In vitro antibiotic susceptibility of bacteria isolated from EUS-affected fishes in India

Letters in Applied Microbiology 2002, 34, 311 316 In vitro antibiotic susceptibility of bacteria isolated from EUS-affected fishes in India D. Saha and J. Pal Department of Zoology, North Bengal University, Siliguri, India 2001/347: received 28 November 2001 and accepted 7 January 2002 D. S A H A A N D J. P A L. 2002. Aims: Twelve antibiotics were evaluated for in vitro sensitivity against 16 bacterial strains isolated from surface lesions of fishes affected with epizootic ulcerative syndrome (EUS). Methods and Results: Disc diffusion assay in Mueller-Hinton agar showed that the pseudomonads and aeromonads were mainly resistant to penicillin, ampicillin and erythromycin. Additionally, some were resistant to gentamycin and amoxycillin. However, resistance towards antibiotics previously recommended for EUS treatment, such as oxytetracycline and chloramphenicol, was not observed. Four aeromonads and two pseudomonads were found to induce ulcers when injected intramuscularly in healthy Anabas testudineus. Conclusions: All six pathogenic isolates were sensitive towards oxytetracycline, chloramphenicol and nalidixic acid. Oxytetracycline seems to be an effective antibiotic, and further investigations to determine the mode of treatment and dose appear to be worthwhile. INTRODUCTION Epizootic ulcerative syndrome (EUS) is one of the most destructive diseases amongst fresh- and brackish-water fish in the Asian Pacific region, causing considerable loss to fish farmers (Bondad-Reantaso et al. 1994; Das 1997). In the initial stages, the disease is characterized by tiny red spots on the skin surface, which gradually grow in size until finally, a circular to oval deep haemorrhagic ulcer exposing the skeletal musculature is visible. Different pathogenic organisms have been reported to be isolated from naturally-infected fish, including bacteria (Pal and Pradhan 1990; Chakraborty and Dastidar 1991; Torres et al. 1993; Karunasagar et al. 1995; Nabi et al. 2000), 1 fungus (Roberts et al. 1993) and virus (Freirichs 1995). The most common bacteria isolated from fish ulcers are pseudomonads and aeromonads (Boonyaratpalin 1989; Pal and Pradhan 1990; Torres et al. 1993; Karunasagar et al. 1995; Nabi et al. 2000). Occasionally, Micrococcus (Pal and Pradhan 1990; Jhingran and Das 1990), Vibrio (Ali and Tamuli 1991), CAN bacteria (Chakraborty and Dastidar 1991) etc. have also been isolated. It has been accepted that EUS is primarily an infectious disease, and that it is a complex condition involving Correspondence to: Dr J. Pal, Department of Zoology, North Bengal University, Siliguri 734430, West Bengal, India (e-mail: jpaulnbu@satyam.net.in). certainly fungal and bacterial elements in its later stages, and probably one or more viruses (Chinabut 1995). The involvement of multiple pathogens, therefore, makes the disease difficult to control using vaccines. Although some attempts have been made to develop a suitable vaccine (Ali et al. 1996), an effective product is not yet available. Application of antibiotics seems to provide an easy alternative to heal the infected fish and control the spread of the disease. Various authors have successfully used antibiotics for the control of fish diseases. According to Jhingran (1990), total recovery was observed in EUS-affected fishes when nalidixic acid and erythromycin, at 50 mg kg )1 body weight per day in a formulated micro-encapsulated feed containing 30% protein and fortified with Vitamin A and C, were supplied for 3 days together with an antibiotic bath (chloramphenicol at 15 ppm). Some of the antibiotics found to be effective are terramycin (Purkait 1990), trimethoprim and sulphamethoxazole (Mahapatra et al. 1996), and oxytetracyclin (Das 1997). However, these are mainly field trials and reports of systematic antimicrobial sensitivity profiles of the pathogens are very few (Pradhan and Pal 1993). In this study, an in vitro screening of a wide range of antibiotics was carried out systematically to investigate their potential against 16 types of bacteria isolated from EUSpositive fish. The antibiotics selected had known antibacterial activity against similar types of bacteria. ª 2002 The Society for Applied Microbiology

312 D. SAHA AND J. PAL MATERIALS AND METHODS Bacteria The 16 bacterial strains used in this study were isolated in 1994 95 from the ulcer region of the skin of three types of fish which were naturally affected during an outbreak of EUS in the State of West Bengal in India (Saha and Pal, 2000). Six types of bacteria were isolated from Channa punctatus while five each were isolated from Puntius sp. and Mystus sp. Four isolates from C. punctatus belonged to the genus Pseudomonas, one belonged to Bacillus and the other belonged to Aeromonas. Among the isolates of Mystus sp., three belonged to Aeromonas, one was found to be a Moraxella sp. and the remaining one belonged to Pseudomonas. Among the isolates of Puntius sp., one belonged to Micrococus, another belonged to Pseudomonas and the rest were identified as motile Aeromonas sp. The isolates were stored in nutrient agar slants containing 1% glucose, at 4 C, and sub-cultured every 3 weeks. For experimental work, sub-cultures were made from the stock cultures and allowed to grow at 30 C for 18 h in Nutrient Broth supplemented with glucose. Induction of ulcer All the bacteria were tested for their ability to induce ulcers when injected intramuscularly in Anabas testudineus 2 according to the method of Pal and Pradhan (1990) with some modifications. The fish, of approximately one year old and weighing about 25 g, were collected from nearby fish farms of Sonapur and Gangarampur of the Darjeeling district of West Bengal that did not have any previous report of an EUS outbreak. All fish were maintained in glass aquaria measuring 90 35 35 cm. Water temperature was maintained at 28 30 C. Intramuscular injection was given with 0Æ05 ml cell suspension containing 1 10 7 cells ml )1 of each isolate in 0Æ85% NaCl. Each isolate was injected into 20 fish. The control set of fish received 0Æ05 ml sterile saline. The fish were observed during 15 days for formation of an ulcer, and the nature of the ulcer formed, if any, was noted. The following antibiotic sensitivity test discs (Hi-Media 3Laboratories, Mumbai, Maharashtra, India) with their concentrations shown in parentheses were used to detect antibiotic sensitivity of the bacterial isolates: penicillin (10 U), streptomycin (10 lg), kanamycin (30 lg), ampicillin (10 lg) chloramphenicol (30 lg), gentamycin (10 lg) norfloxacin (10 lg), cotrimoxazole (trimethoprim:sulphamethoxazole 1:5, 25 lg), erythromycin (15 lg), oxytetracyclin (30 lg), nalidixic acid (30 lg) and amoxycillin (30 lg). Sensitivity test Antibiotic sensitivity was tested in Mueller-Hinton agar plates which were inoculated with 0Æ1 ml of an 18-h-old culture of the test bacterium in glucose-supplemented (1%) nutrient broth. Antibiotic-impregnated discs were placed on the solid medium and the plates were incubated at 30 C for 24 h. Zones of inhibition formed around the discs were measured, and antibiotic sensitivity was assayed from the length of the diameter of the zones (in mm). The zone radius was actually scaled from the centre of the antibiotic disc to the end of the clear zone where bacteria could be seen growing. Zone diameters were interpreted as sensitive, intermediate and resistant according to the manufacturer s instructions. RESULTS The antibiotic sensitivity profile of bacteria isolated from C. punctatus, Mystus sp. and Puntius sp. is listed in Table 1. The results showed that all pseudomonads and aeromonads were resistant to penicillin and ampicillin. Bacillus sp. was resistant to ampicillin only, while Micrococcus sp. was resistant to norfloxacin and nalidixic acid, and Moraxella sp. was resistant to streptomycin and chloramphenicol. Vibrio sp. was susceptible to all the antibiotics tested. Out of the 16 isolated strains, two pseudomonads and four aeromonads were found to produce typical haemorrhagic ulcers at the site of injection in the fish, and mortalities were recorded in all cases. The antibiotic resistance patterns of the bacteria capable of inducing ulcers show that all were resistant to penicillin and ampicillin. In addition, the aeromonads were resistant to erythromycin and P02 was resistant to cotrimoxazole (Table 2). Percentage resistance data showed that 81Æ25% of all the bacteria tested (both pathogenic and non-pathogenic) were resistant to ampicillin, 75% were resistant to penicillin, 56Æ25% were resistant to erythromycin and 31Æ25% were resistant to cotrimoxazole (Table 3). Thus, penicillin, ampicillin, erythromycin and cotrimoxazole cannot be used as chemotherapeutic agents for treating fish ulcers. One non-pathogenic aeromonad isolated from ulcers of Puntius sp. showed multiple resistance to seven antibiotics, including oxytetracyclin. However, none of the other bacteria were resistant to oxytetracyclin, and oxytetracyclin showed 75% sensitivity among the isolates. Other effective antibiotics included kanamycin (62Æ5% sensitivity and 0% resistance), chloramphenicol (68Æ75% sensitivity), norfloxacin (75% sensitivity) and 4streptomycin (56Æ25% sensitivity) ( Table 3). A comparison of data on the inhibition zones of the pathogenic pseudomonads (Fig. 1) and aeromonads (Fig. 2) against five commonly-used antibiotics showed that oxytetracyclin was particularly effective against both types of bacteria.

ANTIBIOTIC SCREENING OF EUS ISOLATES 313 Table 1 Antibiotic sensitivities of the bacteria isolated from the ulcer region of EUS-affected Channa punctatus, Mystus sp. and Puntius sp tested* Bacterial strains AMX AMP CHL COT ERY GEN KAN NAL NFX OTC PEN STM A01 R R S I R R I I S S R I A02 I R S S R R I S S S R I A03 I R S S R I I S S S R S A04 R R I R R R I I I R R I A05 I R S R R R I I I I R I A06 S R S S R I I I S S R S B01 I R I S I S S S S S I S C01 S S S S I S S R R S S S M01 I I R I I S S I S I S R P01 I R I R R S S I I I R S P02 S R S R S S S I S S R S P03 S R I I R S S S S S R S P04 S R S S I S S S S S R I P05 S R S R R S S I S S R S P06 S R S I I S S S S S R S V01 S I S S I S S I S S I I *Abbreviations: AMX ¼ Amoxycillin, AMP ¼ Ampicillin, CHL ¼ Chloramphenicol, COT ¼ Co-trimoxazole, GEN ¼ Gentamycin, NAL ¼ Nalidixic acid, NFX ¼ Norfloxacin, OTC ¼ Oxytetracyclin, PEN ¼ Penicillin, STM ¼ Streptomycin. R ¼ Resistant, S ¼ Sensitive, I ¼ Intermediate. Table 2 Drug resistance patterns of bacteria isolated from EUS-affected fish Isolate number Isolate identity Source fish Resistance profile* Induction of ulcer A01 Aeromonas hydrophila Channa punctatus AMP AMX ERY GEN PEN + A02 Aeromonas sobria Puntius sp. AMP ERY GEN PEN + A03 Aeromonas hydrophila Puntius sp. AMP ERY PEN + A04 Aeromonas hydrophila Puntius sp. AMP AMX COT ERY GEN OTC PEN ) A05 Aeromonas hydrophila Mystus sp. AMP COT ERY PEN ) A06 Aeromonas sobria Mystus sp. AMP ERY PEN + B01 Bacillus sp. Channa punctatus AMP ) C01 Micrococcus sp. Puntius sp. NAL NFX ) M01 Moraxella sp. Mystus sp. CHL STM ) P01 Pseudomonas sp. Channa punctatus AMP COT ERY PEN ) P02 Pseudomonas sp. Channa punctatus AMP COT PEN + P03 Pseudomonas sp. Channa punctatus AMP ERY PEN ) P04 Pseudomonas sp. Channa punctatus AMP PEN ) P05 Pseudomonas sp. Puntius sp. AMP COT ERY PEN ) P06 Pseudomonas sp. Mystus sp. AMP PEN + V01 Vibrio sp. Mystus sp. ) ) *Abbreviations: AMX ¼ Amoxycillin, AMP ¼ Ampicillin, CHL ¼ Chloramphenicol, COT ¼ Co-trimoxazole, GEN ¼ Gentamycin, NAL ¼ Nalidixic acid, NFX ¼ Norfloxacin, OTC ¼ Oxytetracyclin, PEN ¼ Penicillin, STM ¼ Streptomycin. Susceptible to all antibiotics tested. DISCUSSION A review of the literature showed that although reports on the usefulness of antibiotics in treating EUS-affected fish are common in India, the range of antibiotics is mainly limited to nalidixic acid, erythromycin, chloramphenicol, oxytetracyclin and cotrimoxazole (trimethoprim and sulphamethoxazole). These are mainly administered as supplementary feed for the treatment of ulcerated fish. However, due to loss of appetite of the severely affected fish, the antibiotics may

314 D. SAHA AND J. PAL tested Disc content Percentage of bacteria sensitive* Percentage of bacteria resistant Table 3 Percentage distribution of bacterial sensitivity to the chemotherapeutic agents Amoxycillin 30 lg 50 12Æ5 Ampicillin 10 lg 6Æ25 81Æ25 Chloramphenicol 30 lg 68Æ75 6Æ25 Co-trimoxazole 25 lg 43Æ75 31Æ25 Erythromycin 15 lg 6Æ25 56Æ25 Gentamycin 10 lg 62Æ5 25 Kanamycin 30 lg 62Æ5 0 Nalidixic acid 30 lg 37Æ5 6Æ25 Norfloxacin 10 lg 75 6Æ25 Oxytetracyclin 30 lg 75 6Æ25 Penicillin 10 U 12Æ5 75 Streptomycin 10 lg 56Æ25 6Æ25 *Percentage calculated on the basis of 16 isolates. Inhibition zone (mm) 30 25 20 15 10 5 0 CHL COT ERY NAL OTC Fig. 1 Sensitivity of the pathogenic pseudomonads against five recommended antibiotics. (j) PO2; (h) PO6 Inhibition zone (mm) 30 25 20 15 10 5 0 CHL COT ERY NAL OTC Fig. 2 Sensitivity of the pathogenic aeromonads against five recommended antibiotics. (h) AO1; ( ) AO2; (j) AO3; ( ) AO4 be wasted. Moreover, the presence of these antibiotics in water or bottom soil may induce resistance both in the target bacteria and in other bacteria present in the environment. Aoki (1992) reported a considerable overall increase in drugresistant, fish-pathogenic bacteria in parallel with the extensive use of chemotherapeutic agents, which has created a great deal of difficulty in the treatment of bacterial infections in fish culture. Here, it was found that all the aeromonads were resistant to penicillin, ampicillin and erythromycin. Among the pseudomonads, all were resistant to penicillin and ampicillin, and some were resistant to erythromycin. In addition, a particular non-pathogenic strain of Aer. hydrophila (A04) isolated from a skin ulcer of Puntius sp. showed resistance to seven out of the 12 antibiotics tested. Multiple drug resistance in Aer. hydrophila had been reported earlier (Hedges et al. 1985). In many cases, transferable R plasmids were detected in the drug-resistant strains of Aer. hydrophila (Akash and Aoki 1986), Ps. aeruginosa (Levy 1984) and Ps. fluorescens (Aoki et al. 1977). The common type of R plasmids of Aer. hydrophila in Japan had markers resistant to chloramphenicol, sulphonamide and streptomycin (Aoki 1992). Strains isolated in France showed additional resistance towards tetracyclin and kanamycin (Mizon et al. 1978). On the other hand, the present isolates, except A04, were relatively sensitive towards these antibiotics. None of the bacteria were found to be resistant towards kanamycin, while oxytetracyclin, streptomycin and chloramphenicol were among the most effective antibiotics (Table 3). Intramuscular injections with the isolates showed that two pseudomonads and four aeromonads were able to induce ulcers at the site of injection. This result is similar to earlier reports (Saha and Pal, 2000). All the pathogenic pseudomonads and aeromonads showed sensitivity towards chloramphenicol, nalidixic acid and oxytetracyclin (Figs 1 and 2), although these drugs were reported to be efficacious and recommended for treating EUS-affected fish soon after the initial outbreak (Jhingran 1990; Purkait 1990). One reason for the absence of development of resistance may be

ANTIBIOTIC SCREENING OF EUS ISOLATES 315 that although researchers have reported the success of antibiotic treatments, fish farmers in rural Bengal may either not yet be aware of it, or are in a poor economic position and unable to apply them in their ponds. Most of the treatment is carried out using cheaply available water purifiers such as lime (calcium carbonate) and potassium permanganate (Das 1997). Hence, antibiotic resistance may not have reached an alarming stage yet in these areas, as has happened in the more advanced countries of Japan and France. The single Vibrio isolate was in fact susceptible to all the antibiotics tested, whereas multiple-drug-resistant R plasmids were detected in pathogenic V. anguillarum strains in Japan (Zhao et al. 1992). However, isolation of multiple-drug-resistant strains such as A04 shows that development of resistance towards antibiotics is still a fact for bacteria in the aquatic environment. Besides ampicillin, penicillin and erythromycin, the pathogenic isolates also showed resistance towards broad spectrum antibiotics. A02 showed resistance towards gentamycin, and A01 showed resistance towards both gentamycin and amoxycillin, which are not recommended for fish treatment. Therefore, although fish farmers may not apply antibiotics, the resistance patterns still reflect the hazards of antibiotic usage. This may be the reason why only five antibiotics have been found to be effective by previous authors. In the more developed countries, resistance to existing antibiotics has been described for almost every major bacterial fish pathogen. Hence, new classes of antimicrobials, such as fluoroquinolone and third generation cephalosporins, are being used for treatment of bacterial infections (Dixon 1994). The present study has provided a basic indication that it is still possible to use antibiotics like chloramphenicol, nalidixic acid and oxytetracyclin in Indian waters against both aeromonads and pseudomonads associated with EUS. Considering the adverse effect of antibiotics on fish and the environment, oxytetracyclin may be considered to be safer than others. Due to its short halflife both in sea water and fresh water, it is less dangerous to the environment (Choo 1994). In the fish body, it is not immunosuppressive (Karunasagar et al. 1995; Ali et al. 1997) and is rapidly eliminated once the application is 5 withdrawn (Xu and Rogers 1994). It is, in fact, one of the few antibiotics approved by the US Food and Drug Administration for use in the treatment of bacterial fish diseases (Dixon 1994). Therefore, after ascertaining the mode of treatment and dose by necessary field trials, oxytetracyclin may be used by the farmers of West Bengal for treatment of EUS. 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