Browsing by Author "Liyanage, G. Y."

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Bacterial degradation of tetracycline (TET) by TET resistant bacteria; A green solution for antibiotic pollution.
(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Liyanage, G. Y.; Manage, P.M.
Tetracyclines (TET) have been extensively used in aquaculture for chemotherapy against various fish diseases such as fin rot, skin ulcers. Overuse and misuse of antibiotics are widely regarded as two of the major factors promoting antibiotic resistance. Resistance to TET occurs via two primary mechanisms; ribosomal protection and enzyme inactivation. To minimize the development of resistance, excess amount of TET should be removed from the aquatic environment. Therefore, studies on the biodegradability of TET can be taken as a very first step of an environmental risk assessment. The present study reports the biodegradation of TET by Enterobacter sp., Micrococcus luteus, Enterobacter ludwigii, Bacillus sp. and Streptomyces sp., which were isolated as TET resistance, non-pathogenic bacteria. In a previous study, TET resistant bacterial isolates were identified using the 16s rRNA sequencing. Overnight bacterial cultures grown in a medium without TET, were introduced into a medium containing TET at final concentration of 5 μg/ml. Triplicate samples were incubated at 28 °C with shaking at 100 rpm under dark conditions. Subsamples (0.5 ml) were removed at 2 days interval for a period of 14 days. Remaining TET in the subsamples was analyzed using High Performance Liquid Chromatography (HPLC). Complete degradation of TET by M. luteus and E. ludwigii was detected at the end of 14 days of incubation. Descending degradation percentages were followed by Enterobacter sp. (74%), Streptomyces sp. (72%) and Bacillus sp. (70%) at 14 days, respectively. Lowest half-life time of TET was shown by M. luteus and E. ludwigii (6 days), whereas half-life time of 8 days, 11 days and 12 days were recorded for Enterobacter sp, Streptomyces sp. and Bacillus sp., respectively. According to the degradation results obtained, it can be concluded that the selected bacterial strains can be used as potential candidates to be introduced into wastewater effluents to remove TET in effluent water, before reaching natural environments
Detection of antibiotic residues and characterization of antibiotic-resistant potential of bacteria in compost samples in Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) Sewmi, J. A. G.; Liyanage, G. Y.; Wijerathna, P. A. K.; Manage, P. M.
Antibiotic residues in the environment contribute to the survival and growth of antibiotic-resistant bacteria (ARB) by applying selective pressure on microbial populations. This enables the quick spread of antibiotic-resistant factors among various bacterial populations in the environment. This research aimed to detect antibiotic residues in compost and determine the presence of ARB in compost samples. A total number of 11 samples including 6 Municipal Solid Waste (MSW) samples and 5 commercial compost samples were collected for the study. Amoxicillin (AMX), Tetracycline (TET), Cloxacillin (CLOX), and Ciprofloxacin (CIP) were selected as antibiotics to be determined in the samples. The selected antibiotics in the samples were concentrated using the Solid Phase Extraction (SPE) and detected by High-Performance Liquid Chromatography (HPLC). The standard pour plate technique on Nutrient Agar (NA) medium was used to isolate the total viable counts. The ARB was isolated and Minimum Inhibitory Concentrations (MIC) were measured according to the Clinical and Laboratory Standard Institute (CLSI) guidelines, using nutrient broth. The results indicated the absence of antibiotic residues in compost samples. MSW compost had a higher total viable count as well as ARB count (TVC; 224×104–368×104 CFUmL1: ARB; 20×104–47×104 CFUmL-1) compared to commercial compost (TVC; 50×104–198×104: ARB; 2.5×104–18×104). Sixty-eight morphologically different bacterial isolates were counted for the selected antibiotics. Among them 37% exhibited resistance to AMX, 35% to CLOX, 15% to TET, and 13% to CIP. None of the isolates recorded the MIC ranges between 60 and 180 μgmL-1. From the selected 4 antibiotics, 28% isolates for AMX, 12% isolates for CLOX, 22% isolates for CIP, and 20% isolates for TET showed the MIC ranges between 180 and 300 μgmL-1. From the total isolates, 32% bacterial isolates for AMX, 4% bacteria isolates for CLOX, and none of the isolates for CIP and TET showed MIC between 300 μgmL-1 and 420 μgmL-1. Further, 4% bacterial isolates for AMX, 17% bacterial isolates for CLOX, and none of the isolates for CIP and TET showed MIC between 420 μgmL-1 and 660 μgmL-1. Thirty-six percent isolates for AMX, 67% isolates for CLOX, 78% isolates for CIP, and 80% isolates for TET, which showed more than 660 μgmL-1 concentration of MIC. The overall results of this study revealed that the selected compost creates a reservoir for antibiotic-resistant bacteria. There can be a spread of ARB, even under the absence or lower concentrations of antibiotic residues than the instrument detective level (0.05 ppm). Higher MIC values indicate the requirement of greater antibiotic doses for pathogen control, which could create severe health risks.
Determination of Multiple Antibiotic Resistant (MAR) and MAR Index in bacteria isolated from aquaculture farms.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Liyanage, G. Y.; Manage, P.M.
Concurrent resistance to antibiotics of different structural classes has arisen in a multitude of bacterial species and cause complicating the therapeutic management for both human and animal. The objective of the study was to determine the Multiple Antibiotic Resistance (MAR) and MAR index of isolated tetracycline resistance bacteria from aquaculture sources. 42 tetracycline resistance bacteria, which were previously isolated from aquaculture farms were tested against Oxytetracyline (OTC), Tetracycline (TET), Amphicillin (AMP), Amoxicillin (AM.X), Sulfamethaxasol (SMX), Trimethoprim (TMP), Erythromycin (ERM), Cloxacillin (CLOX) and Ciprofloxacin (CJP). The MAR was determined by using Triphenyl Tetrazolium Chloride (TTC) assay and agar dilution method following CLSI guidelines. Then the MAR index was calculated. Among the isolates, 64.3% were resistant to three or more antibiotics and considered as multiple antibiotic resistant according to the WHO and CLSI guidelines. Among the MAR isolates, 93 % were resistant to OTC, 90% to TET, 34% to AMX, 14 % to AMP, 13 % to ERM, 11 % to SMX-TMP and 2% were resistant to CLOX were detected. None of the bacteria were resistance for CIP. The calculated MAR index was ranged from 0.03 to 0.42 for the isolated bacteria and the highest MAR index was recorded by Staphylococcus sp. (0.42), while the lowest was shown by Enterobacter sp. (0.03). The results of the study indicate that the overuse and misuse of antibiotics led to emergence of MAR bacteria and it compromises effectiveness of antibacterial therapy, leading infectious microorganisms to become resistant against antibiotics.
Effect of storage temperature and time on histamine concentration in commercially available fresh fish in Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) Perera, K. B. S. M. H.; Liyanage, G. Y.; Manage, P. M.
People consume fish in order to fulfil their protein requirements. As fish contain high protein and amino acid levels and are susceptible to enzymatic and bacterial processes that result in the formation of biogenic amines like histamine, can lead to histamine fish poisoning. Histamine is a heat-stable compound, resistant to thermal processing and improper storage conditions can elevate its levels in foods. This study evaluated the effect of storage temperature and time on histamine concentration in commercially available fresh fish. Four species of fresh fish (n = 20 each) were examined: frigate tuna (Auxis thazard), Indian scad (Decapterus russelli), skipjack tuna (Katsuwonus pelamis), and mackerel tuna (Euthynnus affinis). Histamine quantification was performed using HPLC-DAD system with a mobile phase consisting of 85% of phosphate buffer and 15% acetonitrile. Fish muscle samples were kept at 27 ℃, 32 ℃, and 37 ℃, and histamine concentrations were measured at these specific temperatures. Study results indicated that, histamine concentration increased with storage temperature. At 37 ℃ all species showed the highest histamine concentrations: frigate tuna (17.99 ± 0.93 mg/kg), Indian scad (14.59 ± 0.97 mg/kg), skipjack tuna (38.92 ± 2.55 mg/kg), and mackerel tuna (32.80 ± 1.37 mg/kg) compared to the histamine concentrations at 27 ℃: 13.05 ± 1.06 mg/kg, 12.82 ± 0.88 mg/kg, 28.72 ± 1.93 mg/kg, and 24.37 ± 0.86 mg/kg respectively. Further, fish muscle samples were kept at 4 ℃ for three consecutive days with samples taken at 24 hours, 48 hours, and 72 hours. The initial histamine concentrations, in frigate tuna, Indian scad, skipjack tuna, and mackerel tuna were 12.91 ± 0.27 mg/kg, 13.99 ± 0.01 mg/kg, 27.17 ± 0.63 mg/kg, and 24.91 ± 0.16 mg/kg respectively. However, after 72 hours, the highest histamine concentration was recorded as 15.24 ± 0.32 mg/kg, 14.49 ± 0.29 mg/kg, 29.31 ± 1.12 mg/kg, and 26.41 ± 0.59 mg/kg respectively in all species, indicating that the histamine concentration has increased with storage time duration. The regression analysis indicated that storage temperature and time had a significant effect on histamine concentration for frigate tuna, skipjack tuna, and mackerel tuna (p < 0.05), but not in Indian scad. This study concludes that histamine concentration increases with the temperature, so it is necessary to store fresh fish at lower temperatures, to prevent bacterial growth and histamine production. This study recommends consuming freshly caught fish without prolonged storage, even in refrigerators.
Prevalence of antibiotic resistance in surface water, ground water and sediment in the transition zone of the Kelani River basin, Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) Meddage, A. K. M. M. K.; Liyanage, G. Y.; Idroos, F. S.; Manage, P. M.
Antibiotics are mainly used to treat and prevent diseases in humans and animals, and they also serve as growth promoters and feed efficiency enhancers in agriculture and animal husbandry. However, antibiotic-resistant bacteria (ARB) have emerged as a result of antibiotic overuse and misuse. The prevalence of ARB becomes more critical, particularly for rivers that fulfil urban drinking water demand. Furthermore, the ARB poses a significant threat to human health by potentially treatments ineffective and leading to the spread of untreatable infections. This study aims to screen the presence of ARB against selected antibiotics such as Ciprofloxacin (CIP), Cefuroxime (CXM), Cloxacillin (CLOX), Amoxicillin (AMX), Co-Amoxiclav (CO-AMX); Tetracycline (TC); Azithromycin (AZT), Erythromycin (ERM); Sulfamethoxazole (SMX) and Gentamycin (GEN) in the transition zone of Kelani River Basin, Sri Lanka. Samples were collected during the first inter-monsoon season in March 2024. Twenty-five water samples (groundwater - 07; surface water - 18) and 12 sediment samples were subjected to determine Total Viable Count (TVC) and ARB using the standard pour plate method at 60 mg/L of antibiotic. The TVC of bacteria ranges between 0.2 × 102 – 4.0 × 102 CFU/mL in collected surface water samples, whereas 0.2 × 102 – 2.2 × 102 CFU/mL, and 0.6 × 102 – 1.2 × 102 CFU/mL for groundwater and sediments, respectively. The resistance bacteria percentage against CXM (16.55%), CLOX (15.82%), AMX (13.98%), AUG (12.90%), SMX (10.46%), GEN (10.22%), ERY (8.76%), AZT (6.60%), CIP (2.68%), and TET (2.19%). According to the obtained results, CXM and CLOX showed the highest resistance, indicating bacterial adaptation in both sediment and water. However, TET and CIP exhibited the lowest resistance rates, suggesting they remain effective options. Moderate resistance was observed for SMX and GEN, highlighting the need for cautious use to prevent further resistance. These findings highlight the notable presence of antibiotic-resistant bacteria in environmental samples. Moreover, the results revealed that a thorough investigation into the presence and spread of antibiotic resistance throughout the Kelani River Basin is urgently required.