Antibiotic resistance among bacteria from Antarctic and Tropics

Introduction Since the discovery of penicillin in 1929, antibiotics have been continuously used to cure diseases caused by bacterial infections. The use of antibiotics has somehow increased the survival rate of human suffering from diseases caused by bacterial infections. Initially, many believed that antibiotics may cure and eliminate infectious diseases. However, diseases that are caused by bacteria such as diarrhea, pneumonia, tuberculosis and other nosocomial infections still remain until today as the bacteria continue to evolve to acquire resistance to the effects of antibiotics (St. Clair and Vissick, 2010). Bacteria that are resistant to antibiotics are commonly found in the natural environment (Kummerer, 2009). Antibacterial resistance that develops in both human pathogens and other microorganisms is sometimes due to the regular exposure to antibiotics or the transmission of resistance traits within and between microbes (Guillemont, 1999). In general, resistance towards antibiotics can be divided into two categories, intrinsic and acquired resistance. Intrinsic resistance occurs naturally in a species and is not due to exposure to antibiotics. Acquired resistance occurs in species that are originally sensitive to an antibiotic, but later develop the resistance. Bacteria can develop antibiotic resistance through mutation or exchange of genetic material among similar or T R A N S A C T IO N S O N S C IE N C E A N D T E C H N O L O G Y E. JAMES & C. M. V. L. WONG. 2015 / Transactions on Science and Technology / 2(2):16 20 17 closely related species. In order to get a better picture of the progression of resistance, it is important to gather more information on the antibiotic resistance capabilities of bacteria from clinical and nonclinical environments (Martinez, 2008). The emergence of antibiotic resistant strains of environmental bacteria and human pathogens is a natural phenomenon that happens when bacteria are constantly exposed to sub-inhibitory concentrations of antibiotics. As a result, many environmental bacteria develop resistance to multiple antibiotics. The increasing usage of antibiotics and consumption without following the prescriptions among patients, and in the food industry are among the main factors that lead to antibiotic resistance. Antibiotic resistance genes that occur naturally in environmental bacteria can potentially be transferred to human pathogens (Martinez, 2008; Martinez, 2009). Environmental bacteria could be a reservoir for antibiotic resistant genes. Despite these, there are relatively limited numbers of studies on the presence of resistance determinants in environmental bacteria, especially those from the tropics and polar regions. Hence, this project was set out to determine the antibiotic resistant profiles of bacteria from the tropics and Antarctic. Methodology Bacterial Samples Origin Thirteen bacteria from the Schirmacher Oasis and King George Island, Antarctica and ten bacterial strains from Universiti Malaysia Sabah were used in this study. Morphology and Physiological Observation The morphological and physiological properties of all bacterial isolates were determined by basic Gram staining and motility test using motility agar techniques. Physical properties of the bacterial colonies were also recorded. Antibiotic Susceptibility Test (AST) AST was carried out using Oxoid TM Antibiotic Susceptibility Disks (Thermo Fisher Scientific Inc.). All bacterial strains were tested against 13 types of antibiotics, namely nitrofurantoin 100 μg, novobiocin 5 μg, rifampicin 5 μg, metronidazole 10 μg, compound sulphonamides 300 μg, ciprofloxacin 5 μg, lincomycin 15 μg, ceftadizime 30 μg, vancomycin 30 μg, vancomycin 30 μg, imepenem 10 μg, streptomycin 10 μg, and trimethroprim 5 μg. The bacteria were grown in broth medium and spread evenly onto the Mueller-Hinton (MHA) agar medium. Antibiotic disks were placed on the inoculated agar and incubated overnight. The diameter of the inhibition zone was measured and the result was scored by referring to the Kirby-Bauer chart. DNA Genomic Isolation Genomic DNA was extracted from the bacterial isolates according to the methods described by Nishiguchi et al. (2002). T R A N S A C T IO N S O N S C IE N C E A N D T E C H N O L O G Y E. JAMES & C. M. V. L. WONG. 2015 / Transactions on Science and Technology / 2(2):16 20 18 Plasmid extraction Alkaline lysis method was used for plasmid extraction. 16S rDNA Amplification Amplification of 16S rDNA was carried out using primers BSF8/20 (5’-aga-gtt-tga-tcc-tgg-ctc-ag-3’) and BSR1541/20 (5’-aag-gag-gtg-atc-cag-ca-3’). The PCR mix consisted of 5μl DNA, 10μl 5x buffer, 3μl MgCl2, 1μl dNTPs, 1μl forward and reverse primer, 0. 25μl polymerase and sterilized distilled water. The PCR conditions were pre-denaturation step for 2 mins at 96°C followed by 30 cycles of 30 sec at 96°C, 40 sec at 52°C, 1 min at 72°C and ended with final extension step at 72°C for 10 min. The PCR product was analyzed using a 0.8% agarose gel, stained with ethidium bromide and visualized under UV trans-illumination. Cloning Purified PCR products were cloned using the TOPO-TATM cloning kit (Invitrogen) according to the manufacturer’s protocol (Thermo Fisher Scientific, Inc.) and sequenced using a Sanger sequencer. Sequencing and DNA Analysis The DNA sequences were aligned using the basic local alignment search tool (BLAST) (https://www.ncbi.nlm.nih.gov/blast) software at National Centre for Biotechnology Information website. Alignments were carried out using ClustalW of MEGA software version 6 (Tamura et al.,