Musa Najiah

Multiple Antibiotic Resistance and Heavy Metal Resistance Profile of Bacteria Isolated from Giant Freshwater Prawn (Macrobrachium rosenbergii) Hatchery

Abstract In this article, antibiogram and heavy metal resistance profile of bacteria isolated from giant freshwater prawn ( Macrobrachium rosenbergii ) hatchery in Malaysia are described. Although giant freshwater prawn was introduced into Malaysia since the 1980s, there was no database information on antibiogram and heavy metal resistance profile of bacteria from giant freshwater prawn ( M. rosenbergii ) hatchery in Malaysia. Therefore, this study was carried out to determine the effectiveness of antibiotic and heavy metal resistance profile to control bacterial diseases in M. rosenbergii hatchery. The results can provide valuable information for local M. rosenbergii post-larval producer. Antibiotic sensitivity test was carried out by disk-diffusion method against 15 types of antibiotics as follows: oxolinic acid (2 μg), ampicillin (10 μg), erythromycin (15 μg), furazolidone (15 μg), lincomycin (15 μg), amoxicillin (25 μg), colistin sulphate (25 μg), doxycycline (30 μg), florfenicol (30 μg), flumequine (30 μg), nalidixic acid (30 μg), tetracycline (30 μg), oleandomycin (15 μg), fosfomycin (50 μg), and spiramycin (100 μg), whereas heavy metal resistance profile of the present bacterial isolates was determined by 2-fold agar dilution technique. In this study, 5 types of bacteria were successfully isolated; they were Aeromonas spp. (n = 77), Escherichia coli (n = 73), Edwardsiella spp. (n = 62), Salmonella spp. (n = 75), and Vibrio spp. (n = 43). The result showed that furazolidone was the most effective antibiotic to control the bacteria isolated in this study, approximately 89.7% of the bacterial isolates were sensitive to this antibiotic. Multiple antibiotic resistance (MAR) index indicated that the hatchery water source and M. rosenbergii post-larval and sediment tanks were at high-risk exposure to the tested antibiotic. Furthermore, all the tested heavy metals (Cd 2+ , Cr 6+ , Hg 2+ , and Cu 2+ ) failed to inhibit the growth of the bacterial isolates. Therefore, it indicated that the water source of the hatchery is contaminated with both antibiotic residues and heavy metal.

Antimicrobial Property of 2-Hydroxypropane-1,2,3-Tricarboxylic Acid Isolated from Citrus microcarpa Extract

Abstract This article described antimicrobial property and structure analysis of 2-hydroxypropane-1,2,3-tricarboxylic acid isolated from the crude extract of Citrus microcarpa. Presently, there was no report on compound from C. microcarpa that possessed antimicrobial property against fish pathogenic bacteria. Therefore, in this study, the bioactive principle in C. microcarpa extract was isolated using thin layer chromatography. Its structure was elucidated based on nuclear magnetic resonance (NMR) spectroscopic data, such as proton NMR (1HNMR), correlation spectroscopy, carbon 13 NMR, and heteronuclear multiple bond correlation data. This study showed that the bioactive compound isolated from C. microcarpa was 2-hydroxypropane-1,2,3-tricarboxylic acid monohydrate. The minimum inhibitory concentration (MIC) values of crude C. microcarpa extract and its bioactive component, 2-hydroxypropane-1,2,3-tricarboxylic acid as well as commercially available synthetic 2-hydroxypropane-1,2,3-tricarboxylic acid, were determined against 18 isolates of Edwardsiella tarda and 7 bacterial reference strains, namely, Escherichia coli (ATCC 25922), Citrobacter freundii (ATCC 8090), Aeromonas hydrophila (ATCC 49140), Pseudomonas aeruginosa (ATCC 35032), Streptococcus agalatiae (ATCC 13813), E. tarda (ATCC 15947), and Yersinia enterocolitica (ATCC 23715), using two-fold microdilution method. The MIC values for both the natural 2-hydroxypropane-1,2,3-tricarboxylic acid and the synthetic one were ranging from 15.6 to 62.5 mg mL−1, whereas that of the crude extract was ranging from 7.8 to 31.3 mg mL−1. These findings showed that both the crude extract and its bioactive component might have potential as antimicrobial agent for aquaculture use.

Phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. in Malaysia.

Mycobacteriosis due to mycobacteria is one of the most common bacterial diseases in ornamental fish. We describe here the phenotypic and genotypic characteristics of Mycobacterium isolates from fighting fish Betta spp. using ATCC Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium chelonae as references. A total of four isolates (M1, M2, M3, M4) were obtained from four out of 106 fish samples using selective agar, and identified to Mycobacterium genus using acid-fast staining and 16s rRNA gene-based genus specific polymerase chain reaction. DNA sequencing and NCBI-BLAST analysis further identified isolate M1 as M. marinum and isolates M2, M3, M4 as M. fortuitum. Morphological, physiological and biochemical tests were carried out for phenotypic characterizations. Universal M13 and wild-type phage M13 RAPD dendogram was generated to illustrate the genetic relationship of the isolates and reference strains.

Effect of Excoecaria agallocha on non-specific immune responses and disease resistance of Oreochromis niloticus against Streptococcus agalactiae

The current study was designed to evaluate the effects of Excoecaria agallocha leaf extracts on immune mechanisms and resistance of tilapia, Oreochromis niloticus, after challenge with Streptococcus agalactiae. Fish were divided into 6 groups; groups 1-5 fed with E. agallocha leaf extracts at 10, 20, 30, 40 and 50mgkg-1 level, respectively. Group 6 were fed without extract addition and acted as control. E. agallocha extracts were administered as feed supplement in fish diet for 28days and the hematological, immunological, and growth performance studies were conducted. Fish were infected with S. agalactiae at a dose of 15×105CFUmL-1 and the total white blood cell (WBC), phagocytosis and respiratory burst activities of leukocytes, serum bactericidal activity, lysozyme, total protein, albumin, and globulin levels were monitored and mortalities recorded for 15days post infection. Results revealed that feeding O. niloticus with 50mgkg-1 of E. agallocha enhanced WBC, phagocytic, respiratory burst, serum bactericidal and lysozyme activities on day 28 pre-challenge and on 3rd, 6th, 9th, 12th and 15th day post-challenge as compared to control. Total protein and albumin were not enhanced by E. agallocha diet. E. agallocha increased the survival of fish after challenge with S. agalactiae. The highest mortality rate (97%) was observed in control fish and the lowest mortality (27%) was observed with group fed with 50mgkg-1 extract. The results indicate that dietary intake of E. agallocha methanolic leaf extract in O. niloticus enhances the non-specific immunity and disease resistance against S. agalactiae pathogen.

Antibiotic Resistance and Heavy Metals Tolerance in Gram-Negative Bacteria from Diseased American Bullfrog (Rana catesbeiana) Cultured in Malaysia

A total of 140 bacterial isolates have been successfully isolated from various organs of diseased American bullfrog (Rana catesbeiana) cultured in Malaysia. The most frequently isolated bacteria was Edwardsiella spp. (46 isolates) followed by Aeromonas spp. (33 isolates), Flavobacterium spp. (31 isolates), and Vibrio spp. (30 isolates). Majority of the bacterial isolates were found sensitive to furazolidone (85.0%), chloramphenicol (85.0%), oxolinic acid (90.0%), florfenicol (95.0%), and flumequine (97.5%). On the other hand, most of the bacterial isolates were resistant to oleandomycin (77.5%) and lincomycin (87.5%). Nitrofurantoin and flumequine can be inhibited the growth of all of Vibrio spp. whereas all isolates of Edwardsiella spp. were found sensitive to florfenicol and flumequine. Multiple antibiotic resistance (MAR) index were in range of 0.30-0.40, indicating that bacterial isolates from cultured bullfrogs may have received high risk exposure to the tested antibiotics. In addition, 90-100% of the isolates were resistant to copper, cadmium, and chromium. These results provided insight information on tolerance level of bacterial isolates from cultured bullfrogs to 21 antibiotics as well as heavy metals.

Molecular identification and histopathological study of natural Streptococcus agalactiae infection in hybrid tilapia (Oreochromis niloticus)

Aim: The main objective of this study was to emphasize on histopathological examinations and molecular identification of Streptococcus agalactiae isolated from natural infections in hybrid tilapia (Oreochromis niloticus) in Temerloh Pahang, Malaysia, as well as to determine the susceptibility of the pathogen strains to various currently available antimicrobial agents. Materials and Methods: The diseased fishes were observed for variable clinical signs including fin hemorrhages, alterations in behavior associated with erratic swimming, exophthalmia, and mortality. Tissue samples from the eyes, brain, kidney, liver, and spleen were taken for bacterial isolation. Identification of S. agalactiae was screened by biochemical methods and confirmed by VITEK 2 and 16S rRNA gene sequencing. The antibiogram profiling of the isolate was tested against 18 standard antibiotics included nitrofurantoin, flumequine, florfenicol, amoxylin, doxycycline, oleandomycin, tetracycline, ampicillin, lincomycin, colistin sulfate, oxolinic acid, novobiocin, spiramycin, erythromycin, fosfomycin, neomycin, gentamycin, and polymyxin B. The histopathological analysis of eyes, brain, liver, kidney, and spleen was observed for abnormalities related to S. agalactiae infection. Results: The suspected colonies of S. agalactiae identified by biochemical methods was observed as Gram-positive chained cocci, β-hemolytic, and non-motile. The isolate was confirmed as S. agalactiae by VITEK 2 (99% similarity), reconfirmed by 16S rRNA gene sequencing (99% similarity) and deposited in GenBank with accession no. KT869025. The isolate was observed to be resistance to neomycin and gentamicin. The most consistent gross findings were marked hemorrhages, erosions of caudal fin, and exophthalmos. Microscopic examination confirmed the presence of marked congestion and infiltration of inflammatory cell in the eye, brain, kidney, liver, and spleen. Eye samples showed damage of the lens capsule, hyperemic and hemorrhagic choroid tissue, and retina hyperplasia accompanied with edema. Brain samples showed perivascular and pericellular edema and hemorrhages of the meninges. Kidney samples showed hemorrhage and thrombosis in the glomeruli and tubules along with atrophy in hematopoietic tissue. Liver samples showed congestion of the sinusoids and blood vessel, thrombosis of portal blood vessel, and vacuolar (fatty) degeneration of hepatocytes. Spleen samples showed large thrombus in the splenic blood vessel, multifocal hemosiderin deposition, congestion of blood vessels, and multifocal infiltration of macrophages. Conclusion: Therefore, it can be concluded that pathological changes in tissues and organs of fish occur proportionally to the pathogen invasion, and because of their high resistance, neomycin and gentamicin utilization in the prophylaxis or treatment of S. agalactiae infection should be avoided.