Abstract
Organisms within the Mycobacterium chelonae-abscessus complex (MCAC) are characterized as rapidly growing, nontuberculosis mycobacteria (NTM). Members of the MCAC complex (Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium immunogenum, Mycobacterium salmoniphilum, Mycobacterium franklinii, and Mycobacterium saopaulense) are ubiquitous in the environment.1,2 Several of these species can cause primary fatal disease in fishes and opportunistic disease in humans.3,4 In addition to a wide host range, environmental isolates can also form disinfectant resistant biofilms in a wide variety of aquatic environments.5 To complicate matters, MCAC isolates are difficult to differentiate by routine diagnostic methods, have differing antimicrobial susceptibilities, and successful treatment protocols are limited to non-existent in aquatic species.6,7 At present, diagnostic laboratories use targeted gene sequencing of rpoB and restriction analysis of the 441 bp fragment of hsp65 to delineate NTM species.6,8 However, classification of MCAC isolates remains unreliable because rpoB may improperly phylogenetically classify isolates and hsp65 does not have a well-defined cut-off point for species designation.
Piscine mycobacteriosis causes significant mortalities in both captive and wild settings and has the potential for zoonotic transmission to humans. Of the numerous species of fishes held in captivity, fishes of the family Syngnathidae (seadragons, seahorses, and pipefish) appear to be uniquely susceptible to mycobacteriosis.9 Piscine mycobacterial isolates are rarely evaluated for antimicrobial susceptibility; however, molecular identification is often pursued. Misidentification of closely related bacterial species can lead to inappropriate antimicrobial treatment.
We hypothesize that analysis of core genomes of MCAC isolates from clinically ill syngnathids (n = 9), fish (n = 1), aquatic biofilms (n = 3), aquatic reptiles (n = 2), and 2 reference strains (ATCC 19977 M. abscessus and ATCC 35752 M. chelonae) will effectively elucidate the accurate phylogenetic position of the isolates and that in vitro antimicrobial susceptibilities will positively correlate with the produced phylogenies. Whole genome sequencing (WGS) was performed on 16 aquatic isolates, previously identified as M. chelonae by hsp65 or 16S sequencing. All genomes underwent assembly, annotation, and multisequence alignment of the extracted core genome. In addition, 14 of the isolates were evaluated for colony morphology variation then tested for in vitro susceptibilities to 15 antibiotics. Phylogenomic analysis revealed 2 syngnathid isolates with less than 92.8% similarity to any species recorded in GenBank, suggesting the presence of an undescribed species. All syngnathid and biofilm core genomes closely grouped with greater than 99.0% identity, but were only 96.4–96.5% similar to the M. chelonae reference strain. One biofilm isolate and 2 syngnathid isolates clustered together with 99.9–100% identity and the same antimicrobial profile. One aquatic reptile isolate had 99.5% identity to ATCC 19977 M. abscessus and the same antimicrobial susceptibility pattern.10 The remaining syngnathid and biofilm isolates clustered together with greater than 99.7% identity and had a susceptibility pattern consistent with M. chelonae.10 All isolates were resistant to trimethoprim/sulfamethoxide. One seadragon isolate and 3 biofilm isolates were resistant to linezolid. We conclude the core genomic phylogenomic position of MCAC isolates positively correlated with antimicrobial susceptibilities. Results of this study will assist diagnosticians and clinicians to accurately identify MCAC isolates and provide treatment recommendations for clinically ill fish patients.
Acknowledgements
The authors would like to thank Ashley Phillips of the Athens Veterinary Diagnostic Laboratory at the University of Georgia College of Veterinary Medicine and Dr. Walt Lorenz of Quantitative Biology Consulting Group at the University of Georgia for their technical support, Dr. Emily Christiansen at the North Carolina Aquarium, Dr. Shane Boylan at the South Carolina Aquarium, and the Georgia Aquarium staff for assistance with sample collection, and Dr. Tom Waltzek for genome sequencing support.
* Presenting author
+ Student presenter
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