Middle East Journal of Applied Science & Technology (MEJAST) (A Referred Quarterly International Journal) Volume 2, Issue 1, Pages 54-61, January-March 2019
Research Article Genotypic Characterization and Resistance Patterns of Flavobacterium columnare from Pond-cultured Clarias gariepinus Ogbonne Fabian Chinedu1, Osegbo Adaora Nwadiobimma2, Nwokwu Chidi Peter1, Ukazu, Ebere Roseline1, Egbe Frances Chinyere1, Akhiromen Dorathy Iniobong1 and Aguta Onuoha Joseph1 1
Nigerian Institute for Oceanography and Marine Research, Victoria Island, Lagos. Durable Crops Research Department (Microbiology), Nigerian Stored Products Research Institute.
2
Article Received: 29 November 2018
Article Accepted: 30 December 2018
Article Published: 26 March 2019
ABSTRACT Flavobacterium columnare is a significant fish pathogen of economic importance. Although, many studies have been done on Flavobacterium columnare in other countries, little or no investigation has been carried out in Nigeria especially on Clarias gariepinus associated Flavobacterium columnare infections. This study was undertaken to investigate the occurrence of Flavobacterium columnare in pond-cultured Clarias gariepinus and to determine their antibiotics susceptibility and resistance patterns. Swabs were aseptically taken from the kidney, liver, gills, skin and intestine of the affected fish samples, and analyzed microbiologically. After series of biochemical test, 58 out of 75 bacterial isolates recovered were presumptively identified as Flavobacterium columnare. Confirmatory identification of the isolates was carried out with previously described primers using multiplex PCR. Standard Kirby-Bauer disc diffusion method was used for antibiotics susceptibility test. Flavobacterium columnare isolates were 100% resistant to Streptomycin, Oxytetracycline, Chloranphenicol, Pefloxacin, Ofloxacin and 90, 70 and 60% resistant to Gentamycin, Amoxycillin and Clindamycin respectively. However, all the isolates were 100% susceptible to Ciprofloxacin and Cotrimoxazole only. The results revealed multiple drug resistance of the isolates to the antibiotics tested as the isolates were resistant to eight out of ten antibiotics used in this study. There are fewer studies on antibiotic resistance in Flavobacterium columnare from aquaculture enterprises and this study provides further support to the view that there is a potential risk of transfer of resistant bacteria and their genes to human pathogen through the food chain. Although, in Nigeria there is no antibiotic product registered for aquaculture usage, yet fish farmers use them off-label for bacterial diseases prevention. Keyword: Flavobacterium columnare, Antibiotic Resistance, Clarias gariepinus, Multiplex PCR.
INTRODUCTION Flavobacterium columnare is the causative agent of columnaris disease, a serious condition affecting numerous freshwater fish species all over the world (Bernardet and Bowman, 2006). Flavobacterium columnare is a Gram-negative bacteria in the family of Flavobacteriaceae. It is one of the most prevalent fish pathogen with a worldwide distribution and causes severe significant economic losses. Studies have shown that there are three different Flavobacterium species that are primary pathogens to freshwater cultured and wild fish populations (Clifford, 2011). They are: Flavobacterium columnare, the cause of columnaris disease, Flavobacterium branchiophilum, the cause of bacterial gill disease, and Flavobacterium psychrophilum, the cause of bacterial Coldwater disease. However, the diseases and mortality caused by these pathogens constitutes one of the broadest host and geographic ranges of any of the bacterial pathogens to fishes. Fish pathogenic Flavobacterium columnare are apparently ubiquitous in tropical and sub-tropical freshwater aquatic environments and occur in water temperatures ranging from 20 - 25°C and above (Chalkoo et al., 2007). A lot of cultured freshwater fish species have been affected by at least one of these pathogens. Many cultured and wild fish species are considered at risk of infection and possible disease outbreak. Columnaris disease affects aquaculture species, particularly the catfish species, as well as many aquarium species. In endemic areas, Columnaris disease outbreaks in aquaculture occur regularly and often in conjunction with increased host stressors (Chalkoo et al., 2007). Although Columnaris disease has been experimentally induced in healthy fish of various ages (Ferguson et al., 1991). Several researchers have noted that this disease typically 54 | P a g e
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Middle East Journal of Applied Science & Technology (MEJAST) (A Referred Quarterly International Journal) Volume 2, Issue 1, Pages 54-61, January-March 2019
occurs in association with certain predisposing factors such as overcrowding, reduced dissolved oxygen, increased ammonia and particulate matter in the water (Bullock, 1990), organic load and high temperature (Decostere et al., 1999). However, in an attempts to eradicate these pathogenic bacterium, various type of chemical and drugs could be applied in cultured ponds and in hatcheries. Nevertheless, during advance condition of disease outbreak several commonly known antibiotics could also be applied and after the long time use of these antibiotics, pathogenic bacterium become resistant against these antibiotics. Therefore, indiscriminate and extensive use of antibiotics, especially in hatcheries and cultured pond as prophylactic and therapeutic agents to prevent the bacterial infection leads to the development of multiple drug resistance (Dinesh et al., 2012). The presumptive diagnosis of columnaris based on the clinical signs of host and the characteristic morphological features of Flavobacterium columnare are more time consuming and strenuous that requires several laboratory testing. However, Polymerase chain reaction (PCR) based diagnostic tests have been used for prompt and accurate detection of Flavobacterium columnare nucleic acid in infected skin samples and in bacterial cultures (Welker et al., 2005). Several Flavobacterium columnare specific PCR protocols have been developed (Darwish et al., 2004). These techniques use species-specific primers to amplify the 16S rRNA gene fragment and to differentiate closely related bacteria such as: F. psychrophilum, F. aquatile, F. branchiophilum and other fish pathogens like Edwardsiella sp., Aeromonas sp. and Streptococcus iniae. A TaqMan-based real-time PCR targeting a base pair nucleotide region of the chondroitin AC lyase gene of F. columnare has also been developed (Panangala et al., 2007) for the detection and quantification of F. columnare in tissues (blood, gills and kidney) of infected fish. This study was undertaken to investigate the occurrence of Flavobacterium columnare in pond-cultured Clarias gariepinus and to determine their antibiotics susceptibility and drug resistance patterns. MATERIALS AND METHODS Sample (Fish) Collection Diseased Clarias gariepinus were collected during a surveillance following a complaint of mass mortalities in one of the farm at Ikorodu fish farm estate in Lagos state, Nigeria. During sampling, the behavioral abnormalities, gross and clinical signs of diseased fish were recorded. Both infected, weak fish with disease signs and symptoms and healthy fish were aseptically collected and transported to the laboratory for further analysis. Isolation and Identification of Flavobacterium columnare Swabs were taken aseptically from the liver, skin, intestine, gills and kidney of the dissected fish samples, and were streaked on petridish containing Tryptone soy agar (LAB M Ltd., United Kingdom) that was prepared according to manufacturers’ instructions. The plates were incubated at 37˚C for 24 hours and were observed for bacterial growth and distinct colonies were further sub-cultured on freshly prepared Tryptone soy agar to obtain pure culture of the isolates. The isolates were then identified using their morphological features, Gram staining reaction and several biochemical test according to (Bergey’s Manual of determinative bacteriology, 7th Edition).
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Middle East Journal of Applied Science & Technology (MEJAST) (A Referred Quarterly International Journal) Volume 2, Issue 1, Pages 54-61, January-March 2019
Antibiotic Susceptibility Testing: A commercially available antimicrobial susceptibility test single disc from Oxoid Ltd, Basingstoke, RG24 8PW, United Kingdom, was used. Pure culture of Flavobacterium columnare were picked from Tryptone soy broth using a sterile wire loop and transferred to tubes each containing 5ml of sterile physiological saline. The suspension was vortexed and adjusted to match 0.5 McFarland turbidity standards. Sterile swab stick was then dipped, rotated and pressed firmly on the tube walls above the culture to remove excess inoculums from the swabs. This was then evenly swabbed on the dried surface of Mueller-Hinton agar (Oxoid Ltd., England) plates ensuring even distribution of the bacterium. Ten (10) different antimicrobial susceptibility single discs were placed on the plates using sterile forceps and were incubated at temperature of 37˚C for 18 to 24 hours, after which the diameter of zone of inhibition was measured with white transparent meter rule in millimeter. The methods describe above and the interpretations of results was done using standard recommended by Clinical Laboratory Standard Institute (CLSI, 2016). The ten antibiotics used have the following concentrations: Amoxycillin (25µg), Streptomycin (25µg), Chloranphenicol (30µg), Gentamycin (10 µg), Ciprofloxacin (10µg), Pefloxacin (5µg), Oxytetracycline (30µg), Cotrimoxazole (25 µg), Clindamycin (10µg) and Ofloxacin (5µg). Extraction of Genomic DNA from Flavobacterium columnare. The DNA was extracted using protocol of bacteria DNA extraction kit from Jena Bioscience Germany (Spin column based genomic DNA purification). Multiplex Polymerase Chain Reaction (PCR) Amplification The PCR of species-specific AC lyase gene fragment was performed using a set of primers previously described by Darwish et al. (2004) as shown in Table 1. Table 1: Primer names, oligonucleotides sequences and the size of Amplicon.
S/N
1
Primer Name
ColF
1
1
Oligonucleotide primer Sequence (5 - 3 )
Target gene
CAGTGGTGAAATCTGGT
Annealing
Size of
Temperature in
Amplicon in
o
C
base pair (bp)
55
675
16S rRNA
2
ColR
GCTCCTACTTGCGTAGT
Col-72F
GAAGGAGCTTGTTCCTTT
Col-1260R
GCCTACTTGCGTAGTG
55
AC lyase
55
860
55
Sequence letters are :C=Cytosine, G=Guanine, A=Adenine, T=Thymine. A multiplex PCR reaction was carried out using the Solis Biodyne 5X HOT FIREPol Blend Master mix. PCR was performed in 20µl of a reaction mixture, and the reaction concentration was brought down from 5x concentration to
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Middle East Journal of Applied Science & Technology (MEJAST) (A Referred Quarterly International Journal) Volume 2, Issue 1, Pages 54-61, January-March 2019
1X concentration containing 1X Blend Master mix buffer (Solis Biodyne), 2.0 mM MgCl2, 200µM of each deoxynucleoside triphosphates (dNTP) (Solis Biodyne), 20pMol of each primer (BIOMERS, Germany), 2 unit of Hot FIREPol DNA polymerase (Solis Biodyne), Proofreading Enzyme, 5µl of the extracted DNA, and sterile distilled water was used to make up the reaction mixture. Thermal cycling was conducted in an Peltier thermal cycler PTC 100 (MJ Research Series) for an initial denaturation of 95°C for 5 minutes followed by 35 amplification cycles of 30 seconds at 95°C; 1 minute at 55°C and 1 minute 30 Seconds at 72°C. This was followed by a final extension step of 10 minutes at 72°C. The amplification product was separated on a 1.5% agarose gel and electrophoresis was carried out at 80V for 1 hour 30 minutes. After electrophoresis, DNA bands were visualized by ethidium bromide staining. 100bp DNA ladder was used as DNA molecular weight standard. RESULTS Table 2 and plate 1 shows morphological, biochemical characteristics and gram staining photomicrograph of the isolates. A total of 58 Flavobacteria strains were recovered out of 75 bacterial isolated from thirty fish samples collected from the fish farm studied. Antibiotics sensitivity test results shows that Flavobacterium columnare isolates were 100% resistant to Streptomycin, Oxytetracycline, Chloranphenicol, Pefloxacin, Ofloxacin and 90, 70 and 60% resistant to Gentamycin, Amoxycillin and Clindamycin respectively (Figure 1). However, all the isolates were 100% susceptible to Ciprofloxacin and Cotrimoxazole only. Table 2: Morphological and biochemical characteristics of the isolated Flavobacterium columnare.
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Biochemical Characteristics
Reaction
Gram stain
+
Morphology
Thin, rod-shape
Gliding motility
-
Production of Indole
-
Catalase
+
Oxidase
+
Hydrogen Sulphide
+
Glucose Fermentation
+
Lactose Fermentation
-
Fermentation of Mannitol
+
Nitrate reduction
+
Citrate
+
Urea hydrolysis
+
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Middle East Journal of Applied Science & Technology (MEJAST) (A Referred Quarterly International Journal) Volume 2, Issue 1, Pages 54-61, January-March 2019
Voges-Proskauer
-
Methyl Red
+
Production of H2S
+
Gelatin hydrolysis
+
Growth on neomycin medium
+
Growth on Polymyxin B medium
+
(+) =positive, (-) = negative.
Percentage of sensitive and resistance isolates
Plate 1: Gram stain photomicrograph of the isolates. Flavobacterium columnare is indicated with an arrow.
100 90 80 70 60 50 40 30 20 10 0
Sensitive % Resistance %
Antibiotics Figure1: The percentage of antibiotics sensitivity and resistant patterns of Flavobacterium columnare isolates.
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Middle East Journal of Applied Science & Technology (MEJAST) (A Referred Quarterly International Journal) Volume 2, Issue 1, Pages 54-61, January-March 2019
Plate 2: PCR amplification of Flavobacterium columnare AC lyase gene at 860 base pair. Lane –ve = negative control (water), lane M = molecular DNA marker, Lane 2, 4, 8, 12, 16, 18, 19 shows amplification of Flavobacterium columnare AC lyase gene at 860 base pair, while lane 1, 3, 5, 6, 7, 9, 10, 11, 13, 14, 15 and 17 were not amplified. DISCUSSION The findings of this study suggest that pond cultured African Catfish (Clarias gariepinus) are susceptible to Flavobacterium columnare. The occurrence and distribution of this bacterium highlight the significant of the organism as a major pathogen in the aquaculture industry. Clarias gariepinus farming is one of the fastest growing industries in Nigeria today. Consequently, there could be an outbreak of F. columnare infection in intensive Clarias gariepinus culture system in the nearest future in Nigeria. Hence, the results from this study will serve as a baseline data for proper diagnostic approach and treatment protocol of F. columnare infection in case of any eventuality. This study demonstrated a Polymerase chain reaction (PCR) method with species-specific primers that uses the AC lyase gene to distinguish Flavobacterium columnare isolates from other closely related yellow pigmented bacteria, and this technique was found to be more sensitive for the detection of Flavobacterium columnare infection in fish than the standard culture techniques because Flavobacterium columnare is often overgrown by the fast-growing bacteria such as Pseudomonas spp. and others (Tiirola et al., 2002). The antimicrobial susceptibility result revealed multiple drug resistance of the isolates to the antibiotics tested as the isolates were resistant to eight out of ten antibiotics used in this study. The incidence of multiple drug resistance by Flavobacterium columnare have been reported by Dinesh-Kumar et al. (2012), and this antibiotics resistance could be attributed to mutation and frequent use of drugs for diseases prevention in fish farming (Ogbonne et al., 2018). However, the PCR confirmed the presence of Flavobacterium columnare in nine of the isolates. Previously, Patra and others provided evidence that the Ac lyase gene was limited to a few species of Flavobacterium and related genera (Patra et al., 2016) and this result is in total agreement with their finding, indicating that the AC lyase gene should be an appropriate target molecule for specific Flavobacterium columnare PCR assay since variation was observed in the conventional biochemical profile of the isolates. The primer combination Col-72F and
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Middle East Journal of Applied Science & Technology (MEJAST) (A Referred Quarterly International Journal) Volume 2, Issue 1, Pages 54-61, January-March 2019
Col-1260R gave a single amplification product of 860bp, which was specific for Flavobacterium columnare identification. Subsequently, the presence of this organism could be a function of the hygienic standard of the fish ponds. In addition, the indiscriminate use of antibiotics for aquaculture portends a significant hazard to public health as there is a global concern of multiple drug resistance and potential risk of transfer of resistant bacteria and their genes to human pathogen through the food chain. Consequently, the implementation of effective, long-term vaccination programs could be the most likely target for future control of the potentially industry-limiting disease caused by F. columnare. Further research is however needed to fully understand this pathogen, principally its serological diversity and epidemiology before vaccination programs can become successful in a global scale. REFERENCES Bernardet, J.F. and Bowman, J.P. (2006). The genus Flavobacterium. In: The prokaryotes: A handbook on the biology of bacteria: Volume 7: Proteobacteria: Delta and Epsilon Subclasses. Deeply Rooting Bacteria. Springer Science + Business Media, LLC. New York. Bullock, G.L. (1990). Bacterial gill disease of freshwater fishes. Fish disease leaflet 84 Fish and Wildlife Service, Washington, DC, U.S. Chalkoo, S R., Qureshi, T.A., Najar, A.M. and Shafi, A. (2007). Bacterial Diseases of Fishes of Wular Lake. Fishing Chimes 26, 41–46. Clifford, E.S. (2011). Bacterial Coldwater disease of fishes caused by Flavobacterium psychrophilum. Journal of Advanced Research 2:97-108. Clinical Laboratory Standards institute (2016). Performance Standards for Antimicrobial Susceptibility Testing; Twenty six Informational supplement M100S-S26 2016; 36 (1). Darwish, A.M., Ismaiel, A.A., Newton, J.C. and Tang, J. (2004). Identification of Flavobacterium columnare by a species-specific polymerase chain reaction and renaming of ATCC 43622 strain to Flavobacterium johnsoniae. Molecular and Cellular Probes 18: 421-427. Decostere, A., Haesebrouck, F., Chalier, G. and Ducatelle, R. (1999). The association of Flavobacterium columnare strains of high and low virulence with gill tissue of black mollies (Poecilia sphenops). Veterinary 67:287 – 298. Dinesh, K., Yogendra, P., Singh, A.K. and Abubakar, A. (2012). Columnaris Disease and its drug resistance in cultured exotic African catfish Clarias gariepinus in India. Biochemical and Cellular Archives 12(2):415-420. Ferguson, H.W., Ostland, V.E., Byrne, P. and Lumsden, J.S. (1991). Experimental production of bacterial gill disease in trout by horizontal transmission and by bath challenge. Journal of Aquatic Animal Health 3:118-123. Ogbonne, F.C., Ukazu, E.R. and Egbe, F.C. (2018). Antibiotics Resistance Pattern and Plasmid Profiling of Edwardsiella tarda Isolated from Heterobranchus longifilis. Journal of Biosciences and Medicines 6:95-105. Panangala, V.S., Shoemaker, C.A. and Klesius, P.H. (2007). TaqMan real-time polymerase chain reaction assay for rapid detection of Flavobacterium columnare. Aquaculture Research 38: 508-517.
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Patra, A., Sudeshna, S., Sayani, B., Harresh, A., Debadyuti, B. and Thangapalam J.A. (2016). Rapid Detection of Flavobacterium columnare Infection in Fish by Species-specific Polymerase Chain Reaction. Journal of Aquaculture Research and Development 7(9):2-5. Robert, S.B., Murray, E.G.D. and Nathan, R.S. (1962). Bergey's manual of determinative bacteriology (7th ed.). Baltimore, Md.: Williams and Wilkins, 1094 pp. Tiirola, M., Valtonen E.T., Rintamaki-Kinnunen, P., Kulomaa M.S. (2002). Diagnosis of avobacteriosis by direct amplification of rRNA genes. Diseases of Aquatic Organisms 51:93-100. Triyanto, K. A. and Wakabayashi, H. (1999). The use of PCR targeted 16S rDNA for identification of genomovars of Flavobacterium columnare. Fish Pathology 34: 217-218. Welker, T.L., Shoemaker, C.A., Arias, C.R. and Klesius, P.H. (2005). Transmission and detection of Flavobacterium columnare in channel catfish Ictalurus punctatus. Diseases of Aquatic Organisms 63: 129-138.
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