Hassan MM
Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University (CVASU), Khulshi, Chittagong-4225, Bangladesh
Ahaduzzaman M
Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University (CVASU), Khulshi, Chittagong-4225, Bangladesh
Alam M
Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University (CVASU), Khulshi, Chittagong-4225, Bangladesh
Bari MS
Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University (CVASU), Khulshi, Chittagong-4225, Bangladesh
Amin KB
Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University (CVASU), Khulshi, Chittagong-4225, Bangladesh
Faruq AA
Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University (CVASU), Khulshi, Chittagong-4225, Bangladesh
Antimicrobial resistance, Effluents, Hospital, Slaughterhouse, E. coli, Salmonella
Faculty of Veterinary Medicine, Chittagong Veterinary and Animal Sciences University (CVASU), Khulshi, Chittagong-4225, Bangladesh
Animal Health and Management
Chittagong is the second largest city, located in southeastern part of Bangladesh. Its estimated population stands at over 5 million and population density per square km is 15276 (http://www.dmb.gov.bd). To provide health care services to her metropolitan civilian, livestock and poultry, it has a number of medical and veterinary hospitals including General hospital, Upazila health complex, Family welfare center, TB hospital, Infectious disease hospital, Diabetic hospital, Mother and children hospital and Police hospital. From which six medical hospitals, five veterinary hospitals and five slaughterhouses were selected randomly. The study was conducted during the period of September to December, 2012. Samples were collected from final effluents of medical hospitals, veterinary hospitals and slaughterhouses. About 250 ml pre-sterilized glass bottles were used to transport the samples to PRTC (Poultry Research and Training Centre) laboratory for analysis. Peptone water (Oxoid Ltd., PH: 6.2±0.0) was used as primary enrichment media for E. coli and Salmonella. Five selective media were used for the isolation of the bacteria. The MacConkey agar (Oxoid Ltd., PH 7.4±0.2) and EMB agar (Merck, PH: 7.1±0.2) were used for E. coli, XLD agar (Oxoid Ltd., PH 7.4±0.2), BGA agar (Merck, PH: 6.9±0.2) and TSI agar (Oxoid Ltd., PH: 7.2±0.2) for Salmonella. Muller Hinton agar (Biotec, PH: 7.3±0.1) was used for determination of antibiotic resistant bacteria. For the isolation of E. coli, 1ml of water sample was inoculated in screw cap test tube containing buffer peptone water (primary enrichment media) and incubated overnight at 37°C. After primary enrichment culture of the buffer peptone water containing bacteria was streaked on MacConkey agar and incubated for another 24 hours at 37°C. After overnight incubation the bacterial growth was observed. The pink color colony suspected for E. coli. Then again sub-culture was done on EMB agar and incubated as the above mentioned time period. The growth of characteristic metallic sheen like colony was confirmed to E. coli positive. It was further confirmed by Gram’s staining and Indole biochemical test. For the isolation of Salmonella, 1ml of water sample was inoculated in screw cap test tube containing buffer peptone water (primary enrichment media) and incubated for 24 hours at 37°C. After primary enrichment sample from buffer peptone was picked up and streaked on both XLD and BGA agar. The agar plates then were incubated at 37°C for 24 hours. After development of characteristic colony the positives were selected for biochemical test (TSI stab) to confirm Salmonella. Gram’s staining was performed as per procedures described by [13] to determine the size, shape and arrangement of bacteria. Therefore, the suspected colonies were taken over a slid to make a thin smear that was done by sliding the edge of another glass slide across the glass slide containing the sample and then allowed it to air dry. The smear was then heat fixed by quickly passing it two to three times through a flame. After fixation the Gram’s staining was done as follows: Crystal violet was used for two minutes, Gram’s iodine for 1 minute, Acetone for 5-7 seconds and finally, Safranin for 1 minute. Rinsing was done gently with tape water after every step. The slide was then observed by microscope under 100X with immersion oil and characterization of bacteria was done. The tube of tryptone broth was inoculated with a small amount of pure culture at 37°C for overnight. A positive Indole test is indicated by the formation of a pink to red color ("cherry-red ring") in the reagent layer on top of the medium within seconds of adding the reagent. A straight inoculating needle was used to pick up isolated colony from culture of isolates. The TSI slant was inoculated by stabbing the butt down to the bottom, and then streaked over the surface of the slant. The TSI slant was then incubated overnight at temperature of 37°C. The positive result for Salmonella and E. coli were detected based on the properties. After confirmation of isolates as E. coli and Salmonella, antimicrobial susceptibility of the isolates was determined by using the micro disc diffusion method, and the method was used according to guidelines established by Clinical and Laboratory Standards Institute [18]. Antibiotics selected for susceptibility testing included a panel of antimicrobial agents of interest to the poultry industry and public health authorities. From the range of antimicrobial drugs, 10 were selected on the basis of their range of activity against entero-bacteria on their use in local poultry farming and human medicine. Veterinary antibiotics were chosen due to their use as therapeutic, prophylactic or growth promoting agents in livestock industry and human antibiotics were selected on the basis of their use and /or importance in human medicine. The following antibiotics and disc potencies were used for E. coli and Salmonella: GEN: Gentamicin (10mcg), DO: Doxycycline (30mcg), CIP: Ciprofloxacin (5mcg), TA: Oxytetracycline (30mcg), ENR: Enrofloxacin (5mcg), AMP: Ampicillin (25mcg), CL: Colistin (10mcg), N: Neomycin (30mcg), E: Erythromycin (15mcg) and PF: Pefloxacin (5mcg). Measurement of the growth inhibition zone permitted the classification of each isolates as susceptible, intermediate and resistant according to data provided by HiMedia Laboratories pvt. Limited, Mumbai. Data analysis Data obtained was imported to the Microsoft Office Excel-2007 and transferred to the software STATA/IC-11 for analysis. Descriptive statistics was done by using the STATA software and expressed as percentages of different variables like resistance, intermediate and sensitivity pattern of antimicrobials.
International Journal of Natural Sciences (2015), 5(2) 52-58
Journal