Mycobacterium marinum Infections in Aquatic Animals and Humans: Diagnosis and Treatment Challenges

Introduction

Mycobacterium marinum is a slow-growing, nontuberculous mycobacterium (NTM) that causes systemic granulomatous disease in a wide range of aquatic animal species and opportunistic infection in humans. First isolated from marine fish in 1926, M. marinum remains a significant pathogen in both wild and captive fish populations, particularly in ornamental fish and finfish aquaculture [1, 2]. The organism is a member of the M. marinum complex, which includes M. ulcerans and M. shottsii [3]. In fish, the disease is termed piscine mycobacteriosis and is characterised by chronic wasting, visceral granulomas, and high mortality in severe outbreaks. In humans, M. marinum is the causative agent of "fish tank granuloma" or "swimming pool granuloma," a cutaneous infection acquired through contact with contaminated water or handling infected fish [4, 5].

The clinical and diagnostic challenges posed by M. marinum stem from its fastidious growth requirements, prolonged incubation period, and intrinsic resistance to many standard antimycobacterial agents [6]. Accurate diagnosis relies on a combination of culture, molecular methods, and histopathology. Treatment of infected aquatic animals is rarely attempted due to cost and efficacy concerns, with depopulation and biosecurity measures often preferred. In human cases, prolonged multidrug therapy is required, often complicated by drug toxicity and lack of standardised breakpoints [7]. This article provides an exhaustive review of the microbiology, pathology, diagnostic strategies, and treatment challenges associated with M. marinum infections in aquatic animals and the comparative zoonotic aspects.

Microbiology and Taxonomy

M. marinum is an acid‑fast, aerobic, pleomorphic rod (often coccoid in older cultures) belonging to the phylum Actinobacteria. Its optimal growth temperature is 28–32 °C, which restricts it to cooler body sites in humans (typically skin of extremities) and to the water temperatures of freshwater and marine environments [8]. On solid media such as Lowenstein‑Jensen or Middlebrook 7H10 agar, colonies appear smooth, cream to yellow, and photochromogenic: exposure to light induces carotenoid pigment production, turning colonies bright yellow [9]. The mycobacterium has a waxy cell wall rich in mycolic acids, conferring acid‑fastness and resistance to disinfectants and desiccation [10].

Genotypically, M. marinum shares high sequence identity with M. ulcerans; the latter is a descendant that acquired a plasmid encoding mycolactone toxin [11]. M. marinum lacks mycolactone and produces a different lipooligosaccharide profile. Whole‑genome sequencing has revealed multiple epidemiologically relevant lineages, with some strains exhibiting enhanced virulence in specific fish hosts [12].

Piscine Mycobacteriosis: Pathology and Clinical Presentation

Pathogenesis

Following entry through the gastrointestinal tract (ingestion of contaminated feed or cannibalism) or through skin abrasions, M. marinum is phagocytosed by macrophages. The organism survives within macrophage phagosomes by inhibiting phagosome‑lysosome fusion [13]. Intracellular replication triggers a chronic granulomatous inflammatory response. Granulomas in fish are typically non‑caseating, consisting of a central core of epithelioid macrophages surrounded by a fibrous capsule, with variable lymphocyte infiltration [14]. Disseminated disease occurs when bacteria travel via blood and lymphatics to the spleen, kidney, liver, and heart.

Clinical Signs in Fish

Piscine mycobacteriosis is primarily a chronic, progressive disease. Clinical signs include:

• Emaciation and spinal curvature (lordosis or scoliosis) due to vertebral osteomyelitis [15]
• Exophthalmos, corneal opacity
• Skin ulcers, scale loss, and fin erosion
• Coelomic distension from organomegaly or ascites
• Anemia and pale gills
• Lethargy, anorexia, and eventual mortality

In acute outbreaks, especially in juvenile fish, mortality may approach 80–90% without external signs [16]. Asymptomatic carriers are common and contribute to within‑facility transmission.

Host Range

More than 150 species of marine and freshwater fish are susceptible. Major economic impacts are reported in salmonid aquaculture, ornamental production (zebrafish, guppies, cichlids), and in public aquarium displays [17]. Co‑infections with other aquatic pathogens such as Aeromonas hydrophila (see Aeromonas hydrophila in Aquaculture: Pathogenesis, Antimicrobial Resistance, and Vaccine Development) and Streptococcus agalactiae (see Streptococcus agalactiae in Farmed Tilapia: Diagnosis, Virulence Factors, and Vaccine Development) have been documented [18].

Diagnostic Strategies

Sampling and Specimen Handling

Samples for culture and PCR should be collected from moribund fish or freshly dead carcasses. Target organs include kidney, spleen, liver, and granuloma‑containing tissues. Swabs of external ulcers are less sensitive [19]. For histopathology, tissues are fixed in 10% neutral‑buffered formalin. For molecular analysis, samples should be frozen at −80 °C or placed in RNA‑stabilizing solutions.

Ziehl‑Neelsen Staining and Histopathology

Acid‑fast bacilli (AFB) can be visualised by Ziehl‑Neelsen or Fite‑Faraco stains on impression smears or tissue sections. In haematoxylin‑and‑eosin (H&E)‑stained sections, granulomas are evident, but AFB staining is required for genus‑level confirmation [20]. Sensitivity of direct smear microscopy is low (approximately 10³–10⁴ CFU/mL) [21].

Culture

M. marinum is cultured on Lowenstein‑Jensen agar, Middlebrook 7H10/7H11 agar with oleic acid‑albumin‑dextrose‑catalase (OADC) enrichment, or in liquid media such as BACTEC MGIT (generic mycobacterial growth indicator tube systems). Incubation at 28–30 °C for 4–8 weeks is required due to the organism’s doubling time of 10–12 hours [22]. Positive cultures are confirmed by acid‑fast staining, photochromogenicity, growth characteristics, and molecular identification.

Culture remains the gold standard but has several drawbacks: long turnaround time (weeks), low sensitivity (especially after antimicrobial treatment), and the need for specialised laboratory infrastructure [23].

Nucleic Acid Amplification Tests (NAATs)

PCR assays targeting the 16S rRNA gene, hsp65, or the ITS region are widely used for detection and species identification [24]. Real‑time PCR (qPCR) using probes or melting curve analysis offers higher sensitivity (detection limit ≤ 10 CFU per reaction) and reduced turnaround time (2–4 hours) [25]. Multiplex PCR panels capable of differentiating M. marinum from other NTM are available commercially and in‑house [26].

Whole‑genome sequencing and metagenomic approaches are increasingly used for outbreak investigation, mixed infections, and direct detection from water samples and biofilms [27]. Bioinformatics workflows compare sequence reads to curated NTM databases.

Serology

Serological assays such as enzyme‑linked immunosorbent assays (ELISA) and agglutination tests have been developed for piscine mycobacteriosis, but cross‑reactivity with other mycobacteria and environmental bacteria reduces specificity [28]. Commercial ELISA kits for M. marinum are not widely available. For human anti‑M. marinum antibodies, no validated serological test exists; diagnosis relies on culture and PCR [29].

Comparative Diagnostic Performance

Diagnostic Decision Workflow

flowchart TD
    A[Fish showing signs of chronic wasting, granulomas, or unexplained mortality], > B{Sampling}
    B, > C[Fresh tissue: kidney, spleen, liver]
    B, > D[Tissues in formalin for histology]
    C, > E{Fish cut / carcass integrity?}
    E, >|Good for culture| F[Decontaminate (NALC‑NaOH) or direct]
    E, >|Poor / contaminated| G[Alternate PCR direct from tissue]
    F, > H[Inoculate Lowenstein‑Jensen at 28°C]
    H, > I[Check weekly for 8 weeks]
    I, > J{Colonies observed?}
    J, >|No growth| K[Report negative after 8 weeks]
    J, >|Suspicious colonies| L[Acid‑fast stain & photochromogenicity test]
    L, > M[PCR (16S rRNA or hsp65)]
    M, > N{Species identification}
    N, > O[*M. marinum* confirmed]
    O, > P[Submit to susceptibility testing (broth microdilution at 28°C)]
    G, > Q[DNA extraction (bead‑beating + column)]
    Q, > R[qPCR targeting *M. marinum*‑specific regions]
    R, > S{Positive?}
    S, >|Yes| O
    S, >|No| T[Consider histology or additional NTM targets]
    D, > U[Section, H&E, and Fite‑Faraco stain]
    U, > V[Granulomas + AFB?]
    V, >|Yes| O
    V, >|No| W[Keep suspect; consider PCR on remaining paraffin block]

Drug Susceptibility Testing and Treatment Challenges

In Vitro Susceptibility Methods

Broth microdilution using the CLSI M24‑A2 method is the reference standard for M. marinum. Panels are prepared in cation‑adjusted Mueller‑Hinton broth supplemented with OADC and incubated at 28–30 °C for 7–14 days [30]. MIC breakpoints for NTM are extrapolated from M. avium complex data [31]. Clinically relevant drugs include clarithromycin, rifampin, ethambutol, doxycycline, minocycline, amikacin, and sulfamethoxazole‑trimethoprim.

Resistance Patterns

M. marinum is intrinsically resistant to isoniazid and pyrazinamide, and to most beta‑lactams due to the mycolic acid barrier and inducible beta‑lactamases [32]. Acquired resistance to clarithromycin (due to mutations in the 23S rRNA gene) and to rifampin (due to rpoB mutations) has been reported in clinical isolates from both fish and humans [33, 34]. Heteroresistance (mixed populations of susceptible and resistant subclones) complicates susceptibility testing [35].

Fish Treatment Considerations

Treatment of clinically affected fish in aquaculture settings is generally not recommended because of:

• Cost and prolonged duration (2–6 months of daily antibiotics in feed)
• Poor feed intake in anorectic fish
• Risk of developing antimicrobial resistance (AMR) (see Salmonella enterica Serovar Typhimurium in Backyard Poultry Flocks: Zoonotic Risk, Antimicrobial Resistance, and Biosecurity for parallels in livestock)
• Inability to eliminate intracellular reservoirs
• Lack of regulatory approval for antimicrobials in aquatic species [36]

Antibiotic‑medicated feed supplemented with rifampin (10–20 mg/kg feed) and clarithromycin (20–30 mg/kg feed) has been used in experimental settings but carries risk for resistance selection [37]. Depopulation, thorough disinfection, and fallowing are the cornerstones of control.

Zoonotic Transmission

Human infection occurs after direct contact with contaminated water, handling infected fish, or cleaning aquarium tanks. M. marinum enters through skin abrasions and initially produces a solitary papulonodular lesion at the inoculation site (usually the hand or arm). Without treatment, infection can ascend along lymphatics (sporotrichoid spread) and invade deeper structures, causing tenosynovitis, arthritis, or osteomyelitis [38, 39].

Zoonotic risk is highest in aquarium hobbyists, fish shop employees, aquaculturists, and laboratory workers. Molecular typing has confirmed identical strains in fish and human cases from the same facility [40]. Immunocompromised individuals (e.g., those on TNF‑alpha inhibitors) are at greater risk for disseminated disease [41]. Human treatment typically involves combination therapy with clarithromycin and ethambutol, with or without rifampin, for 3–6 months [42]. Surgical debridement is required when tendon sheaths or bone are involved.

Prevention and Biosecurity in Aquaculture

Prevention strategies include:

• Sourcing fish only from certified specific‑pathogen‑free stocks
• Quarantining new arrivals for 30 days with observation and diagnostic testing
• Maintaining optimal water quality and temperature to reduce stress
• Avoiding overstocking and providing adequate nutrition
• Regular disinfection of equipment with 2% glutaraldehyde or 1% peracetic acid [43]
• Not feeding raw fish or unpasteurised offal to cultured fish [44]

In ornamental fish facilities, UV sterilisation and ozone treatment of recirculating water can reduce bacterial load [45].

Current Research and Future Directions

Efforts to develop improved diagnostics include loop‑mediated isothermal amplification (LAMP) assays for rapid field detection [46] and matrix‑assisted laser desorption/ionisation‑time of flight (MALDI‑TOF) mass spectrometry for species‑level identification from culture [47]. Whole‑genome sequencing is aiding understanding of host‑specific virulence factors and transmission dynamics [48]. Vaccine research has focused on inactivated whole‑cell preparations and recombinant antigens targeting M. marinum secreted proteins, but no licensed fish vaccine exists [49]. Additionally, the application of biological foundation models (see Biological Foundation Models for Veterinary Virology: Predicting Host Tropism and Pathogenicity) may accelerate discovery of pathogenicity determinants and drug targets.

Conclusion

M. marinum remains a challenging pathogen in aquatic animal medicine and an important zoonotic agent. Its fastidious nature, slow growth, and intrinsic resistance to several antibiotics complicate diagnosis and treatment. Culture followed by molecular confirmation remains the diagnostic standard, but qPCR and metagenomic approaches offer faster and more sensitive alternatives, particularly for screening fish populations and detecting asymptomatic carriers. Treatment of infected aquatic animals is rarely viable, placing emphasis on biosecurity and prevention. Human infections demand prolonged multidrug therapy guided by susceptibility testing. Continued research into rapid diagnostics, alternatives to antibiotics, and effective vaccines will be critical for reducing the impact of this pathogen in aquaculture and for protecting at‑risk human populations.

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