Variation among human, veterinary and environmental Mycobacterium chelonae-abscessus complex isolates observed using core genome phylogenomic analysis, targeted gene comparison, and anti-microbial susceptibility patterns

Susan B. Fogelson; Alvin C. Camus; W. Walter Lorenz; Ravikiran Vasireddy; Sruthi Vasireddy; Terry Smith; Barbara A. Brown-Elliott; Richard J. Wallace Jr.; Nabeeh A. Hasan; Udo Reischl; Susan Sanchez

doi:10.1371/journal.pone.0214274

Abstract

Mycobacterium chelonae is a member of the Mycobacterium chelonae-abscessus complex and a cause of opportunistic disease in fish, reptiles, birds, and mammals including humans. Isolates in the complex are often difficult to identify and have differing antimicrobial susceptibilities. Thirty-one previously identified rapidly-growing, non-tuberculous Mycobacterium sp. isolates cultured from biofilms, fish, reptiles, mammals, including humans, and three ATCC reference strains were evaluated with nine M. chelonae-abscessus complex whole genome sequences from GenBank by phylogenomic analysis, targeted gene comparisons, and in-vitro antimicrobial susceptibility patterns to assess strain variation among isolates from different sources. Results revealed minimal genetic variation among the M. chelonae strains. However, the core genomic alignment and SNP pattern of the complete 16S rRNA sequence clearly separated the turtle type strain ATCC 35752^T from the clinical isolates and human reference strain “M. chelonae chemovar niacinogenes” ATCC 19237, providing evidence of two distinct subspecies. Concatenation of the partial rpoB (752 bp) and complete hsp65 (1,626 bp) sequence produced the same species/subspecies delineations as the core phylogeny. Partial rpoB and hsp65 sequences identified all the clinical isolates to the appropriate species level when respective cut-offs of 98% and 98.4% identity to the M. chelonae type strain ATCC 35752^T were employed. The human strain, ATCC19237, was the most representative strain for the evaluated human, veterinary, and environmental strains. Additionally, two isolates were identified as Mycobacterium saopaulense, its first identification in a non-fish or non-human host.

Citation: Fogelson SB, Camus AC, Lorenz WW, Vasireddy R, Vasireddy S, Smith T, et al. (2019) Variation among human, veterinary and environmental Mycobacterium chelonae-abscessus complex isolates observed using core genome phylogenomic analysis, targeted gene comparison, and anti-microbial susceptibility patterns. PLoS ONE 14(3): e0214274. https://doi.org/10.1371/journal.pone.0214274

Editor: Yoshihiko Hoshino, National Institute of Infectious Diseases, JAPAN

Received: July 18, 2018; Accepted: March 11, 2019; Published: March 25, 2019

Copyright: © 2019 Fogelson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: A total of 34 whole genome sequences produced from this study have been deposited in GenBank under Bioproject: PRJNA347845, Biosamples: SAMN05897971-SAMN05898003.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Mycobacterium chelonae is a nontuberculous mycobacteria (NTM) within the Mycobacterium chelonae-abscessus complex, which also includes the closely related Mycobacterium abscessus subspecies abscessus, Mycobacterium immunogenum, Mycobacterium salmoniphilum, Mycobacterium franklinii, and Mycobacterium saopaulense [1–4]. Individual members cause disease in fish, reptiles, birds, and mammals, including humans [5–7]. Due to their phenotypic, biochemical, and genetic similarity, species identification can be problematic.

M. chelonae-abscessus complex members have been identified in municipal water supplies, soil, and biofilms, and cases of mycobacteriosis have been linked to environmental sources [8–10]. Zoonotic disease is also a significant concern [11, 12]. Although considered an opportunistic pathogen, M. chelonae, is being increasingly reported in both healthy and immune deficient human patients [13, 14]. M. chelonae is similarly concerning to the veterinary community, especially in aquatic species such as fish. Susceptibility varies among families of fish, but a link has also been made between disease and immune system compromise [15–17]. Highly dependent on correct identification, treatment regimens for M. chelonae infections exist for human patients, while effective treatments for fish are largely non-existent.

Accurate identification of M. chelonae poses a challenge to human and veterinary diagnostic laboratories. Reliability has improved as identification methods have evolved from biochemical testing to molecular typing, restriction fragment length polymorphism analysis of hsp65 (hsp65 PRA), DNA strip assays, and matrix-assisted ionization time of flight mass spectrometry (MALDI-TOF) [18, 19]. However, ambiguity remains due to deficiencies in public databases, inconsistencies in restriction patterns for hsp65 PRA gel electrophoresis versus in-silico analysis, and a lack of consensus among laboratories regarding percent identity breakpoints used to differentiate closely related species [20].

In recent years, decreasing costs and increasing availability of molecular tools has enabled labs to investigate M. chelonae-abscessus complex isolates by whole genome sequencing (WGS) and target the most reliable genes for identification purposes [3, 10, 19]. While 16S rRNA gene sequencing is useful for identifying NTM isolates [21], partial 16S rRNA sequencing fails to separate M. chelonae and M. abscessus subsp. abscessus [22–24]. Other genes purported to differentiate closely related bacterial species include regions 3 and 5 of the β-subunit of the RNA polymerase gene (rpoB), the Telenti sequence of the 65 kDa heat shock protein gene (hsp65), DNA gyrase subunits A (gyr A) and B (gyr B), translation elongation factor Tu (EF-Tu), manganese dependent superoxide dismutase (Mn-SodA), Escherichia coli secretion gene (SecA), and the 16S-23S internal transcribed spacer region (ITS) [24–27]. However, the diagnostic utility of many of these genes has not been evaluated for the M. chelonae-abscessus complex. At present, diagnostic laboratories employ a combination of gene targets to identify closely related species. The Nocardia/Mycobacteria Research Laboratory (Tyler, TX) uses targeted sequencing of erm(41) and rpoB, but uncertainty remains for M. chelonae isolates, as breakpoints for rpoB have not been established [28]. Many laboratories simply identify isolates to the M. chelonae-abscessus complex level [29]. This poses a risk to patients, as antibiotic susceptibilities vary among members of the complex [28, 30, 31].

Reports describe M. chelonae infections in individual hosts and epizootics within the same species [32–34]. Yet, little is known regarding strain variability among different animal species and the environment. In this study, a One Health approach investigating the genetic variation among 31 rapidly-growing Mycobacterium sp. isolates from biofilms, humans, diseased animals, and three ATCC reference strains were compared following WGS and core genome extraction. Isolates were evaluated by core phylogenomic analysis, targeted gene sequence phylogenetic analysis, hsp65 PRA, in-silico dDNA-DNA hybridization, and antimicrobial minimum inhibitory concentration (MIC) determination. Results provide insight into strain variation between sources and the basis for a standard method for M. chelonae identification.

Materials and methods

Sample preparation

The analysis included 31 isolates previously identified as M. chelonae or Mycobacterium sp. from biofilms, fish, reptiles, and mammals, including humans, from the United States and Puerto Rico supplied by the Athens Veterinary Diagnostic Laboratory and the Mycobacteria/Nocardia Research Laboratory (MNRL), as well as three American Type Culture Collection (ATCC) reference strains (Table 1). Genomic DNA was extracted from Middlebrook 7H11 grown cultures using the UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories, Inc, Carlsbad, CA) following the manufacturer’s protocol. Approximately 15–28 ng/μL of DNA was submitted from each isolate to the Georgia Genomics Facility (The University of Georgia, Athens, GA) for DNA library preparation using Illumina TruSeq adaptors. Paired end (PE) 300-base reads were generated on an Illumina MiSeq PE300 sequencer (Illumina Inc., San Diego, CA).

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Table 1. Mycobacterium chelonae-abscessus sequenced isolates.

https://doi.org/10.1371/journal.pone.0214274.t001

Sequence preparation and assembly

Sequence read quality was assessed using FastQC [35]. Raw reads were trimmed using Trimmomatic software [36] run with the following settings: ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:20 TRAILING:10 SLIDINGWINDOW:4:20 MINLEN:50. Draft level genomes were assembled from trimmed reads using SPAdes software (version 3.6.2) [37]. Assembly metrics were evaluated using the Quality Assessment Tool for Genome Assemblies (QUAST) [38]. Automated genome annotation was performed using the RAST (Rapid Annotations using Subsystems Technology) server [39].

Core genome alignment and phylogenomic analysis

A pair-wise genome content distance matrix was produced for the WGS assemblies of the 31 samples, three reference strains, and nine sequences in GenBank: M. chelonae ATCC 35752^T (turtle), M. abscessus subspecies abscessus ATCC 19977^T (human), M. abscessus subsp. massiliense CCUG48898 (human), M. abscessus subsp. bolletii MC1518 (human), M. chelonae 1518 (human), M. franklinii DSM 45524^T (human), M. immunogenum CCUG 47286^T (drinking water), M. salmoniphilum ATCC 13758^T (chinook salmon), M. saopaulense EPM 10906^T using Progressive Mauve aligner [40]. Extraction of a core genome containing genes present in all 43 whole genomes was performed and the genes were concatenated using a custom perl script. Two outliers were identified and removed to perform core sequence analysis of the remaining 41 genomes. Phylogenomic analysis of a 3,204,105 bp core sequence, composed of 3,141 annotated regions, was performed to assess phylogenomic position using RAxML, employing GTR Gamma rapid bootstrapping and search for best scoring Maximum Likelihood model with 1000 bootstrap replications [41].

Sequence analyses and phylogenetic comparisons

All assembled and annotated genomes were imported into Geneious for in-silico targeted gene evaluation [42]. Keyword searches identified genes of interest whose DNA sequences were then extracted from the annotated genomes. For the partial rpoB (752 bp), partial hsp65 (441 bp), and partial ITS (245–257 bp), published primers were utilized in-silico [9, 18, 24, 43]. A multisequence nucleotide alignment for 16S rRNA (1,526 bp), rpoB (752 bp), hsp65 (1,626 bp), hsp65 (441 bp), gyrA (2,118 bp), gyrB (1,935–2,013 bp), EF-Tu (1,259 bp), Mn sodA (624 bp), recA (1,041 bp), ITS (245–257 bp), and erm(41) (673 bp) was performed and percent identity between sequences achieved using default settings in the MUSCLE program with a maximum of 10 iterations [44]. GenBank sequences for M. abscessus subsp. abscessus ATCC 19977^T, M. chelonae ATCC 35752^T, M. abscessus subsp massiliense CCUG 48898, M. franklinii DSM 45524 or D16R27, M. saopaulense EPM 10906, M. salmoniphilum ATCC 13758, and M. immunogenum CCUG 47286 were included for partial rpoB, partial hsp65, and ITS alignments when available.

The rpoB, hsp65 (441 bp), and 16S rRNA (1,526 bp) loci were further evaluated by multisequence alignment with 22 Mycobacterium sp. clinical isolates from Nogueira et al. [19]. Furthermore, 170 human sequences contributed by the MNRL were included in evaluation of the sequences for potential sequevars by evaluation of single nucleotide polymorphisms (SNPs) in the 752 bp sequence. The M. chelonae ATCC 35752^T reference strain was designated as sequevar 1 and subsequent sequevars were identified by SNPs in relation to it. These sequences were then translated for evaluation of amino acid discrepancies at loci of nucleotide difference.

RAxML (version 7.2.8) was used to estimate phylogenies and produce phylogenetic comparison matrices [41]. Phylogenetic trees were obtained from DNA sequences by GTR Gamma rapid bootstrapping and search for best scoring Maximum Likelihood model with 1000 bootstrap replications. In addition, concatenated sequences, partial hsp65 (441 bp) and rpoB, as well as the concatenated complete hsp65 (1,626 bp) and rpoB (752 bp) were evaluated as described above and compared to the core genomic phylogeny for evaluation of potential for diagnostic use.

Erm (41).

All isolates were evaluated for presence of erm(41) by generating a custom BLAST database for each individual assembly followed by BLASTn using the 673 bp erm(41) GenBank M. abscessus subsp. abscessus ATCC 19977^T NC 010397 as a query sequence [45].

hsp65 and PCR-restriction fragment length polymorphism analysis of hsp65 (hsp65 PRA).

Extraction of the partial hsp65 (441 bp) from the annotated genome assemblies was performed in-silico. Primers Tb11 and Tb12 [18] were used to identify and extract a 441 bp region of interest including flanking sequence. Primer sequences were included in the analysis as minor variation in primer binding areas of sequences did occur.

In-silico restriction length polymorphism analysis of the partial hsp65 sequence was performed targeting restriction sites for enzymes BstEII and HaeIII. A virtual gel was used to evaluate fragments larger than 35 bp. Using an algorithm similar to Taylor et al. [46], additional reference Mycobacterium species (M. abscessus subsp. bolletii MC 1518, M. abscessus subsp. massiliense CCUG 48898, M. franklinii DSM 45524, M. fortuitum CT6, M. immunogenum CCUG 47286, M. septicum DSM 44393, M. farcinogenes DSM 43637, M. salmoniphilum ATCC 13758, and M. saopaulense EPM 10906) were selected for comparison to other closely related species. Fragments were also compared to sequences in the database contained by http://app.chuv.ch/prasite.

dDNA-DNA Hybridization.

Whole genome assemblies of 31 samples, three reference strains, and seven GenBank sequences were submitted to the Genome to Genome distance calculator [47] using M. chelonae ATCC 35752^T and M. chelonae ATCC 19237 as reference isolates. Formula 2 (identities/HSP length) was used to calculate a digital DNA-DNA hybridization (dDDH) estimate using a GLM-based method.

Minimum inhibitory concentrations (MIC) and colony morphology.

Antimicrobial susceptibility testing was performed for 30 isolates harvested from Middlebrook 7H11 plates using a Sensititre RAPMYCO panel (Thermofisher Thermo Scientific, Oakwood Village, OH), following Clinical and Laboratory Standards Institute recommendations [48]. Clarithromycin was evaluated on days 3 and 14 of incubation. Sensititre RAPMYCO uses a standard-ordered broth microdilution panel for susceptibility testing and previously established breakpoints for rapidly growing mycobacteria (RGM) [49, 50]. In addition, colony morphologies were recorded.

GenBank accessions.

Accessions used: NC_010397 M. abscessus subsp. abscesssus ATCC 19977^T, CP010946 M. chelonae ATCC 35752^T, CP007220 M. chelonae CCUG 47445^T, GCA_000523895.1 M. chelonae MC 1518, NZ_HG964481 M. farcinogenes DSM 43637, NZ_CP011269 Mycobacterium fortuitum CT6, GCA_002013895.1 M. franklinii CV002 DSM 45524^T, AY550238 M. fuerthensis DSM 44567 (hsp65 partial), NZ_CP011530 M. immunogenum CCUG 47286^T, NZ_AP014547.1 M. abscessus subsp. massiliense CCUG 48898 ^T, CP009613.1 M. abscessus subsp. bolletii MC1518, NZ_HG322951 Mycobacterium septicum DSM 44393, GCA_002086715.1 M. saopaulense EPM10906 ^T, GCA_002013645.1 M. salmoniphilum ATCC 13758^T. Sequences from Noguiera et al. [19]: (hsp65) KT779818, KT779821-KT779824, KT779826-KT779827, KT779844, (rpoB) KT779876, KT779879-KT779882, and KT 779884-KT 779885, KT 779887-KT779902, (16S rRNA) MAEQ00000000 M. chelonae 96–1705, MAER00000000 M. chelonae 96–1717, MAES00000000 M. chelonae 96–1720, MAET00000000 M. chelonae 96–1724, MAEU00000000 M. chelonae 96–1728, MAEV00000000 M. sp. D16R24, MAEP00000000 M. franklinii D16R27, MAEW00000000 M. sp. D16Q13, MAEX00000000 M. sp. D16Q14, MAEY00000000 M. sp. D16Q16, MAFS00000000 M. franklinii D16Q19, MAEZ00000000 M. sp. D16Q20, MAFA00000000 M. chelonae D16Q24, MAFB00000000 M. sp. D17A2, MAFC00000000 M. sp. D16R12, MAFD00000000 M. sp. D16R18, MAFE00000000 M. salmoniphilum D16Q15, MAFF00000000 M. chelonae D16R2, MAFG00000000 M. chelonae D16R3, MAFH00000000 M. chelonae D16R7, MAFI00000000 M. chelonae D16R9, MAFJ00000000 M. chelonae D16R10, MAFK00000000 M. chelonae D16R14, MAFL00000000 M. chelonae D16R19, MAFM00000000 M. chelonae D16R20, MAFN00000000 M. sp. 96–892. Thirty-four whole genome sequences from this study have been deposited in GenBank under Bioproject: PRJNA347845, Biosamples: SAMN05897971-SAMN05898003.

Results

Core genomic analysis

Phylogenetic comparison of isolates using core genes observed in all genomes separated and identified species within the M. chelonae-abscessus complex, as well as two outliers, seahorse1 and pipefish. The outliers were 99.4% identical to each other, but the closest reference strain, M. chelonae ATCC 35752^T, shared only 75.1% identity. BLASTn searches of the NCBI database placed the two closest to NZ_CP011269.1 Mycobacterium fortuitum strain CT6 and CP009914.1 Mycobacterium sp. VKM Ac-1817D, with only 88% identity and were removed from further analysis. The core genomes of the remaining 41 strains produced a 3,204,105 bp in length sequence with 3,141 coding sequences (CDS). Of the CDS, 2,367 were confirmed by RAST as genes, 683 were hypothetical protein CDS, and the remaining 91 were probable CDS. Within the core CDS, 16S rRNA, rpoB, hsp65 partial, hsp65 whole, gyrA, EF-Tu, Mn-sodA, and recA were present. The sequenced reference strain, M. chelonae ATCC 35752^T, was100% identical to the GenBank strains M. chelonae ATCC 35752^T. There was 100% identity between the reference strain M. abscessus subsp. abscessus ATCC 19977^T, GenBank sequences M. abscessus subsp. abscessus ATCC 19977^T, M. abscessus subsp. bolletii MC 1518, and M. chelonae 1518, demonstrating the presence of improper sequence designations in GenBank. Since the GenBank M. chelonae ATCC 35752^T and M. abscessus subsp. abscessus ATCC 19977^T downloaded sequences were identical to the sequenced isolates, hereafter, M. chelonae ATCC 35752^T and M. abscessus subsp. abscessus ATCC 19977^T will represent the sequenced and downloaded sequences for each strain.

Twenty-nine strains grouped closely with M. chelonae ATCC 35752^T using the core genomic comparison. However, four isolates were determined to be members of the M. chelonae-abscessus complex, but not M. chelonae. These isolates included seakrait, cow, turtle, and H9 (Fig 1).

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Fig 1. Phylogenomic comparison of Mycobacterium chelonae-abscessus isolates.

Phylogenomic comparison of 32 Mycobacterium chelonae-abscessus. sequences relative to nine GenBank genome sequences using a core genome from all 41 sequences. Phylogeny was produced using the best scoring Maximum Likelihood model with 1000 bootstrap replications. Dotted box delineates M. chelonae clinical isolates clustered with “M. chelonae chemovar niacinogenes” ATCC 19237 and breakdown into 4 subclusters. Scale bar represents average number of nucleotide substitutions per site. 0.004 represents approximately 13,000 nucleotides that are not identical. ^T Denotes Type strain * Denotes sequence used from GenBank. ^# Denotes ATCC isolate sequenced in study.

https://doi.org/10.1371/journal.pone.0214274.g001

Twenty-five of the 31 clinical isolates clustered with the sequenced M. chelonae ATCC 19237 with 98.4–99.6% identity (Fig 1). A mixture of human, fish, reptile, and biofilm isolates clustered in this large group, all with greater than 98.1% identity to each other. The current type strain M. chelonae ATCC 35752^T branched separately, with no greater than 96.5% identity to the 25 M. chelonae isolates. Minimal genetic variation was present within the isolates, although four distinct subclusters were present.

Targeted gene analysis

Gene targets evaluated by multisequence alignment produced an identity matrix for comparison of sequences. Alignments of 16S rRNA, gyrA, gyrB, EF-Tu, recA, and Mn-sodA produced erroneous clustering or separation of the isolates and/or reference strains evidenced by inaccurate phylogenetic placement of the human isolates (EF-Tu, Mn-sodA, gyrA, gyrB) or lack of species separation (16S rRNA, recA) when compared to the core genomic results. Evaluation based on these alignments was not pursued further. However, the sequences for the clinical isolates and ATCC 19237 had at least three single nucleotide polymorphisms in the complete 16S rRNA sequence that distinctly separated them from the type strain ATCC 35752^T (S1 Fig and S1 Table). Furthermore, inclusion of 13 M. chelonae and 9 M. sp. isolates from Germany and Belgium revealed higher similarity to”M. chelonae chemovar niacinogenes” ATCC 19237 and M. salmoniphilum ATCC 13758^T, respectively (S2 Fig).

ITS.

A 257 bp ITS sequence was extracted for the M. chelonae-abscessus isolates. However, different ITS extraction product lengths were observed for isolate H9, M salmoniphilum ATCC 13758 (256 bp), M immunogenum CCUG 47286 (267 bp), and pipefish and seahorse1 (245 bp). Multi-sequence alignment of the clinical isolates and reference strains revealed adequate grouping into species-specific branches, but the high percent identity (99.1%) between H9 and the cow and turtle strains did not provide an accurate separation of the identities of the three isolates. For this study, isolates with greater than 98.8% (254/257bp) identity at the ITS locus to M. chelonae ATCC 35752^T were considered M. chelonae (S3 Fig).

hsp65.

Targeted extraction of the 441bp partial hsp65 gene sequence reproduced the main M. chelonae ATCC 35752^T clusters generated by core genome analysis (S4 Fig). Isolates with greater than 98.4% identity (434-441/441 bp) to M. chelonae ATCC 35752^T were considered M. chelonae. Although minimal sequence diversity is present at this locus (0–7 bp difference), two large sub-clusters, each containing strains 99.8–100% identical to each other are present. One sub-cluster contained exclusively human isolates (H7, H10, H11, H15, H18, H19, H20) and the other a mixture of M. chelonae ATCC 19237, human (H8, H12, H13, H14), fish (cichlid, trumpetfish, seadragon1, seadragon2, seahorse2, seahorse3, seahorse4, seahorse5), and biofilm (biofilm1, bioflm2, biofilm3) isolates. The partial hsp65 sequence of human isolate H9 was 98.4% identical (434/441 bp) to M. franklinii DSM45524. The turtle and cow isolates also branched separately from the M. chelonae cluster and were 99.5% identical (439/441bp) to M. saopaulense EPM 10906. Inclusion of M. chelonae and M. sp. isolates from Nogueira et al. [19] showed a similar distribution where human M. chelonae isolates clustered together with 100% similarity to a mixture of environmental isolates, veterinary isolates, and “M. chelonae chemovar niacinogenes” ATCC 19237.

The complete 1,626 bp hsp65 multisequence alignment was more discriminating than the partial sequence and produced some clusters mirroring the core genome phylogeny (S5 Fig). All isolates with greater than 95.3% identity (1,550/1,626 bp) to M. chelonae ATCC 35752^T at the complete hsp65 were considered M. chelonae. As with the core genome and partial hsp65 phylogenies, the same group of human isolates branched together (H7, H10, H11, H15, H18, H19, H20) and shared 99.9–100% (1,625–1,626/1,626 bp) identity, but all M. chelonae isolates were greater than 99.1% identical to each other, showing minimal genetic variation in the group at this locus.

rpoB

Phylogenetic analysis of rpoB (752 bp) produced similar phylogenetic positioning as the core genome (S6 Fig). Isolates with identities greater than 97.9% identity (736/752 bp) to M. chelonae ATCC 35752^T were considered as M. chelonae. The largest grouping consisted of multiple fish, biofilm, water, and human isolates, all of which had 99.9–100% identity to each other and contained ATCC 19237, but not ATCC 35752^T.

One hundred and seventy rpoB sequences from the MNRL were evaluated with the 31 clinical isolates for SNPs, which ranged from zero in M. chelonae ATCC 35752^T up to 5 in some clinical isolates. Seventeen sequevars were recognized based on SNPs consistently identified at positions 24 (A-to-G), 36 (C-to-G), 90 (C-to-T), 99 (C-to-T), 100 (C-to-T), 102 (C-to-G), 123 (C-to-T), 126 (C-to-A), 204 (G-to-A), 237 (T-to-C), 363 (T-to-C), 384 (C-to-T), 385 (C-to-T), 430 (G-to-A), 444 (G-to-A), 480 (C-to-G), 559 (C-to-T), 654 (C-to-A), and 723 (G-to-T). However, sequence translations revealed only one amino acid change in a single human isolate from the sequence database, where a G-to-A substitution at codon 430 resulted in a glutamic acid substitution for lysine. Multisequence alignment of the additional rpoB sequences showed greater than 99.2% identity to M. chelonae ATCC 35752^T.

hsp65 whole sequence and rpoB

Concatenation of partial hsp65 (441 bp) and rpoB (752 bp) sequences produced a 1,193 bp sequence. The phylogenetic positioning of several isolates was not consistent with that of the core genome and no further analysis was performed. A concatenation of the complete hsp65 (1,626 bp) and partial rpoB (752 bp) created a 2,378 bp sequence (S7 Fig). Clustering of clinical isolates was almost identical to the core genome phylogeny. However, unlike the core phylogeny, M. chelonae ATCC 35752^T branched at a different location. Isolates with greater than 96.1% (2,285/2,387 bp) identity to M. chelonae ATCC 35752^T were considered M. chelonae.

16S rRNA, rpoB, and partial hsp65

Concatenation of 16S rRNA (1,521–1,526 bp), rpoB (752 bp), and partial hsp65 (441 bp) sequences from the present study and the Nogueira et al. [19] isolates revealed similar phylogenetic positioning to the core genome (S8 Fig). Human, veterinary, and environmental M. chelonae isolates grouped together with more than 97.2% similarity. However, M. chelonae ATCC 35752 and M. chelonae ATCC 19237 are 99.7% identical and grouped differently than the core phylogeny.

Erm (41)

The erm (41) gene was only observed in GenBank reference strains M. abscessus subsp. abscessus ATCC 19977^T, M. chelonae 1518, M. abscessus subsp. bolletii strain MC1518, and the seakrait isolate. All other clinical isolates and reference strains lacked this genetic sequence.

Restriction fragment length polymorphism analysis (hsp65 PRA). The partial 441 bp hsp65 sequences were evaluated to produce two-step BstEII and HaeIII in-silico digestion reference patterns to compare the accuracy of identification in relation to the core genome phylogeny (S9 Fig) using fragments over 60 bp. In addition, fragments over 35 bp were also evaluated for pattern of fragmentation. BstEII produced three groups, each with 2–4 fragments: 310/131 bp, 231/210 bp, and 231/116/84 bp. If these groupings are followed, M. franklinii, isolate H9 and M. salmoniphilum are considered within the grouping for M. chelonae. HaeIII did not separate M. salmoniphilum from M. chelonae ATCC 35752^T unless fragments under 35 bp were considered. Additionally, human isolates H7, H10, H11, H15, H18, H19, and H20 were separated from other M. chelonae isolates. The patterns between these groups differ at 60 bp and under. The pattern for the M. chelonae 1518 GenBank sequence was the same as M. abscessus subsp. abscessus ATCC 19977^T.

dDNA-DNA Hybridization.

DNA-DNA relatedness for M. chelonae-abscessus members and clinical isolates were tested using M. chelonae ATCC 35752^T and “M. chelonae chemovar niacinogenes” ATCC 19237 as a reference (S2 Table). As expected, all M. chelonae isolates had a higher percent relatedness to M. chelonae ATCC 19237, ranging from 77.8% (CI 74.9–80.6%) to 95.7% (CI 94.2–96.8%), than to M. chelonae ATCC 35752^T, which ranged from 63.3% (CI 60.4–66.1%) to 66.3% (CI 63.4–69.2%).

MIC susceptibility and colony morphology

Twenty-seven non-genetically identical clinical isolates and three ATCC strains were evaluated using the Sensititre RAPMYCO panel (S3 Table). Subtle phenotypic differences in colony morphologies were observed when isolates were viewed simultaneously. The majority (22/30) were nonpigmented, smooth, glossy, and raised. The cow and turtle isolates produced similar colonies, but turned the 7H11 media brown after 7 days. The pipefish and seahorse1 outliers grew as nonpigmented, granular, glossy, raised, colonies, different from all others. Isolates H12, H13, H17, seahorse5 and python1 produced nonpigmented, rough, crusty, raised colonies.

MICs of the NTM isolates were classified as susceptible, intermediate, or resistant. A high degree of antimicrobial resistance was observed among all isolates, but the greatest resistance was found in the aquatic biofilm and fish isolates. However, 93% (28/30) were susceptible to the macrolide clarithromycin (S3 Table). Only M. abscessus subsp. abscessus ATCC 19977^T and isolate H10 were resistant to clarithromycin after 14 days. For the M. chelonae isolates, 70% (21/30 isolates) and 60% (18/30 isolates) were susceptible to the aminoglycosides tobramycin and amikacin, respectively. Only 50% of the M. chelonae isolates were susceptible to linezolid, the majority of which were of human origin (n = 9). Susceptibilities of M. chelonae were low for cefoxitin, trimethoprim/sulfamethoxide, imipenem, moxifloxicin, and ciprofloxacin at 3%, 10%, 3%, 13% and 20% (1/30, 3/30, 1/30, 4/30, 6/30), respectively. The human ATCC 19237 had a more resistant antimicrobial pattern than ATCC 35752^T. The “M. chelonae chemovar niacinogenes” ATCC 19237 strain had a pattern more like the fish (cichlid, seahorse2, seahorse3, seahorse4, seahorse5, seadragon1), human (H10, H11, H12, H14, H17, H19, H20), and biofilm (biofilm1, biofilm2, biofilm3) isolates than ATCC 35752^T.

Discussion

Disease caused by members of the M. chelonae-abscessus complex in healthy and immunocompromised humans is increasing [14, 51–53]. M. chelonae infections are common in aquatic species and cause significant losses in certain groups of fish, particularly syngnathids (seahorses, seadragons and pipefish) [15, 54, 55]. Since M. chelonae-abscessus complex organisms are a human and veterinary health concern, characterization and appropriate identification methods are key to understanding the delicate balance of NTM interactions among humans, veterinary species, and the environment for disease control. Whole genome sequencing and core genome analysis was used to characterize NTM from fish, reptiles, mammals, and aquatic biofilms to investigate their genetic variation. High sequence homology was observed across M. chelonae isolates. Genetically similar strains infected a range of hosts and existed within environmental samples. A correlation between the environmental presence of M. chelonae and human disease has been established [56]. Similar strain characteristics and the low genetic variability of M. chelonae isolates from fish and biofilms suggests an environmental source of infection, a theory supported by a study of diseased pompano Trachinotus carolinus [12].

Certain human isolates tended to cluster using the different gene targeted sequencing methods, while others were more genetically similar to the aquatic animal or biofilm isolates. The consistent clustering of isolates H7, H10, H11, H15, H18-H20, suggests an epidemiologic link, although they share no known geographic or environmental associations. Human isolates H12, H13, H14, and H16 were genetically similar to fish and biofilm isolates, and to human “M. chelonae chemovar niacnogenes” ATCC 19237. It is reasonable to speculate that they may have originated from aquatic sources [57–59].

Core genomic comparison accurately identified closely related species in the M. chelonae-abscessus complex, as well as two divergent outliers (pipefish and seahorse1) cultured from syngnathid fish. Additional targeted gene sequencing, dDDH, and PRA analysis (S2 Table and S7 Fig) established the two outliers as a novel species, Mycobacterium syngnathidarum [60]. Core genome analysis of the remaining 41 whole mycobacterial genomes separated the human “M. chelonae chemovar niacinogenes” ATCC 19237 and turtle M. chelonae type strain ATCC 35752^T into subgroups. Clinical isolate sequences were more similar to ATCC 19237 (98.4–99.6%) than to ATCC 35752^T (96.5–96.6% identity). Adékambi et al. found similar results when comparing human clinical isolates with ATCC 19237 and ATCC 35752 ^T [61]. M. chelonae ATCC 35752^T also had a slightly different antimicrobial sensitivity profile than ATCC 19237 and the other M. chelonae isolates (S3 Table). Likewise, dDDH showed a difference in relatedness between the clinical isolates and M. chelonae ATCC 35752^T. The genomic and antimicrobial data support recognition of two M. chelonae subspecies and indicate that use of M. chelonae ATCC 35752^T as a type strain may not be optimal for phylogenetic studies of M. chelonae isolates.

Core genome comparison revealed that earlier identification methods lacked fidelity for identification of M. chelonae isolates. Power of the core comparisons was high, because over half of the bacterial genome consisting of 4,898,027 bp and 4,489 CDS for M. chelonae ATCC 35752^T [62], was used for analysis. In the core alignment, 65.4% of the genome and 70% of the conserved coding regions were analyzed, including common housekeeping genes that are employed independently for species identification, such as EF-Tu, SecA, gyrA, Mn-SodA, 16S rRNA, rpoB, and hsp65. As a result, two human mycobacterial sequences in GenBank previously identified as M. chelonae 1518 and M. abscessus subsp. abscessus MC 1518 were found to be incorrect. The core alignment and presence of erm (41) delineate the sequences as M. abscessus subsp. abscessus ATCC 19977^T. Isolates originally identified by hsp65 or phenotyping as M. chelonae and Mycobacterium sp. (seahorse5, cow, turtle, and seakrait) were more precisely identified as M. chelonae, M. saopaulense and M. abscessus subsp. abscessus.

Similar to other published studies, WGS provided the greatest discrimination of M. chelonae-abscessus complex isolates, but is not yet practical in diagnostic settings where multilocus sequence analysis offers a practical alternative [10, 19, 63]. Comparison of commonly targeted genes to the core genome indicated that concatenated complete hsp65 and partial rpoB sequences were diagnostically useful. Isolates with identities greater than 98.4% to turtle reference strain M. chelonae ATCC 35752^T were considered M. chelonae. While promising for species identification, there is no published data to support the proposed threshold and a larger sample size is needed to validate the method. Using the concatenated complete hsp65 and partial rpoB sequences, the turtle type strain M. chelonae ATCC 35752^T and human reference strain M. chelonae ATCC 19237 both had greater than 99.1% identity to the main M. chelonae group of isolates, making differentiation between the potential subspecies difficult.

As previously reported, 16S rRNA analysis did not adequately differentiate species in the M. chelonae-abscessus complex [22] (S1 and S2 Figs and S1 Table). However, similar to that stated by Ballard et al. [64], SNPs patterns of the tested isolates designated M. chelonae were the same as ATCC 19237, not the turtle type strain ATCC 35752^T (three nucleotides different), further supporting the two as subspecies of M. chelonae. The genes gyrA, gyrB, EF-Tu, RecA, and Mn-Sod did not reliably identify species or produced inaccurate phylogenetic positioning, while the ITS, partial and complete hsp65, and rpoB loci were the most discriminating and identified isolates similarly to the core genomic analysis (S3, S4, S5 and S6 Figs). Partial hsp65, complete hsp65, and rpoB sequences identified the cow and turtle isolates as M. saopaulense, while rpoB and partial hsp65 delineated H9 as M. franklinii. However, contradictory to the core genome analysis, hsp65 (partial and complete), and the rpoB phylogenies, the ITS sequences of M. salmoniphilum ATCC 13758^T and H9 (M. franklinii) were 98.1% identical, which may not differentiate the species.

Regardless of phylogenetic differences produced by hsp65 (partial and complete), partial rpoB, and the core genome, these methods can identify M. chelonae and closely related species when specified breakpoints are employed[19]. With other bacterial genera this is widely done for the16S rRNA locus where a 98.7% identity is applied as a cut-off level [65]. Breakpoints of 98.4% for partial hsp65 (441 bp), 95.4% for complete hsp65, and 97.9% for rpoB or greater will identify M. chelonae when compared to the turtle type strain M. chelonae ATCC 35752^T. Furthermore, inclusion of M. chelonae isolates from Germany and Belgium to the partial hsp65 and rpoB analyses provides additional support for these breakpoints and the representative nature of ATCC 19237 to the current clinical isolates being evaluated worldwide, potentially making it a better candidate for comparison and identification purposes. Although a breakpoint was found for hsp65, additional partial and complete sequences are needed to confirm their validity.

Examination of a 170 sequence dataset provided by the Mycobacteria/Nocardia Research Laboratory confirmed the 97.9% rpoB breakpoint differentiates M. chelonae from other closely related species, but does not agree with Adékambi et al., which found intraspecies homology was 98.3–100% for the partial rpoB [24, 61, 66]. This discrepancy may be the result of comparison to M. fortuitum rather than M. chelonae strains in the earlier study. Further evaluation of SNPs from the rpoB sequences separated isolates into sequevars. Translation of the sequences confirmed that gene function was likely not affected, as amino acid sequences were unchanged in all but one sequence. Identifying rpoB sequevars may be useful for epidemiologic tracking of outbreaks, but no such connection could be made from the data set.

Replacement of PRA by targeted gene sequencing is supported by findings in this study. Comparisons of the partial hsp65 PRA algorithm of Telenti et al. [18] and revised by Taylor et al. [46] and Chimera et al. [67] using in-silico digested fragments confirms the inability of PRA to differentiate species closely related to M. chelonae, likely a result of the greater discriminating power of “in-silico” analysis (1 bp) versus human interpretation of agarose gels (up to 10 bp). The fragments produced were 9–15 bp different than those derived using previously reported algorithms. For example, the PRA pattern for M. chelonae is 320/130 bp for BstEII and 200/60/55 bp for HaeIII, compared to the “in-silico” restriction pattern of 310/131 bp and 197/60/58/54 bp, respectively [46]. PRA analysis should not be used to identify mycobacteria in the M. chelonae-abscessus complex without revision of the algorithm to accommodate in-silico fragment sizes and fragments less than 60 bp in length, which were not assessed in the earlier studies that used traditional methods.

Susceptibility patterns, including significant antimicrobial resistance, have been reported for Mycobacterium chelonae-abscessus isolates and a multitude of acquired resistance mechanisms exist [31, 68–70]. One such example is the MspA gene, which, when expressed, has shown differential resistance of M. chelonae 9917 and M. chelonae ATCC 35752^T to rifampin (rifampicin), vancomycin, ciprofloxacin, clarithromycin, erythromycin, linezolid, and tetracycline. Investigation into specific resistance genes was not pursued for this study; however, the observed variable resistance to amikacin, ciprofloxacin, moxifloxacin, trimethoprim/sulfamethoxide, imipenem, cefoxitin, and linezolid among genetically similar isolates suggests differential expression of regulatory genes. The evaluated clinical isolates exhibited multidrug resistance, but biofilm isolates had the broadest resistance patterns [30, 49, 69]. Regardless of their origin, 96% of M. chelonae strains were susceptible to clarithromycin. Isolate H10 was resistant to clarithromycin and a gene mutation associated with resistance is suspected.

The erm (41) sequence in strains M. abscessus subsp. abscessus and M. abscessus subsp. bolleti MC 1518^T, but not M. chelonae, can indicate inducible macrolide resistance [45, 71]. The presence of erm (41) in isolates originally identified as M. chelonae (M. chelonae 1518, M. abscessus subsp. bolletii MC1518, and seakrait), support their identification as M. abscessus subsp. abscessus by complete genome sequencing. Although erm (41) in a bacterial genome does not necessarily convey macrolide resistance, sensitivity to macrolides could serve as an aide in the identification of M. chelonae-abscessus complex species.

Colony morphology and phenotypic traits can aid conventional and molecular diagnostics [72, 73], but as demonstrated here, rarely provide sufficient evidence for definitive identification. Most isolates produced similar raised nonpigmented colonies that were smooth to dry and flaky, and virtually impossible to distinguish without side by side observation. Exceptions were the novel pipefish and seahorse1 isolates, which produced granular rough colonies, and M. saopaulense, which turned agar brown after several days of incubation [2]. This morphologic variance supported identification of the turtle and cow isolates as M. saopaulense, not M. chelonae as originally determined.

This whole genome evaluation of environmental, non-mammalian, and mammalian M. chelonae-abscessus isolates provides insight into the diversity of isolates within the complex and similarity of M. chelonae isolates. Identification of isolate similarity throughout different sources supports the necessity to understand the intricate relationship and interactions of the bacteria with humans, animals, and the environment. Especially because the high sequence homology among isolates from different geographic locations and host origin suggest an epidemiologic link. Core genome, dDDH, and 16S rRNA sequences indicate that M. chelonae is not a homogeneous species and that the current turtle type strain ATCC 35752 ^T and human ATCC 19237 represent two M. chelonae subspecies. Core genome comparison was the most discriminatory method for species identification, but concatenation of the complete hsp65 and partial rpoB genes produced similar results and could be used for identification purposes.

Supporting information

S1 Fig. Phylogenetic comparison of Mycobacterium chelonae-abscessus isolates by 16S rRNA analysis.

Phylogenetic comparison of Mycobacterium chelonae-abscessus complex isolates relative to eight GenBank sequences using the 16S rRNA 1,522 bp locus and two M. syngnathidarum outliers as an outgroup. Phylogeny was produced using the best scoring Maximum Likelihood model with 1000 bootstrap replications. Scale bar represents average number of nucleotide substitutions per site. 0.002 represents 2–3 nucleotides which are not identical.

T Denotes Type strain.