Research Article
Prevalence of Antibiotic Resistance among the Gut Associated Bacteria of Indigenous Freshwater Fishes Aplocheilus lineatus and Etroplus maculatus 
,
Neethu K.P.1 ,
Athira V.1 ,
Nashad M.1,2 ,
Mohamed H.A.1 
2 Nansen Environmental Research Centre, India (NERCI), 6A, Oxford Business Centre (6th Floor), Sreekandath Road, Ravipuram, Kochi 682016, Kerala, India
Author Correspondence author
International Journal of Aquaculture, 2016, Vol. 6, No. 3 doi: 10.5376/ija.2016.06.0003
Received: 16 Feb., 2016 Accepted: 29 May, 2016 Published: 12 Sep., 2016
Asla V., Neethu K.P., Athira V., Nashad M. and Mohamed Hatha A.A., 2016, Prevalence of Antibiotic Resistance Among the Gut Associated Bacteria of Indigenous Freshwater Fishes Aplocheilus lineatus and Etroplus maculatus, International Journal of Aquaculture, 6(3): 1-9 (doi: 10.5376/ija.2016.06.0003)
1 Introduction
Aquarium keeping is amongst the most popular of hobbies with millions of enthusiasts worldwide. With the high demand and pricing of many beautiful species, ornamental fish are being harvested at greater volumes and higher rates, threatening the viability or sustainability of the fishery (Lunn and Moreau, 2004). Hatchery rearing is an alternative to overcome this situation. However, disease outbreaks during the early developmental stages results in economic loss and is the main constraint in preventing the success (Verschuere et al., 2000). Aeromonas, Citrobacter, Edwardsiella, Flavobacterium, Mycobacterium, Pseudomonas, Vibrio,Yersinia and Streptococcus are the bacterial pathogens commonly involved in infections in ornamental fishes (Musa et al., 2008).
Infections are treated with antibiotics as therapeutic, but there has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the cumulative drug resistance in bacteria associated with these fishes (Rose et al., 2013). Aquaculture ponds and ornamental fish farms have been concerned as potential environment to the progress and selection of resistant bacteria and the source of these pathogens to other animals and humans (Madhuri et al., 2012). It can also act as a pool of antimicrobial resistance genes that may eventually be transferred to clinically significant bacteria (Miranda et al., 2013). Detection of antibiotics and antibiotic resistant bacteria from aquatic system all around the world alarms its future impacts (Karki et al., 2013). In addition to the development and spread of drug resistance, the use of antibiotics in the ornamental fish industry and aquaculture can lead to the accretion of residual antibiotics in aquatic environments (Cabello, 2006; Hoque, 2014), accumulation in the food chain (Chen et al., 2010) and detrimental effect on the microbial biodiversity (Zhou et al., 2010).
Transfer of resistance genes, between pathogenic to non-pathogenic bacteria make the situationworseand act as a pool of resistance genes from which genes can be further distributed and may ultimately end up in human pathogens (Bogaard and Stobberingh, 2000). Plasmid-borne resistance genes have been transferred by conjugation from the fish pathogen Aeromonas salmonicida to Escherichia coli, a bacterium of human origin, some strains of which are pathogenic for humans (Romero et al., 2012).Reportsonthe prevalence of resistant bacteria from water, sediments or wild animals are increasing (Schmidtet al., 2001; Giraud, 2004) and pose potential danger to human (Rose et al., 2013).Multi drug resistance was noted in many Aeromonasstrainsfrom ornamental fish and fresh water fish farms, (Hatha et al., 2005; Sreedharan et al., 2012; Nifty and Hatha, 2012).
Kerala, the southernmost state in India is bestowed 44 rivers and extensive backwater systems which support good fish fauna, both edible and ornamental. Many of the indigenous ornamental fishes are being identified for potential use in ornamental fish industry and breeding technologies are being developed as pre-requisite to commercial exploitation. This has led to a spurt in freshwater ornamental industry and trade in Kerala during the last five years. However, many of the natural waters are subjected considerable organic pollution which might provide right kind of environment for the emergence of drug resistant mutants. In this regard, we have analysed the prevalence of antibiotic resistance among the gut associated bacteria from two potential indigenous freshwater ornamental fishes such as A. lineatus and E. maculatus in order to get a snapshot of prevalence of drug resistant mutants.
2 Materials and Methods
2.1 Description of the collection site and fishes selected for the study
Fresh water fishes namely Etroplus maculatus (Orange chromide) and Apocheilus lineatus (Striped panchax) (Figure 1 a; Figure 1 b) were collected from backwaters at Edavanakad, (10o15’20” N; 76o34’20” E) using scoop net and brought alive to the laboratory. These backwaters are an extension of Cochin estuary which has undergone considerable organic pollution from anthropogenic activities. E. maculatus and A. lineatus thrive in these polluted waters and we identified them as potential candidates for the study of gut associated bacteria.
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Figure 1 Indigenous freshwater ornamental fishes analysed in this study Note: a: Etroplus maculatus; b: Aplocheilus lineatus |
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After taking the morphometric measurements such as total length (TL) and standard length (ST) the fishes were dissected out aseptically using sterile surgical blade. The entire gut region was aseptically removed, weighed and homogenized using sterile glass homogenizer, and serially diluted up to 10-6 using 10% phosphate buffer solution of pH 7.2 Aliquots of 0.2 ml samples from each dilution were spread plated in duplicate on nutrient agar (Hi media- Mumbai) for the enumeration of heterotrophic bacteria. The plates were then incubated at 30° C for 24- 48 hours. Colonies developed on the plate were counted and expressed as colony forming units (cfu) / gm of fish gut. Well separated and morphologically different colonies were picked up using a sterile inoculation needle and transferred to sterile nutrient agar slants. The isolates were purified by quadrant streaking and were stored in nutrient agar slants for further study.
Characterisation of microbiota of the gut was done using standard methods (Ringo et al., 1995). The isolated strains were identified up to generic level using the taxonomic key by Buchanan and Gibbons (1984). For generic level classification of the isolates they were subjected to various tests such as Gram stain, spore stain, motility, Kovac’s oxidase test, catalase activity, and oxidation/ fermentation test.
2.2 Antibiotic susceptibility testing
The antibiotic sensitivity of the bacterial isolates were analysed using the disc diffusion method (Bauer et al., 1966). Antibiotic impregnated discs (Himedia, India) of 8-mm diameter were used for the test. Antibiotic discs of carbenicillin (Cb – 100 μg), cephalothin (Cep- 30 μg), cefpodoxime (Cpd- 10 μg), chloramphenicol (C – 30 μg), ciprofloxacin (Cip - 5 μg), gentamicin (G -10 μg), nalidixic acid (Na- 30 μg), streptomycin (S - 10 μg), sulfafurazole (Sf- 300 μg), tetracycline (T - 30 μg) and trimethoprim (Tr- 5μg)were then placed on the sterile Mueller Hinton agar plates swabbed with enriched bacterial culture (equivalent to 0.5 McFarland standard) and incubated at 37° C for 16-18 hours. The antibiotics belonged to eight different classes according to their chemical structure: aminoglycocides (streptomycin, gentamicin), quinolones (nalidixic acid), fluorquinolones (ciprofloxacin), tetracyclines (tetracycline), penicillin (carbenicillin), cephalosporins (cephalothin, cefpodoxime), sulphonamides (sulfafurazole, trimethoprim), phenicol (chloramphenicol).
After incubation, the diameter of the zone of inhibition was measured and the results were interpreted based on recommendations of Clinical Laboratory Standards Institute (CLSI, 2007). E.coli isolate (ATCC 25922) is used as referebce strain. Isolates that are resistant to three or more antibiotics were grouped as multiple antibiotic resistant isolates. Images of the antibiotic sensitivity testing are presented as Figure 2.
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Figure 2 Plates showing antibiotic resistance of isolates from the gut of Aplochileus lineatus and Etroplus maculatus |
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2.3 Multiple Antibiotic Resistance (MAR) indexing
The MAR indexing of the isolates was determined by calculating the ratio between the number of antibiotics to which an isolate is resistant and the total number of antibiotics to which the isolate was exposed (CLSI, 2007).
3 Results and Discussion
3.1 Enumeration and Characterisation of Gut associated bacteria
Total viable count (TVC) of heterotrophic bacteria ranged between 0.2 x 104 to 0.65 x 107 cfu/ml and 1.7 x106 to 6.8 x106 cfu/mlin the gut of Aplocheilus lineatus and Etroplus maculatus respectively. A total of fifty six isolates (26 from the gut of A. lineatus and 30 from the gut of E. maculatus) were selected for characterisation and further analysis. Characterisation of the isolates revealed that Lactobacillus sp. is the predominant genus in the gut of A. lineatusfollowed by genera such as Bacillus, Micrococcus. Aeromonas sp. Micrococcus sp. was found to be the dominant genera in the gut of E. maculatusfollowed by genera Bacillus, Lactobacillus, and Vibrio (Figure 3).In both these fishes the gut associated bacterial flora was found to be dominated by Gram positive forms. Results are in agreement with the microbiota that has been reported previously from the guts of different species of fishes (Ray et al., 2012). Predominance of Corynebacterium sp. and Bacillus sp. in the gut of freshwater ornamental fishes such as Puntius filamentosus and Barilius bakerirespectively, have been reported (Nashad et al., 2015). However, Sakata (1990) reported that freshwater fish species tend to be dominated by members of the genera Aeromonas, Plesiomonas, members of the family Eneterobacteriaceae, and obligate anaerobic bacteria of the genera Bacteroides, Fusobacterium, and Eubacterium. Aeromonas veronii has been isolated from the ascitic fluid of Astronotus ocellatus (Oscar) showing signs of infectious dropsy in India (Sreedharan et al., 2012). Hathaet al. (2005) reported A. hydrophila to be the predominant species followed by fish A. caviae and A. sobria in the gut of cultured freshwater fishes. However, the aerobic heterotrophic bacteria in the gut of these fishes were found to be dominated by Gram positive forms. These variations are accounted due to factors like bacterial host specificity, food type, and water resource (Verner et al., 2003).
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Figure 3 Distribution of various genera of heterotrophic of bacteria in the gut of a) A. lineatus b) E. maculatus |
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The fishes selected in the study are indigenous to the back water systems of Cochin estuary, which has undergone considerable organic pollution during the last decade. The number of people living along the waterfront has increased dramatically and domestic waste often finds their way to these water bodies. Bacillus is often found to be a dominant genera of heterotrophic bacteria associated with waters that are organically rich as the copious extracellular hydrolytic enzymes produced by them help utilize these nutrients much more effectively than the other ones. E. maculatus and A. lineatus found thriving in these waters and in close association with the bacterial flora of these waters. It is proven fact that gut microflora of the fish is considerably influenced by that of the water and food where it lives. Dominance of Bacillus in the gut of these fishes might be a reflection of such as association.
3.2 Antibiotic resistance of the bacterial isolates from fish gut of Etroplus maculatus and Aplocheilus lineatus
Overall antibiotic resistance among the heterotrophic bacteria from the gut of A. lineatus and E. maculatus is presented in Figure 4. Majority of the isolates from the gut of both fishes exhibited resistance to cefpodoxime, meanwhile none of the isolates were resistant to gentamicin. These results corroborate with the findings of (Sahoo and Mukherjee, 1997; Bharathkumar and Abraham, 2011). In contrast, gentamicin resistant bacteria were isolated from the fish and the aquatic environment by DePaola et al. (1995). Resistance to cephalothin, ciprofloxacin, tetracycline, chloramphenicol, nalidixic acid, sulfafurazole and trimethoprim was found to relatively less prevalent among the gut associated bacteria.
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Figure 4 Overall antibiotic resistances among the gut associated bacteria from A. lineatus and E. maculatus |
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Prevalence of antibiotic resistance among the gut associated bacteria from A. lineatus and E. maculatus is given in Table 1; Table 2 Lactobacillus sp., isolated from the gut of A. lineatus exhibited resistance to 8 antibiotics tested (Table 1) and they are the only isolates shows resistant to ciprofloxacin (Cip). Bacillus sp., exhibited resistance to 6 antibiotics among 11 tested and showed maximum resistance against Cefpodoxime and Nalidixic acid (Na) and moderate resistance against antibiotics like cephalothin (Cep), cefpodoxime (Cpd), trimethoprim (Tr) and chloramphenicol (C). Aeromonas sp., exhibited resistance to cephalothin, cefpodoxime, nalidixic acid and carbenicillin. This result is in contrast to the study of Peterson and Dalsgaard (2003), they observed poor resistance of Aeromonas to all antibiotics.Some isolates of Micrococcus exhibited resistance to 4 antibiotics, while, Pseudomonas sp., exhibited resistance to 3 antibiotics with 100% resistance. All bacterial isolates shows resistance to cephalothin other than Corynebacterium, they shows resistance only against carbenicillin (Cb) and cefpodoxime (Cpd). Except Vibrio spp., all the other species exhibited resistance to carbenicillin with high degree of resistance, while they show 100% resistance against streptomycin (S). Among the various genera of bacteria encountered in the gut of E. maculatus, members of the family Enterbacteriaceae were found to have acquired relatively higher resistance (Table 2). Bacillus which was found to be the dominant genus among the gut associated bacteria of E. maculatus also showed considerable resistance to various classes of antibiotics.
.png) Table 1 Variation in prevalence of antibiotic resistance among different bacterial genera from the gut of fresh water fish Aplocheilus lineatus
Note: *Number of bacterial isolates.
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.png) Table 2 Variation in prevalence of antibiotic resistance among different bacterial genera from the gut of fresh water fish Etroplus maculatus
Note: (Lac* - Lactobacillus; Aer* - Aeromonas; Bac*- Bacillus; Mic*- Micrococcus; Vib*-- Vibrios spp.; Ent*- Enterobacterium; Cor*- Corynebacterium.)
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4 Multiple antibiotic resistance among the gut associated bacteria
Table 3 represents the MAR index and resistance pattern of bacterial isolates from the gut of A. lineatus. MAR index varied from 0.09 to 0.36. Thirteen different resistance patterns were exhibited by these isolates of which the most frequently observed pattern was CepCpd (27%) followed by CepCpdTr (11.5%). Nearly 38% of the isolates were resistant to more than two antibiotics. MAR index of these isolates were relatively low when compared to our previous observations in the gut associated bacteria of freshwater aquarium fishes and farm raised shrimps (Nifty and Hatha, 2012)
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Table 3 MAR index, resistance pattern and percentage of occurrence of each pattern among the bacterial isolates from the gut of Aplocheilus lineatus |
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MAR index and resistance patterns of gut associated bacteria from E. maculatus are given in Table 4. Prevalence of multidrug resistance among the bacteria from this fish gut was considerably higher when compared to those from A. lineatus. MAR index ranged from 0.09 to 0.63. Gut associated bacteria from the gut of E. maculatus collectively exhibited 18 different resistance patterns. While CbCpd was the most frequently encountered resistance pattern (20%), nearly 70% of them were multidrug resistant with 13 diverse patterns. The most common multi resistance patterns were CbCepCpd, CbCpdSf and CepCpdSf, with each one of them contributing 6.66% to overall resistance patterns. Unlike A. lineatus, E. maculatus is a bottom feeder and pick up decaying vegetation and detritus from the bottom along with the associated microbiota. Our studies have revealed relatively higher load bacteria in the sediment samples of the study area (Deborah et al., 2012). Antibiotic resistance among the sediment borne bacteria was also found to be higher as we found sediment offers better selection pressure for drug resistant mutants (Hatha et al., 2015).
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Table 4 MAR index, resistance pattern and percentage of occurrence of each pattern among the bacterial isolates from the gut of Etroplus maculatus |
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5 Conclusion
Different patterns of antibiotic resistance were noted in bacterial isolates from the gut of A. lineatus and E. maculatus, some of them showed multiple antibiotic resistance, indicating that fish gut bacteria may influenced by its habitat. Further research is required to find out the source of selection pressure for antibiotic resistance observed among these fish gut bacteria. Future research prospects include investigation of the gene responsible for the antibiotic resistance and strategies to control/ reduce the selection pressure for the emergence of drug resistant mutants in the natural environment.
Acknowledgments
The authors wish to thank Kerala State Council for Science, Technology and Environment (KSCSTE), Govt. of Kerala for funding support under the SARD programme. The authors wish to thank Head, Department of Marine Biology, Microbiology and Biochemistry, CUSAT for providing the facilities to carry out the research.
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