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Occurrence of Antibiotic Resistant Bacteria in Pond Water Associated with Integrated Poultry-fish Farming in Bangladesh

FARZANA ASHRAFI NEELA*
Department of Botany, Rajshahi University Rajshahi 6205, Bangladesh

MST. NASRIN AKHTER BANU
Department of Botany, Rajshahi University Rajshahi 6205, Bangladesh

MD. ATIQUR RAHMAN
Department of Botany, Rajshahi University Rajshahi 6205, Bangladesh

M. HABIBUR RAHMAN
Department of Zoology, Rajshahi University Rajshahi 6205, Bangladesh

M. FIROZ ALAM
Department of Botany, Rajshahi University Rajshahi 6205, Bangladesh

Abstract/ Executive Summary:

The use of antibiotics in poultry feed can cause antibiotic resistance in an integrated poultry-fish farming pond environment. Therefore, the goal of this study was to monitor the prevalence of drug-resistant bacteria in pond water associated with poultry-fish farming in Bangladesh. The results showed that the prevalence rate of tetracycline (TC) and ampicillin (AMP) resistant bacteria were 0.24 to 2.59% and 0.16 to 1.0%, respectively. The higher prevalence rate of TC and AMP-resistant bacteria were found in site 3 followed by site 4 and 2 compared with control site 1. Site 2, 3 and 4 were adjacent to the integrated poultry farm. On the other hand, higher TC and AMP-resistant bacteria were found in the rainy season than in the winter. The minimum inhibitory concentration result showed a quite high level of TC and AMP-resistant bacterial distribution in the pond water. Seven genera of resistant bacteria¡a were identified in pond water samples by 16S rRN Agene analysis. Among them Acinetobacter sp. was predominant followed by Enterococcus sp., Pseudomonas sp., Bacillus, Staphylococcus sp., Enterobacter sp. and Brevibacillus sp.

Keywords: Antibiotics; Integrated fish farm; Poultry; Resistant bacteria

Location: Bangladesh

Start Date:

End Date:

Program Area: Pest Management

Commodity: Bacteria

Objectives:

his study was carried out to monitor the occurrence of drug-resistant bacteria in pond water associated with poultry-fish farming in Bangladesh. The results of this study will provide scientific information on the present status of antibiotic-resistant bacteria in the aquatic environment of Bangladesh.

Methodology:

SAMPLING SITE AND SAMPLES Water samples were collected in two different seasons (winter and rainy) from four ponds in Puthia (24° 22′ 30′′ N, 88° 51′ 0′′ E) of Bangladesh, where three ponds were associated with poultry farms and another one was considered as control site (Site-1) which was not adjacent with poultry. Water samples were collected using a sterile bottle and placed in an icebox and transported to the laboratory for further experiment. VIABLE BACTERIAL COUNTS In order to enumerate viable bacteria, water samples were mixed with sterile phosphate-buffered saline (PBS) and a 10 fold serial dilution was done. Plates were counted on nutrient broth plus 1.5% Bactrim agar (BD and Co., Sparks, MD) containing 32 μg/mL of tetracycline (TC) and ampicillin (AMP), respectively. The media without antibiotics was considered as control. The plates were incubated at 25°C for 3 days. Duplicate counting was performed and colony-forming unit (CFU)/mL was calculated. MINIMUM INHIBITORY CONCENTRATION (MIC) Thirty-eight isolates in winter and 40 isolates in the rainy season were found resistant to 32 μg/mL of TC and AMP, respectively and were selected for determining resistance level by MIC assay. The MIC was determined by agar dilution method (Neela et al. 2007). A bacterial cell suspension was prepared in PBS and cell density was adjusted to Macfar land No. 1.0. Ten microliters of bacterial cell suspension were spotted on nutrient agar medium containing 2-fold dilution of TC and AMP concentration of 0, 16, 32, 64, 128 and 256 μg/mL, respectively. Plates were incubated at 25°C for 24 h. Resistance to an antibiotic was defined as ≥ 32 μg/mL (Walsh 2003). DENSIFICATION OF RESISTANT BACTERIA A total of 18 resistant strains were considered for classification by 16S rRNA gene analysis. Polymerase chain reaction (PCR) amplification of 16S rRNA gene was performed using the set of primers of 10f (5’-AGTTTGATCCTGGCTCAG -3’) and r907 (5’-CCGTCAATTCCTTTRAGTTT-3’). The PCR amplification was performed with a PCR Thermal Cycler Dice (TaKaRa, Shuzo Co. Japan). The reaction mixture for PCR contained 1 × Ex Taq reaction buffer (TaKaRa, Otsu, Japan), 2 μL, each of 0.2 mM dNTP, 25 pmol of each primer, 0.62 U of Ex Ta qDNA polymerase (TaKaRa, Otsu, Japan) and 20-90 ng of template DNA in a final volume of 25 μL. PCR cycles consisted of initial denaturing at 95°C for 2 min, denaturing at 95°C for 30 s, annealing at 54°C for 45 s and elongation at 72°C for 1.30 min. The reaction was performed for 22 cycles. Five μL of PCR product was analyzed on electrophoresis of a 1.5% (w/v) agarose gel. Gels were stained with ethidium bromide and visualized on an Epi-Light UV FA1100 system with a luminous Imager version 2.0 (Aisin Cosmos R&D, Aichi, Japan). The PCR products of 16S rRNA were purified by ethanol precipitation and sequencing were performed for both strands with a BigDye Terminater version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) by a 3130× -AB1 PRISMDNA sequencer (Applied Biosystems, Foster City, CA, USA). On-line similarity searching was performed using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information web site (NCBI, http://www.ncbi.nlm.nih.gov/).

Source of Information: Sains Malaysiana 44(3)(2015): 371–377

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

In conclusion, these results suggested that aquatic environments associated with integrated poultry farms in this region could be consider as a reservoir for the antibiotic-resistant bacteria.

Knowledge Management: Journal