anophelis
Elizabethkingia anophelis (a.no.phe′lis. N.L. gen. n. anophelis of/from a mosquito of the genus Anopheles, as the type strain was isolated from the midgut of Anopheles gambiae).
Cells are aerobic Gram-reaction-negative, non-motile, non-spore-forming rods, approximately 1 µm in width and 2 µm in length. Catalase-positive. Good growth occurs after 48 h on NA, R2A agar and TSA (all Oxoid) at 11–36 °C. Growth on MacConkey agar (Oxoid) at 28 °C is strain-dependent. Unable to grow at temperatures below 10 °C or above 37 °C. Two growth optima are detected on LB medium: 30–31 °C with a doubling time of 50 min; and 37 °C with a doubling time of 42 min. Colonies on NA are smooth, yellowish, circular, translucent and shiny with entire edges. The non-diffusible and non-fluorescent yellow pigment is not of the flexirubin-type (KOH test-negative). Resistant to a number of antibiotics; MICs in LB medium are >400 µg ml−1 for ampicillin, >250 µg ml−1 for kanamycin, >250 µg ml−1 for streptomycin, >30 µg ml−1 for chloramphenicol and >10 µg ml−1 for tetracycline. Indole is produced. Acid is produced from trehalose. No acid is produced from adonitol, d-arabitol, dulcitol, erythritol, i-inositol, methyl α- d-glucoside, raffinose, salicin, or d-sorbitol. Acid production from d-melibiose, d-cellobiose, d-glucose, lactose, d-mannitol, maltose, d-mannitol, d-xylose, lactose, sucrose and arabinose is variable. Indole production from tryptophan and β-galactosidase activity (ONPG) are positive. Aesculin hydrolysis, nitrate reduction, urease and oxidase activity is variable; Hydrolysis of casein, starch, DNA and tyrosine, activity of arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase, and utilization of malonate are negative. Hydrogen sulfide and gelatinase production is strain dependent. The following compounds are not utilized as sole sources of carbon: N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, l-arabinose, l-arbutin, cellobiose, d-fructose, d-glucose, maltose, d-galactose, gluconate, glycerol, d-mannose, d-mannitol, maltitol, α-melibiose, l-rhamnose, d-ribose, sucrose, salicin, trehalose, d-xylose, adonitol, inositol, d-sorbitol, putrescine, acetate, propionate, cis-aconitate, trans-aconitate, 4-aminobutyrate, adipate, azelate, fumarate, glutarate, dl-3-hydroxybutyrate, itaconate, dl-lactate, 2-oxoglutarate, pyruvate, suberate, mesaconate, l-alanine, β-alanine, l-ornithine, l-phenylalanine, l-serine, l-aspartate, l-histidine, l-leucine, l-proline, l-tryptophan, 3-hydroxybenzoate, 4-hydroxybenzoate and phenylacetate. Utilization of citrate as the sole source of carbon is strain-dependent. The chromogenic substrates p-nitrophenyl (pNP)-β-d-glucopyranoside, pNP-β-d-galactopyranoside, pNP-α-d-glucopyranoside, bis-pNP-phosphate, bis-pNP-phenyl-phosphonate, bis-pNP-phosphoryl-choline, 2-deoxythymidine-2′-pNP-phosphate, l-alanine-p-nitroanilide (pNA), γ-l-glutamate-pNA and l-proline-pNA are hydrolysed but not pNP-β-d-xylopyranoside or pNP-β-d-glucuronide. Major cellular fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 4 (iso-C15 : 0 2-OH and/or C16 : 1ω7 c/t). The only menaquinone is MK-6. The major polar lipids are diphosphatidylglycerol, phosphatidylinositol, a characteristic unknown phospholipid, and unknown polar lipids and glycolipids.
The type strain is R26T (= CCUG 60038T = CCM 7804T), isolated from the midgut of Anopheles gambiae G3, originating from McCarthy Island, The Gambia, and deposited by Dr William Collins at Malaria Research Reference Resource Centre. This emended species description is represented in the Digital protologue by taxon number TA00064.
Emended description of Elizabethkingia meningoseptica
Elizabethkingia meningoseptica (me.nin.go.sep′ti.ca. Gr. n. meninx, meningos meninges, membrane covering the brain; Gr. adj. septikos putrefactive; N.L. fem. adj. meningoseptica apparently referring to association of the bacterium with both meningitis and septicaemia, but not septic meningitis as the name implies).
Basonym Flavobacterium meningosepticum King (1959) (Approved Lists 1980).
Cells are Gram-negative, non-motile, non-spore-forming rods (0.5 × 1.0–2.0 μm). Growth on MacConkey agar is strain-dependent. Oxidase, gelatinase, H2S and indole are produced. Aesculin is hydrolyzed. Acid is produced from ethanol, d-glucose, glycerol, lactose, d-maltose, d-mannitol and trehalose, but not from l-arabinose, d-cellobiose, raffinose, sucrose, salicin or rhamnose, d-xylose. Urea hydrolysis, use of citrate as a sole carbon source, and acid production from fructose is strain-dependent. The fatty acid profile consists largely of 15 : 0 iso (43.9 ± 2.0 %), 17 : 0 iso 3-OH (14.6 ± 1.0 %) and summed feature 4 (15 : 0 iso 2-OH and/or 16 : 1 ω7 c/t; 19.6 ± 1.0%). The G+C content of the DNA is 37.2 ± 0.6 mol% (37.1 mol% for the type strain).
The type strain is ATCC 13253T (= KC1913T = NCTC 10016T = LMG 12279T = CCUG 214T). This emended species description is represented in the Digital protologue by Taxon Number TA00060.
Emended description of Elizabethkingia miricola
Elizabethkingia miricola [mi.ri′co.la. N.L. neut. n. mirum derived from mir (peace) (name of Russian space station); L. suff.—cola from L. masc. or fem. n. incola inhabitant; N.L. masc. or fem. n. miricola inhabitant of the Mir space station].
Basonym Chryseobacterium miricola Li et al. (2003).
Cells are Gram-negative, non-motile, non-spore-forming rods (0.5 × 1.0–2.5 μm). Hydrolysis of urea and gelatin, nitrate reduction, growth on MacConkey agar, H2S production and use of citrate as a sole carbon source are strain dependent. Indole is produced. Aesculin and oxidase are positive. Acid is produced from d-fructose, d-mannitol and trehalose, but not from l-arabinose, raffinose, sucrose, salicin, rhamnose, or d-xylose. Acid production from d-glucose, lactose, d-maltose, and d-cellobiose is strain dependent. The fatty acid profile consists largely of 15 : 0 iso (46.4 ± 2.2 %), 17 : 0 iso 3-OH (15.3 ± 0.2%) and summed feature 4 (15 : 0 iso 2-OH and/or 16 : 1 ω7 c/t, 17.0 ± 1.3%). The G+C content of the DNA is 35.3 ± 0.3 mol% (35.0 mol% for the type strain).
The type strain is DSM 14571T (=JCM 11413T = GTC 862T). This emended species description is represented in the Digital protologue by Taxon Number TA00061.
Description of Elizabethkingia bruuniana sp. nov.
Elizabethkingia bruuniana (bruun.i.a’na. N.L. fem. adj. bruuniana, named in honour of Brita Bruun).
Cells are Gram-stain negative, non-motile, non-spore-forming rods (0.5 × 1.0–2.5 μm). Good growth is observed on TSA and nutrient agar at 28–37 °C, but no growth is observed at 5 or 42 °C. Colonies are white or yellow, semi-translucent, circular and shiny with entire edges. Catalase, phosphatase, gelatinase, and β-galactosidase activities are positive. Nitrate is not produced. Oxidase, aesculin, H2S production, and ability to use citrate as a sole carbon source are strain-dependent. Indole is produced. Casein is hydrolysed, but starch is not. Malonate is not utilized. Acid is produced from d-mannitol, glucose, and maltose but not produced from arabinose, lactose, rhamnose, sucrose, xylose, galactose, melezitose, raffinose, sucrose, adonitol, dulcitol, sorbitol, inositol, or salicin. Acid production from cellobiose, fructose, mannitol, and trehalose is strain-dependent.
The type strain is G0146T (= DSM 2975T = CCUG 69503T = CIP 111191T). The species description is represented in the Digital protologue by taxon number TA00058.
Description of Elizabethkingia ursingii sp. nov.
Elizabethkingia ursingii (ur.sing’i.i. N.L. gen. n. ursingii, of Ursing, named in honour of Jan Ursing).
Cells are Gram-stain negative, non-motile, non-spore-forming rods (0.5 × 1.0–2.5 μm). Good growth is observed on TSA and nutrient agar at 28–37 °C, but no growth is observed at 5 or 42 °C. Colonies are white or yellow, semi-translucent, circular and shiny with entire edges. Aesculin, oxidase, catalase, phosphatase and β-galactosidase activities are positive. H2S and Indole are produced. Casein is hydrolysed, but starch is not. Malonate is not utilized and nitrate is not reduced. Gelatinase, urease activity, use of citrate as the sole carbon source and growth on MacConkey agar are strain dependent. Acid is produced from fructose, glucose, maltose, and mannitol but not produced from cellobiose, rhamnose, sucrose, xylose, galactose, melezitose, raffinose, sucrose, adonitol, dulcitol, sorbitol, or inositol, or salicin. Acid production from lactose is strain dependent.
The type strain is G4122T (=DSM 2974T = CCUG 69496T = CIP 111192T). The species description is represented in the Digital protologue by Taxon Number TA00059.
Description of Elizabethkingia occulta sp. nov.
Elizabethkingia occulta (oc.cul’ta. L. fem. adj. occulta hidden, to reflect that it was hiding in plain sight, and had been previously masquerading as Sphingobacterium mizutae or Elizabethkingia genomospecies 4).
Cells are Gram-stain negative, non-motile, non-spore-forming rods (0.5 × 1.0–2.5 μm). Good growth is observed on MacConkey agar, TSA and nutrient agar at 28–37 °C, but no growth is observed at 5 or 42 °C. Colonies are white or yellow, semi-translucent, circular and shiny with entire edges. Aesculin, catalase, oxidase, phosphatase, urease, and β-galactosidase activities are positive. Indole is produced and nitrate is reduced. Casein is hydrolysed, but gelatin and starch are not. Malonate is not utilized, and citrate cannot be used as the sole carbon source. H2S production is strain-dependent. Acid is produced from cellobiose, glucose, lactose, maltose, mannitol, and trehalose, but not produced from arabinose, fructose, rhamnose, galactose, melezitose, raffinose, sucrose, adonitol, dulcitol, sorbitol, or inositol, or salicin. Acid production from xylose is strain-dependent.
The type strain is G4070T (= DSM 2976T = CCUG 69505T = CIP 111193T). The species description is represented in the Digital protologue by Taxon Number TA00062.
We thank John McInroy for providing strain JM-87, and the State Health Departments of Wisconsin, Minnesota, Illinois, Michigan, Florida, Arizona, Texas, South Carolina, and California in the USA for providing Elizabethkingia clinical specimens. We also thank Aharon Oren for nomenclature advice, Barry Holmes for supplying phenotypic data on some of the strains included in this study, and Aaron Villarma for technical assistance.
Compliance with ethical standards
CDC research was supported by the Advanced Molecular Detection (AMD) initiative, and work done at the Institut Pastuer was funded by the French government’s Investissement d’Avenir program Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (Grant ANR-10-LABX-62-IBEID).
Conflict of interest
All authors report that they have no conflicts of interest.
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