Abstract 

Several members of the Mycobacterium genus cause invasive infections in humans and animals. According to a recent phylogenetic analysis, some strains of Mycobacterium salmoniphilum (Msal), which are the main culprit in bacterial outbreaks in freshwater fish aquaculture, have been assigned to a separate branch containing Mycobacterium franklinii (Mfra), another species that causes infections in humans. However, this genus is little studied in an aquaculture context. Here, we isolated four Mycobacterium spp. strains from freshwater cultures of Atlantic and coho salmon in Chile and performed whole-genome sequencing for deep genomic characterization. 

In addition, we described the gross pathology and histopathology of the outbreaks. Several bioinformatic analyses were performed using the genomes of these four Mycobacterium isolates in conjunction with those of Msal strains, four Msal-like strains, and one Mfra strains, plus 17 other publicly available Mycobacterium genomes. We found that three isolates are clustered into the Msal branch, whereas one isolate clustered with the Mfra/Msal-like strains. 

We further evaluated the presence of virulence and antimicrobial resistance genes and observed that the four isolates were closely related to the Msal and Msal-like taxa and carried several antimicrobial resistance and virulence genes that are similar to those of other pathogenic members of the Mycobacterium clade. Altogether, our characterization Msal and Msal-like presented here shed new light on the basis of mycobacteriosis provides quantitative evidence that Mycobacterium strains are a potential risk for aquaculture asetiological agents of emerging diseases, and highlight their biological scopes in the aquaculture industry.

Introduction 

Mycobacteriosis refers to infections caused by some members of the bacterial genus Mycobacterium. In fish, mycobacteriosis is characterized as a chronic progressive disease, with several external signs such as emaciation, inflammation of the skin, exophthalmia, ulceration, and open lesions (Austin and Austin 2016). Commonly, Mycobacterium are found in a wide range of environmental niches, particularly in aquatic environments, and only a small percentage of these species cause diseases in humans and animals. Fish mycobacteriosis may take several years to progress from an asymptomatic state to clinical illness and can affect more than 150 fish species, both farmed and wild (Decostere et al. 2004; Gauthier and Rhodes 2009; Heckert et al. 2001; Zanoni et al. 2008). For example, Mycobacterium pseudoshottsii has recently been identified as the etiological agent of outbreaks in three farmed fish species (Dicentrarchus labrax, Sparus aurata, and Sciaenops ocellatus), thus indicating that various farmed fish species are affected by Mycobacterium (Mugetti et al. 2020).

Currently, more than 170 distinct species have been identified within the Mycobacterium genus (Forbes 2017), and three of these, namely M. marinum (Mma), M. fortuitum (Mfor), and M. chelonae (Mche), have been recognized as major causes of mycobacteriosis in fish (Gauthier and Rhodes 2009; Gcebe et al. 2018; Heckert et al. 2001; Rhodes et al. 2001; Whipps et al. 2008; Talaat et al. 1999). The typical gross pathology of mycobacteriosis in fish includes non-specific phenotypic changes, such as lethargy and anorexia, cutaneous ulcers, and decolouration (Bruno et al. 1998; Brocklebank et al. 2003; Luo et al. 2018). Several case studies have identified the presence of multiple granulomas in the skin and internal organs (Bruno et al. 1998; Brocklebank et al. 2003; Luo et al. 2018; Keller et al. 2018).

Recently, the list of species causing piscine mycobacteriosis has been expanded to include Msal, a taxonomically controversial specie. Msal was first classified in 1960 following its isolation from the kidneys of salmonid fishes and was described as an acid-fast bacillus (Ross 1960). However, owing to its biochemical similarities with M. fortuitum (Gordon and Mihm 1959) and the fact that two species could not be distinguished from each other, Msal was subsequently omitted as a separate Mycobacterium species (Skerman et al. 1989). Thereafter, following subsequent advances in molecular techniques, Whipps et al. (2007) have performed a phylogenetic analysis of the SSU rRNA genes of the strains isolated from salmonid fishes and confirmed that Msal is a monophyletic species, which is included within the Mche– M. abscessus (Mabs) complex (MCAC), which contains many clinically relevant human pathogens but Msal has not been implicated as a cause of disease in humans (Simmon et al. 2011).

Advances in molecular techniques allowed genomic analysis with single-nucleotide resolution and thus propelled the characterization and identification of Msal. Zerihun et al. (2011a) were the first to identify an Msal strain isolated from infected Norwegian salmon. By aligning the partial sequences of the 16S rRNA, rpoB, and Hsp65 genes, the isolated strain was found to be 95–99% similar to Msal ATCC 13758. Later, Aro et al. (2014) utilized a similar strategy to identify the strain they isolated from an infected Chilean Salmo salar, and they showed that the 16S rRNA gene sequence obtained from Msal isolates shared 100% similarity with those from the Msal DQ866770 and Msal DQ866766 strains. In addition, these studies have found that the infected fish displayed significant macroscopic symptoms, such as external ulcers, poor body condition, pale gills, and erosion in the pectoral fin. Internal lesions, loss of adipose tissues, liver discolouration, and swelling of the internal organs have also been reported (Zerihun et al. 2011b, a; Aro et al. 2014). Nevertheless, both studies lacked of a genome-wide characterizations of the isolated strains. whole-genome.

The ability of Msal to infect mammals was first tested in vitro by Harriff et al. (2008). They observed that several Msal strains could infect and grow in both mouse and human macrophage cell lines. More recently, Msal infection has been reported in mice, in which an outbreak in an animal facility at Uppsala University was confirmed through deep DNA sequencing to have been caused by Mycobacterium species (Behra et al. 2019). The authors identified one as Msal, and the other three strains as Msal-like, which they proposed as new species of Mycobacterium after performing comparative genomics with 36 other MCAC members. Another recent study detected the presence of Renibacterium salmoninarum and Mycobacterium spp. in wild brown trout in Austria during summer. Owing to the importance of R. salmoninarum in aquaculture as an etiological agent of bacterial kidney diseases, the presence of both pathogens in wild fish could present a potentially serious infectious disease risk for the aquaculture industry (Delghandi et al. 2020).

The objective of this study was characterizing the macroscopic and microscopic traits of mycobacteriosis and performing whole-genome sequencing on the four Mycobacterium strains isolated from S. salar and Oncorhynchus kisutch salmon aquaculture facilities, presenting both kind of results, in silico and in vivo findings simultaneously that further describe the pathogenicity of mycobacteriosis. In addition, we compared the genomic and functional features of these strains with those found in several publicly available Mycobacterium genomes. In overall, this study gives novel insight regarding the identification and deep characterization of Mycobacteryum isolates using a combination of several macroscopic, microscopic, phenotypic, and genomic approaches.