Discussion 

This study comprehensively analysed the genomes of serveral Mycobacterium strains. The presence of this genus had been previously reported years ago in Chile (Aro et al. 2014). Here, we expand a previous the report of mycobacteriosis in Chile, characterising the whole-genome of 4 isolates derived from salmon freshwater aquaculture facilities. This new description includes whole-genome analyses of four isolated bacterial strains, three of which (myc161, myc182 and myc162) were from S. salar, and one of which (myc151) was from O. kisutch. All four were isolated from freshwater systems, which suggests that these Mycobacterium strains specifically affect the freshwater stage of salmonid farming.

 First, macroscopically, the fish displayed biological characteristics consistent with previous descriptions of infections caused by members of the Mycobacterium genus(Aro et al. 2014; Bruno et al. 1998; Brocklebank et al. 2003; Keller et al. 2018; Luo et al. 2018; Parikka et al. 2012). Second, our bacteriological findings and 16S rRNA PCR results confirmed that the isolates belong to the Mycobacterium clade. Whole-genome sequencing confirmed that three of the isolates: myc161, myc182, and myc162, belonged to the Msal species, while the isolate myc151 was closely related to the new Mfra/Msal-like phylogenetic branch previously described by Behra et al. (2019), wherein the authors identified significant genomic differences between the strains. As such, these Msal-like strains belong to a single species according to their ANI score.

In accordance with previously reported data, we observed that the Mfra strains displayed patterns that were similar, but not identical, to those of the Msallike strains in terms of their 16S rRNA, wholegenome, and core- and pangenome sequences as the Msal-like strains. This supports the existence of an independent Mfra/Msal-like species closely related to Mfra. Interestingly, only the strain isolated from coho salmon was identified as belonging to the Mfra/Msallike species, whereas those from the Atlantic salmon were identified as Msal (Figs. 3, 4 and Supplementary Fig. 2).

The bacterium Mfra was first classified as a member of the Mche-Mabs complex (MCAC) by Simmon et al. (2011), who isolated it from patients with underlying lung conditions. Although the exact source of the disease-causing pathogen is unknown, another study (Van Ingen et al. 2010) has detected Mfra in municipal water sources, suggesting an environmental origin that may be region-specific. Collectively, these studies suggest that terrestrial Mfra/Msal-like infections may directly originate from nearby water sources. Hence, this is the first study to report a newly proposed species, which potentially cause mycobacteriosis in salmonids, belonging to the Mfra/Msal-like cluster from a Chilean freshwater breeding system, highlighting the need to monitor these mycobacteriosis-related diseases in aquaculture settings. 

On the other hand, Msal has been previously reported to cause mycobacteriosis outbreaks in S. salar and other fish species (Whipps et al. 2007; Zerihun et al. 2011b, a; Austin and Austin 2016). However, no evidence has thus far suggested that Msal causes infections in terrestrial animals. Interestingly, the mycobacteriosis described here manifested differently in coho and Atlantic salmon species. Mycobacteriosis in S. salar was characterized by internal granulomas, whereas O. kisutch mainly exhibited chronic peritonitis (Fig. 1). Nevertheless, we cannot eliminate the possibility that different Mycobacterium species cause distinct disease manifestations.

Recent advances in sequencing technology facilitated the identification of several Mycobacterium species and subspecies based on phylogenetic algorithms from multiple genomic databases, thus generating more accurate and robust phylogenetic classification. One such example is Das et al. (2018), who analysed the genome sequences of 19 Mma strains using comparative genomics, ANI analysis, and phylogenetic trees to cluster the strains into two distinct branches. Similarly, Behra et al. (2019) performed several comparative genomic analyses and concluded that Msal-like isolates and Mfra isolates are similar enough to constitute a single species. 

Recently, a study performed on isolates recovered from gilthead seabream identified Mma as an etiological agent of mycobacteriosis using molecular and mass spectrometry (MALDI-TOF) analyses (Davidovich et al. 2020). In this work, in addition to 16S rRNA analysis for phylogenetical classification, we utilized multiple genomic tools to assign the identities and characterise the strains isolated. Altogether, this work highlights the importance of reviewing previous classifications of infectious Mycobacterium species using novel scientific tools and several bioinformatic approaches.

One limitation of our study was the low number of Chilean isolates analysed. However, the identification of new bacterial isolates from aquaculture facilities may confirm the presence of the Msal-like strains. Our findings pave the way for the further investigation and monitoring of this bacterial genus as a potential etiological agent of an emerging disease, namely mycobacteriosis in aquaculture systems. Interestingly, virulence factors were found in all the strains studied that have been reported in other mycobacterium spp.; phoP (Ryndak et al. 2008), mbtH (Baltz 2011), ideR (Zondervan et al. 2018), among others. 

These last two genes, regulate iron homeostasis through various mechanisms, and play a crucial role in mycobacterium tuberculosis virulence (Pandey and Rodriguez 2014). According to, this VFs has been used as an important target to guide the search for new drugs (Wang et al. 2016; Rohilla et al. 2017; Salimizand et al. 2017). If strategies are found that successfully inhibit these targets, they may be used to treat multiple other mycobacteriosis of human and animal origin. In addition, the presence of AR genes in myc162 and myc182 that encode genes for resistance to carbapenem, points to the need to study the minimum inhibitory antibiotic concentrations for these and other new isolates. Finally, our findings will serve as guide to future studies aiming to provide insights into mycobacterial biology and pathogenicity using a combination of several tools simultaneously.