Introduction
Fish mycobacteriosis is a chronic progressive disease caused by several species of the Mycobacterium genus. Mycobacterial species are capable of causing serious diseases in most vertebrates, including humans (28), and infect a wide range of tissue and organ types, with pulmonary infections being the most frequent (57). According to the List of Prokaryotic Names with Standing in Nomenclature, there are over 150 recognised species of mycobacteria (42), all of which other than those in the M. tuberculosis complex and M. leprae are nontuberculous mycobacteria (NTM), also known as environmental mycobacteria, atypical mycobacteria and mycobactaria other than tuberculosis (MOTT). These are generally free-living organisms ubiquitous in the environment and are known to infect a number of aquatic animals, including fish. The host range of this disease is correspondingly broad and includes over 150 species of both marine and freshwater ornamental fish (e.g. Astronotus ocellatus, Carassius auratus, Colisa lalia, Cyprinus carpio subsp. haematopterus, Danio rerio, Helostoma temminckii, Hyphessobrycon serape, Labidochromis caeruleus, Microgeophagus ramirezi, Paracheirodon innesi, Poecilia reticulata, Symphysodon discus, Trichogaster lalius, Xiphophorus helleri and X. maculatus) (15, 19, 23, 31, 44, 45). The most common NTM pathogens of fish include M. marinum, M. fortuitum, M. peregrinum and M. chelonae. Other species isolated from fish include M. abscessus, M. arupense, M. avium, M. chesapeaki, M. conceptionense, M. flavescens, M. gordonae, M. haemophilum, M. kansasii, M. montefiorense, M. mucogenicum, M. neoaurum, M. nonchromogenicum, M. parascrofulaceum, M. porcinum, M. pseudoshottsii, M. salmoniphilum, M. saopaulense, M. scrofulaceum, M. senegalense, M. septicum, M. shimoidei, M. shottsii, M. simiae, M. terrae, M. szulgai, M. triviale, M. triplex, M. ulcerans and M. xenopi (18, 23, 34, 41, 43, 44, 45). In recent years, human nontuberculous mycobacterial infections and diseases have significantly increased (32). There are approximately 30 NTM that are pathogenic to humans, who commonly acquire infections if they are aquarium staff and tropical fish breeders.
The clinical signs of fish mycobacteriosis are nonspecific and include dermal ulceration, scale loss, pigmentary changes, abnormal behaviour, spinal defects, and emaciation. Ascites and granulomas may appear in all internal organs, e.g. the kidneys, liver and spleen (17). Bacterial species such as M. fortuitum, M. marinum, M. smegmatis, M. flavescens, M. peregrinum and M. chelonae, which are well-known pathogens in fish and humans, have been isolated from apparently healthy fish (23, 50). Mycobacterial infections in fish are a risk factor for the human population; nevertheless, relatively few studies have investigated large collections of ornamental fish for the presence of mycobacteria (26, 31, 34, 44).
Regarding food fish rather than ornamental fish and antimycobacterial therapy rather than mycobacterial presence, Kawakami and Kusuda (29) reported that rifampicin, streptomycin, and erythromycin were effective in reducing mortality associated with Mycobacterium spp. in cultured yellowtail (Seriola quinqueradiata). However, there are no treatments for mycobacteriosis in cultured food fish approved by the US Food and Drug Administration, nor are there any unapproved products which have been proven effective in application in the field. If antibiotics are to be used in fish mycobacteriosis treatment, their appropriacy must be validated in terms of the benefit against the risk, because the development and spread of antimicrobial resistance has become a global public health problem that is impacted by both human and non-human antimicrobial usage (37, 65). In this study, the drug susceptibility of 99 isolates of Mycobacterium spp. isolated from diseased ornamental fish to nine antibiotics was investigated.
Discussion
Nontuberculous mycobacteria are known to be ubiquitous in the environment. Mycobacterium marinum, M. chelonae, M. peregrinum, M. gordonae, M. fortuitum, M. abscessus, M. mucogenicum, M. neoaurum, M. septicum, M. senegalense and M. szulgai have been isolated from diseased fish (1, 11, 16, 44, 45, 63, 64) and have been shown to be the causative agents of pulmonary, skin, soft tissue and disseminated diseases in humans (1, 10, 11, 16, 36, 46, 54, 55). As in other countries, in Poland also Kwiatkowska et al. (32) observed an increased frequency of NTM isolated from clinical samples. Several isolates in the present study, namely M. abscessus, M. chelonae, M. fortuitum, M. gordonae, M. marinum, M. mucogenicum, M. neoaurum, M. peregrinum and M. szulgai, were also identified by Kwiatkowska et al. (32) in human clinical samples.
At present, standard therapeutic strategies for treating NTM infections are yet to be laid down. In this study, nine antimicrobial agents were tested against 99 NTM pathogens isolated from diseased ornamental fish (44). The growth of most NTM isolates was inhibited by AMK and KAN, only a few isolates showing a multidrug resistance profile which rendered these antimicrobials ineffective. Similarly, Yakrus et al. (67) also found that only 1 of 75 strains of M. abscessus and M. chelonae was resistant to AMK, and Swenson et al. (53) observed AMK to be most active against M. fortuitum. In the present study, the M. abscessus, M. chelonae, M. fortuitum, M. mucogenicum, M. saopaulense and M. szulgai isolates showed a multidrug resistance profile to at least three different classes of antimicrobials.
Mycobacterium marinum is intrinsically resistant to pyrazinamide and INH. Antibiotic agents that have been shown to be active against M. marinum include CLR, RMP, SMX, ethambutol, tetracyclines, some of the quinolones, and those used in combination therapy, i.e. ethambutol and RMP (27). The MICs of CIP, trimethoprim, azithromycin, telithromycin, quinupristin/dalfopristin, gemifloxacin, ofloxacin, and levofloxacin are above the concentrations usually obtained in vivo, and consequently, M. marinum may be considered resistant to them (5, 7, 47, 60). Chang and Whipps (11) showed that six strains of M. marinum isolated from diseased zebrafish were susceptible to AMK, CLR and RMP. In the present study, the majority of the M. marinum isolates were susceptible to AMK, CLR, KAN, DOX, CIP and SMX, whereas most isolates were resistant to RMP and INH.
There is currently no effective and definitive treatment for M. gordonae infection. Ethambutol, rifabutin, linezolid, CLR and new quinolones are active in vitro as antibiotics, but in vivo data are still insufficient (22, 56). Treatment with RMP, INH, pyrazinamide, and ethambutol was successfully used by Tsankova et al. (56). In a study by Goswami et al. (21), most of the M. gordonae isolates were sensitive to CLR and AMK and resistant to the first-line antitubercular drugs INH, RMP, ethambutol and streptomycin. In the present study, the majority of M. gordonae isolates were susceptible to AMK, KAN, DOX, CLR and CIP and SMX, but resistant to INH and RMP.
Mycobacterium abscessus, M. chelonae, M. salmoniphilum and M. saopaulense are members of the M. chelonae-abscessus complex (35, 40, 53). Natural susceptibility to AMK, cefoxitin and imipenem and resistance to many other chemotherapeutic agents are characteristics of M. abscessus (35). Current treatment recommendations for M. abscessus pulmonary infections include therapy combining two or more intravenous drugs (AMK, tigecycline, imipenem and cefoxitin) with one or two oral antimicrobials, including macrolides, linezolid, clofazimine and, occasionally, a quinolone (38). Almost all M. abscessus strains tested by Shen et al. (51) were found to be resistant to SMX, vancomycin, oxacillin, clindamycin, and all fluoroquinolones, and more than 50% of the isolates were resistant to tetracyclines, carbapenems, and aminoglycosides, except for amikacin. The lowest resistance rates to cefoxitin (10%), azithromycin (10%), AMK (10%), and CLR (20%) (51) were demonstrated by M. abscessus. In this study, M. abscessus was susceptible to AMK, KAN and CLR, and resistant to TOB, DOX, SMX, RMP and INH. These findings are comparable to those described in other studies (14, 35, 43).
Regimens for the treatment of M. chelonae infections may include TOB, CLR, CIP, DOX and AMK. Hatakeyama et al. (25) showed that M. chelonae was susceptible to AMK, TOB, CLR, SMX, imipenem, linezolid and tigecycline. In this study, the majority of the M. chelonae isolates were susceptible to AMK, CLR and KAN, but resistant to DOX, CIP, SMX, RMP and INH.
The antimicrobial pattern of M. saopaulense is characterised by susceptibility to CLR and resistance to DOX, TOB and cefoxitin. Variable results, intermediate or resistant, were obtained with AMK, CIP, minocycline and moxifloxacin (40). In this research, the M. saopaulense isolate was susceptible to AMK, KAN and CLR, and resistant to DOX, SMX, RMP and INH.
Nogueira et al. (40) found that M. salmoniphilum was susceptible to AMK, CLR, and CIP and resistant to DOX, which is consistent with our results finding the test strain to be susceptible to AMK, KAN, TOB, CLR, CIP and SMX, and resistant to DOX, RMP and INH.
Mycobacterium fortuitum, M. peregrinum, M. septicum and M. senegalense are members of the M. fortuitum complex. M. fortuitum isolates are usually susceptible to multiple antimicrobial agents, including AMK, CIP, CLR, DOX, sulphonamides, cefoxitin, and imipenem (6, 53, 61). Hatakeyama et al. (25) showed that M. fortuitum was susceptible to AMK, CIP, moxifloxacin, imipenem, linezolid, meropenem and tigecycline. In our research, most of the M. fortuitum isolates were found to be susceptible to AMK, KAN, CIP and SMX, and resistant to TOB, DOX, RMP and INH. The results of this study correlate well with those of other investigators (33), differing only from those reported by Lee et al. (33), who found strains susceptible to CLR (93%) and DOX (84%).
At present, little information is available on antibiotic activity against M. peregrinum. In a study by Guz et al. (23), test strains of M. peregrinum were found to be susceptible to AMK, ofloxacin and capreomycin, and resistant to RMP, INH, streptomycin, ethambutol, sulfamethoxazole/trimethoprim, clofazimine and erythromycin cyclocarbonate. Santos et al. (48) showed that the new fluoroquinolones with the C8-methoxy group, especially moxifloxacin, exhibit greater activity against this species. In the present study, most strains were susceptible to AMK, KAN, CIP, SMX, CLR and TOB, which correlates well with the results reported by Hatakeyama et al. (25).
Most of the M. septicum strains tested by Lian et al. (36) were found to be susceptible to AMK, CIP, SMX, KAN, ofloxacin and levofloxacin. The strains of M. septicum described by Schinsky et al. (49) were susceptible to AMK, CIP, DOX, SMX, TOB, KAN, amoxicillin-clavulanate, erythromycin, imipenem, minocycline, trimethoprim-sulfamethoxazole, vancomycin, gentamicin and neomycin but resistant to ampicillin, cefamandole, cefotaxime, ceftriaxone and streptomycin. Go et al. (20) found M. septicum isolates to be susceptible to AMK, CIP, imipenem, linezolid, moxifloxacin, and trimethoprim-sulfamethoxazole but universally resistant to CLR and DOX. In our research, the M. septicum isolates were susceptible to AMK, KAN, TOB, CLR and SMX, but resistant to RMP and INH.
Talavilikar et al. (54) showed that M. senegalense was susceptible to AMK, CLR, CIP, DOX, cefoxitin, imipenem and trimethoprim/sulfamethoxazole, which is consistent with previously published results (2, 62). In the present study, most M. senegalense isolates were susceptible to AMK, KAN, TOB, DOX, CLR and SMX and resistant to RMP and INH.
The majority of M. neoaurum isolates tested by Brown-Elliott et al. (9) were susceptible to AMK, TOB, CIP, DOX, SMX, cefoxitin, gatifloxacin, imipenem, linezolid, moxifloxacin, tigecycline and trimethoprim/ sulfamethoxazole. In our research, the M. neoaurum isolates were susceptible to AMK, KAN, TOB, DOX, CLR, CIP, SMX and INH and resistant to RMP and INH.
Rapidly growing mycobacteria are usually resistant to standard antimicrobial therapy, but M. mucogenicum is generally more susceptible to antimicrobials. Isolates of this species are susceptible to most antibacterial agents, i.e. AMK, KAN, CLR, CIP, imipenem, cefoxitin, linezolid, cephalothin, polymyxin B, and fluoroquinolones, but like other RGM, they are resistant to first-line antitubercular agents, i.e. RMP, INH, and pyrazinamide (8, 24, 52). Han et al. (24) reported that 100% (25/25) of M. mucogenicum isolates were susceptible to AMK, CLR, cefoxitin, imipenem and trimethoprim-sulfamethoxazole. In addition, 88% of isolates were susceptible to CIP, and 67% were susceptible to DOX, whereas 45% of strains were resistant to minocycline. Furthermore, Van Ingen et al. (58) tested 15 M. mucogenicum strains against a panel of 11 antibiotics and found the majority to be susceptible to AMK, CLR, CIP and rifabutin. The current NTM practice guidelines do not explicitly state a specific treatment protocol for M. mucogenicum but do state that most isolates are susceptible to multiple antimicrobial agents, including AMK, CLR, KAN, DOX, quinolones and imipenem (4, 24). In our research, the M. mucogenicum isolate was susceptible to AMK, KAN and SMX but resistant to CIP, RMP and INH.
Isolates of M. szulgai are often susceptible in vitro to most antitubercular agents. The most common regimen includes RMP, ethambutol and macrolides and/or quinolones (59). Lung disease induced by M. szulgai was successfully treated with RMP, CLR and ethambutol by Kempisty et al. (30). In the present study, the M. szulgai isolate was susceptible to AMK, KAN, DOX, CLR, SMX and INH and resistant to RMP, CIP and TOB.
In summary, this study determined the antibiotic susceptibility of ornamental fish mycobacteria. The antimicrobial resistance rate of Mycobacterium spp. isolated from ornamental fish is high and thus needs to be monitored. Tested in this investigation for effect against isolates from diseased fish, KAN, AMK, CLR and SMX were the most inhibitory of rapidly growing mycobacteria, while AMK, KAN, SMX, CLR, DOX and CIP were the most efficacious against slowly growing mycobacteria. Our results confirm that antibiotic-resistant bacteria can be found in fish, which has potential consequences for public health. Consequently, continued monitoring of Mycobacterium spp. for antibiotic resistance should be performed in ornamental fish to help to establish strategies for the treatment of mycobacteriosis.