DOI: 10.15468/zef4w8
title: Emendation of the Genus Auritidibacter Yassin et al. 2011 and Auritidibacter ignavus Yassin et al. 2011 based on features observed from Canadian and Swiss clinical isolates and wholegenome sequencing analysis

description: This dataset contains the digitized treatments in Plazi based on the original journal article Bernard, K. A., Pacheco, A. L., Burdz, T., Wiebe, D., Beniac, D. R., Hiebert, S. L., Booth, T. F., Jakopp, B., Goldenberger, D., Seth-Smith, H. M. B., Egli, A., Bernier, A-M (2020): Emendation of the Genus Auritidibacter Yassin et al. 2011 and Auritidibacter ignavus Yassin et al. 2011 based on features observed from Canadian and Swiss clinical isolates and wholegenome sequencing analysis. International Journal of Systematic and Evolutionary Microbiology 70 (1): 83-88, DOI: 10.1099/ijsem.0.003719, URL: http://dx.doi.org/10.1099/ijsem.0.003719AbstractAuritidibacter ignavus is a Gram-stain-positive bacillus derived from otorrhea. Four strains derived from ear discharges in Canada and Switzerland, with features consistent with but distinguishable from Auritidibacter ignavus IMMIB L-1656 T (accession number FN554542) by 16S rRNA gene sequencing (97.5% similarity), were thought to represent a novel species of the genus Auritidibacter. Auritidibacter ignavus DSM 45359 T (=IMMIB L-1656 T) was acquired to compare with Canadian and Swiss strains by whole-genome sequencing (WGS). Unexpectedly, those isolates were observed to be consistent with A. ignavus DSM 45359 T by WGS (ANIb scores&gt;98%), MALDI-TOF (Bruker), cellular fatty acid analysis and biochemically (some differences were observed). A nearly full 16S rRNA gene sequence could not be readily prepared from A. ignavus DSM 45359 T, even after multiple attempts. A 16S rRNA gene chimeric consensus sequence created from the genome assembly of A. ignavus DSM 45359 T had only 97.5% similarity to that of A. ignavus IMMIB L-1656 T, implying that 16S rRNA sequence accession number FN554542 could not be replicated. We concluded that our isolates of members of the genus Auritidibacter were consistent with A. ignavus DSM 45359 T, did not represent a novel species, and that the sequence corresponding to FN554542 was not reproducible. By WGS, A. ignavus DSM 45359 T had genome of 2.53× 106 bp with a DNA G+C content of 59.34%, while genomes of Canadian and Swiss isolates ranged from 2.47 to 2.59× 106 bp with DNA G+C contents of 59.3–59.52%. A. ignavus NML 100628 (=NCTC 14178=LMG 30897) did not demonstrate a rodcoccus cycle. Emendation of Auritidibacter ignavus was proposed based on these results.Auritidibacter ignavus gen. nov. sp. nov. of the family Micrococcaceae was described in 2011 to encompass a biochemically minimally-reactive, Gram-stain-positive coccus to rod-shaped bacterium recovered from a case of fulminant otitis externa; whole-genome sequencing (WGS) analysis had not been performed on the type strain [1]. Four isolates recovered from 2010 to 2013 from ear infections of patients in Canada and Switzerland had, by 16S rRNA gene sequence analysis 99.8–99.9 % similarity to each other, indicating that these isolates represented a single taxon which was close to, but distinguishable from (97.5% sequence similarity) the type strain of A. ignavus by that method. National Microbiology Laboratory (NML) identifier 100628 from the ear of a 57year old male and NML 120636 isolated from a left mastoid biopsy from a 51 year old female, recovered in 2010 and 2012 respectively, were referred from Vancouver British Columbia Canada (49.2827° N, 123.1207° W), with no other patient information being available. A third Canadian strain isolated in 2012 (NML 120779) was from a 76 year old male from Winnipeg Manitoba (49.8951° N, 97.1384° W) with chronic otitis media and otorrhea who had been repeatedly treated with topical ciprofloxacin. Respective provincial laboratories found these isolates to be unidentifiable using standard methods and so had forwarded isolates to the NML for additional characterization. The fourth isolate was recovered at University Hospital Basel (USB), Switzerland (400516/2013=NML 130574, described below as NML 130574) (47.5596° N, 7.5886° E). The patient was a 30 year old HIV-positive male immigrant from Hungary who had recurrent otitis, with at least one infection per year, and had recurrent tympanoplasties (seven on the left side and one on the right side) resulting in presbycusis. Superinfected chronic otitis perforata after tympanoplasty had been resolved with the use of high doses of ciprofloxacin ear drops, two times per day for seven days.Author affiliations: 1 National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg Manitoba, Canada; 2 Department of Medical Microbiology, University of Manitoba, Winnipeg Manitoba, Canada; 3 Division of Infectious Diseases and Hospital Epidemiology, University Hospital, Basel, Switzerland; 4 Division of Clinical Bacteriology and Mycology, University Hospital Basel, Basel, Switzerland; 5 Applied Microbiology Research, Department of Biomedicine, University of Basel, Basel, Switzerland; 6 Department of Biology, Université de Saint-Boniface, Winnipeg Manitoba, Canada. *Correspondence: K. A. Bernard,kathy.bernard@canada.caKeywords: Auritidibacter ignavus; otitis media; Genus Auritidibacter.Abbreviations: ANIb, average nucleotide identity using BLASTn; CFA, cellular fatty acids; dDDH, digital DNA–DNA hybridization; GGDC, genome to genome distance calculator; HPLC, high-pressure liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry; MSP, mass spectral profile; NML, National Microbiology Laboratory; USB, University Hospital Basel Switzerland; WGS, wholegenome sequence.†Present address: Infectious Diseases Unit, Katonsspital Aarau, Aarau, Switzerland.The GenBank/EMBL/DDBJ accession numbers for 16S rRNA gene sequences derived in this study are KC669621, KC669622 and KC669623, forNML 100628, NML 120636 and NML 120779, respectively, KF360241 for the Swiss isolate 400516/2013 (=NML 130574) and MK069432 for A. ignavus DSM 45359 T. Illumina Sequencing Reads for strains NML 100628, NML 120636, NML 120779 and A. ignavus DSM 45359 T were assigned the accession numbers SRX4212137, SRX4212136, SRX4212138 and SRX5335920, respectively. Whole-genome sequencing shotgun projects for strains of members of the genus Auritidibacter have been deposited under the accession numbers QHLP00000000 (NML 100628), QHLO00000000 (NML 120636), QHLN00000000, (NML 120779) and CP031746 (400516/2013=NML 130574). The WGS project prepared here for Auritidibacter ignavus DSM 45359 T (=IMMIB L-1656 T) has been deposited under the accession number RDQR00000000.Three supplementary figures and two supplementary tables are available with the online version of this article.The observation of 16S rRNA gene similarity of these isolates to the sequence from the A. ignavus type strain (accession number FN554542) prompted characterization of these four bacteria as representing a single taxon which was thought to possibly represent a novel species of the genus Auritidibacter. Strains were characterized biochemically, for antimicrobial susceptibilities, by cellular fatty acid (CFA) composition, matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI–TOF MS, Bruker Daltronics) and by WGS analyses. Auritidibacter ignavus DSM 45359 T (=IMMIB L-1656 T) was acquired from the Leibniz-Institut DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) to compare with genomes derived from Canadian and Swiss strains. The methodology and features derived from a complete genome prepared from the Swiss isolate (400516/2013=NML 130574) has been described separately [2].The four clinical isolates were found to be generally consistent with Auritidibacter ignavus IMMIB L-1656 T [1] morphologically after growth on SBA or by Gram-stain appearance [Fig. S1a and Fig. 1b (available in the online version of this article), respectively]. Cell morphology including observation of rod-like forms (at 17 h old) or cocci (at 108 h old) had been previously described for A. ignavus IMMIB L-1656 T [1]. Therefore, NML 100628 was grown in brain–heart infusion broth at 35 °C and evaluated for type of morphology observable at different time intervals. Samples drawn at 0, 7.5, 24, 48, 72 and 96h were tested. Each aliquot was gently mixed with fixative containing buffered 2% glutaraldehyde and 1% paraformaldehyde and was then prepared for transmission electron microscopy as previously described [3]. Length measurements were made using the Image J software package using the free hand line tool and the analyse/measure function [4]. Measurements were made in Image J, collated and analysed using Microsoft Excel. Straight or curved rods were seen at all time intervals and coccoidal forms were not observed after 96 h (interval set of electron micrographs not shown). The rod-like morphology of NML 100628 after 48 h of growth was selected as being typical; it was (N, number of measurements) 1.85±0.45 µm long (N =35) with a width of 0.44±0.05 µm (N =58) (Fig. S1c), dimensions similar to those observed for A. ignavus [1].Phenotypically, the four isolates were consistent with A. ignavus IMMIB L-1656 T [1] except for the following features, also summarized in Table S1. Strains were observed to grow at 42 °C on 5% Columbia blood agar base with 5% sheep blood (SBA) as well as under strictly anaerobic conditions, at 35 °C. Strains were tested by tube motility and by the hanging drop method [5]; strains were non-motile by the tube test but positive by hanging drop. Isolates assimilated substrate at pH 6 but not pH 5 (Biolog), could not reduce nitrite and were negative for acetoin, DNase and starch hydrolysis. Pyrolidonyl arylamidase (PYRa) was detected for two strains. API Coryne (Biomérieux) codes generated were 2110004 (two strains) or 6110004 (two strains), which were associated with poor confidence identification scores to members of the genera Rhodococcus, Arthrobacter or Brevibacterium. Strains were found to be non-reactive with API 50 CH panel substrates. By API Zym (Biomérieux), isolates were reactive for esterase and leucine arylamidase but not napthol-AS-DI phosphohydrolase. Biolog reactions (Biolog) were not performed on A. ignavus IMMIB L-1656 T [1] but were performed on Canadian and Swiss strains, described in detail in Table S1.MALDI–TOF MS (Bruker Daltronics) analysis was applied to these isolates but they were found to be unidentifiable, as Bruker Biotyper library versions 6903, 7311 or 7854 did not have an entry for A. ignavus. In-house mass spectral profiles (MSPs) were prepared for the Canadian and Swiss isolates using methods as described previously [6]. A. ignavus DSM 45359 T was then tested by MALDI–TOF, whereby scores greater than 2.1 were obtained only towards Canadian or Swiss MSPs but it had no significant scores (&lt;1.7) towards any taxon in Biotyper library version 7854. MALDI–TOF results indicated that a single taxon group, consistent with A. ignavus DSM 45359 T, existed and that this method could be used to unambiguously identify strains of this species.Antimicrobial susceptibility testing was done using broth microdilution as described previously [6], Trek Sensititre GPN3F and STP6F plates (Thermofisher), with methods, ranges and breakpoints as described in CLSI guidelines 100- S23 and CLSI M45-2 [7, 8] for Corynebacterium species and allied genera. MICs [µg ml−1 (interpreted)] of the four strains ranged from: penicillin [0.25–1 (susceptible)]; cefepime [1–4 (two susceptible; one intermediate; one resistant)]; cefotaxime [0.25–1 (susceptible)]; ceftriaxone [0.5–2 (three susceptible; one intermediate)]; meropenem [0.5–1 (susceptible)]; vancomycin [all ≤0.5 (susceptible)]; daptomycin [0.25–0.5 (susceptible)]; gentamicin [≤2 (two susceptible) or&gt;16 (two resistant)]; erythromycin [1 (one intermediate) to&gt;4 (three resistant)]; ciprofloxacin [all&gt;2 (resistant)]; tetracycline [≤1 (three strains susceptible) to 8 (one intermediate)]; clindamycin [all&gt;2 (resistant)]; trimethoprim/sulfamethoxazole [0.5/9.5–1/19 (susceptible)]; rifampin [≤0.5 (susceptible)], quinupristin/dalfopristin [0.5–1 (susceptible)] and linezolid [≤0.5–1 (susceptible)]. In contrast, Yassin et al. found that the type strain was susceptible to all antibiotics tested including penicillin and clindamycin using the disc diffusion method [1]. Interestingly, several of the cases linked to these isolates had been repeatedly treated topically with courses of ciprofloxacin, but strains here were all observed to have high MICs to that antibiotic using CLSI test conditions and broth microdilution. It was conjectured that detection of the nor B locus was possibly linked to the quinolone resistance observed here [2].Table 1. Assembly metrics and genome characterizationAccession number Number of contigs Mean coverage DNA G+C content (mol%) Assembly size (bp) # CDS N50 NML100628 QHLP00000000 40 90.8 59.48 2596136 2244 245104 NML120636 QHLO00000000 24 125 59.52 2472452 2113 139719 NML120779 QHLM00000000 172 126 59.42 2498540 2088 18511 NML130574 CP031746.1 1 243 59.48 2656163 2265 2656163 DSM 45359 T RDQR00000000 53 206 59.23 2589795 2266 92097Cellular fatty acid composition analyses (CFA) were done after growth on 5% Columbia blood agar base with 5% sheep blood at 35 °C in 5% CO 2 for 48 h, with analysis being done on the Sherlock system (MIDI) as described previously except that operating system 4.5 was used [9]. CFAs obtained after averaging data for the Canadian and Swiss isolates were qualitatively identical to those found for the A. ignavus type strain, with saturated branched-chain CFAs predominating (Table S2) [1].16S rRNA gene sequencing and analysis by BLAST was done as previously outlined [10]. Canadian sequences 100628, 120636 and 120779 were deposited in Genbank with the accession numbers KC669621, KC669622 and KC669623 respectively and the Swiss isolate with the accession number KF360241. Strains had 99.8–99.9 % similarity to each other but 97.5– 97.7% similarity to A. ignavus IMMIB L-1656 T (accession number FN554542). Standard 16S rRNA gene sequencing was applied to A. ignavus DSM 45359 T as a quality measure. Two attempts using the same protocol on two separate DNA extractions were done but an approximately 1450–1500 bp sequence could not be assembled de novo using SeqMan Pro version 11.2.1. In each instance, the first approximately 700bps and last approximately 200 bps of the gene were found to be essentially identical to A. ignavus IMMIB L-1656 T (accession number FN554542) but the middle approximately 600 bps could not be assembled into a full-length sequence. Sanger reads could not be aligned to the A. ignavus IMMIB L-1656 T (accession number FN554542), leaving a gap of approximately 600 bps between the aligned 5′ and 3′ ends of the 16S rRNA gene sequence.The Canadian isolates and A. ignavus DSM 45359 T were subjected to whole-genome sequencing using the Illumina MiSeq platform as outlined previously [6]. Coverage, genome assembly statistics and annotation data using Prokka (Galaxy version 1.13) [11] are summarized in Table 1. DNA extraction, sequencing, and assembly for NML 130574, resulting in a single scaffold, has been described separately [2]. Draft or complete genomes of A. ignavus DSM 45359 T, the Canadian and Swiss strains were deemed to be consistent with a single taxon group based on average nucleotide identity (ANI) by Blast+ [12, 13] (ANIb) values of 97.6–98.7% as calculated by JSpeciesWS [12] (Table 2). Digital DNA–DNA hybridization (dDDH) values of 86.2 to 92% to each other [14] as calculated using Formula 2 of the genome-to-genome distance calculator (GGDC) [13] (Table 2), which supported a single taxon grouping for the five strains. Genome drafts of these isolates ranged from 2.472 Mb to 2.596 Mb in size with DNA G+C contents of 59.4–59.5%. A. ignavus DSM 45359 T was found to be 2 589 795 bp in size with a DNA G+C content of 59.3% based on results from this study, which was consistent with the value of 59.7% as calculated using high-pressure liquid chromatography (HPLC) [1].A partial 16S rRNA gene sequence derived from the draft genome of A. ignavus DSM 45359 T was found on contig 389 (length 983 bp, coverage 430). That contig was blasted against the Genbank nucleotide database and was found to have 100% similarity to the three NML strains and 99.9% similarity to the Swiss isolate (785 out of 786 nucleotides). It also had 100% similarity to A. ignavus IMMIB L-1656 T 16S rRNA gene sequence (accession number FN554542), however only 750 to 785 bps lengths aligned. We could not identify a nearly full length 16S rRNA gene sequence in the genome assembly even after several attempts. In order to identify the missing portions of the 16S rRNA gene, all filtered contigs from the draft genome of A. ignavus DSM 45359 T were reblasted against A. ignavus IMMIB L-1656 T (accession no. FN554542) 16S gene sequence. Contig 389 aligned to the first 766 bp, and contig 39 (length 4554, coverage 606) aligned to bps 1208 to 1466. Two contigs were identified that matched the middle portion of the 16S gene: contig 896 (length 696, coverage 209) and contig 897 (length 696, coverage 294). These two contigs were blasted to each other and found to have 95% similarity to each other (665 out of 698 bp with 4 gaps), indicating that two different 16S rRNA gene sequences which differed over this region, existed in the genome. When blasted to the nucleotide database, contig 897 had 100% similarity to the NML and Swiss strains of members of the genus Auritidibacter. The closest match to contig 896 was to Brevibacterium ravenspurgense 26CB1 at 98% similarity. Neither contig matched the A. ignavus IMMIB L-1656 T (FN554542) 16S gene sequence. Therefore, a consensus 16S rRNA gene sequence for Auritidibacter ignavus DSM 45359 T was assembled by alignment of the four isolated contigs to the 16S sequence derived from the complete WGS sequence of NML 130574 [2] using SeqMan Pro. The final sequence contained 1491bp and had one bp difference with the 16S sequence derived from NML 130574. This consensus chimeric sequence produced by alignment to the WGS-derived 16S rRNA gene of NML 130574 has been deposited in Genbank with the accession number MK069432. The 16S rRNA gene chimeric sequence, accession number MK069432, created from the A. ignavus DSM 45359 T genome assembly, and 16S rRNA gene sequences derived from the Canadian and Swiss isolates were then compared with that from A. ignavus IMMIB L-1656 T, accession number FN554542 (=NR_117005). Alignment was done using Clustal W and the relationship was inferred using neighbour-joining, maximum-likelihood and maximum-parsimony algorithms found in MEGAX [15] after 1000 resampled datasets. All sequences, including that derived from DSM 45359 T, had 99.8–99.9% similarity to each other but had 97.5–97.7% similarity to A. ignavus IMMIB L-1656 T (NR_117005) (Fig. 1). Therefore, the sequence associated with the type strain of A. ignavus and deposited under the accession number FN554542 (=NR_117005) could not be readily reproduced here and was deemed inappropriate for use as a tool for identification. During this study, we also found that sequences for members of the genus Auritidibacter derived from blood culture isolates (accession numbers LR135781 and KP230542), deposited by laboratories in Marseille (France) and Madrid (Spain) respectively, also had&gt;99.7% similarity to DSM 45359 T (accession number MK069432) but had 97.6% similarity to A. ignavus IMMIB L-1656 T accession number FN554542. Therefore, this anomaly led at least four different laboratories located in four countries to deposit sequences for what each perceived to be novel Auritidibacter- like bacteria in Genbank over several years.Table 2. Comparative whole-genome sequence analysis results using GGDC for i s DDH values (upper right shaded area) and JSpeciesWS for ANIb values (lower left unshaded area)NML100628 NML120636 NML120779 NML130574 DSM 45359 T NML100628 * 87.3 86.3 86.2 86.4 NML120636 98.41 * 89.4 92.00 93.4 NML120779 97.59 97.74 * 88.5 88.7 NML130574 98.10 98.72 98.33 * 92.00 DSM 45359 T 98.15 98.91 98.56 98.84 *In summary, difficulties in reproducing 16S rRNA gene sequence with high degrees of similarity to that of A. ignavus IMMIB L-1656 T [accession number FN554542 (=NR_117005)] initially caused us to falsely infer that the Canadian and Swiss strains represented a novel species within the genus Auritidibacter. Re-testing A. ignavus DSM 45359 T, as well as additional clinical isolates, using WGS, MALDITOF MS, CFA analysis and biochemical analyses was critical here in clarifying biochemical and genomic features of this auditory tract pathogen. On the basis of the results presented in this report, we propose an emendation of the genus and species Auritidibacter ignavus.