ABSTRACT
ß-lactam antibiotics are the most frequently prescribed class of drugs worldwide, due to its efficacy and good safety profile. However, the emergence of ß-lactamase producing bacterial strains eliminated the use of ß-lactam antibiotics as a chemotherapeutic choice. To restore their usability, a non-antibiotic adjuvant in conjunction with ß-lactam antibiotics is now being utilised. Cholic acid potentially acts as an adjuvant since it can blunt the pro-inflammatory activity in human. Our main objective is to scrutinise the inhibition of ß-lactamase-producing bacteria by adjuvant cholic acid, synergism of the test drugs and the primary mechanism of enzymatic reaction. Antibacterial effect of the cholic acid-ampicillin (CA-AMP) on 7 ß-lactamase positive isolates were evaluated accordingly to disc diffusion assay, antibiotic susceptibility test, as well as checkerboard analysis. Then, all activities were compared with ampicillin alone, penicillin alone, cholic acid alone and cholic acid-penicillin combination. The CA-AMP displayed notable antibiotic activity on all test bacteria and depicted synergistic influence by representing low fractional inhibitory concentration index (FIC ≤ 0.5). According to kinetic analyses, CA-AMP behaved as an uncompetitive inhibitor against beta lactamase, with reducing values of Michaelis constant (Km) and maximal velocity (Vmax) recorded. The inhibitor constant (Ki) of CA-AMP was equal to 4.98 ± 0.3 µM, which slightly lower than ampicillin (5.00 ± 0.1 µM).
Subject(s)
Cholic Acid/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Gram-Negative Bacteria/growth & development , Gram-Positive Bacteria/growth & development , beta-Lactamase Inhibitors/pharmacology , Ampicillin/pharmacology , Drug Synergism , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/enzymology , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/enzymology , Microbial Sensitivity Tests , Microbial Viability/drug effects , Penicillins/pharmacology , beta-Lactamases/metabolismABSTRACT
Human erythropoietin (huEPO) is a glycoprotein with important physiological functions, such as erythropoiesis, angiogenesis, and wound healing. A therapeutic protein, huEPO is commonly used to treat patients suffering from renal and non-renal anemia. Recombinant human erythropoietin (rhuEPO) and endogenous huEPO are similar with respect to their biological and chemical properties. In this study, we describe the construction of synthetic huEPO gene to produce rhuEPO. The synthetic huEPO gene was constructed by overlapping oligonucleotides assembly and amplified by polymerase chain reaction (PCR). Twenty oligonucleotide sets, covering the huEPO gene sequence and two newly introduced restriction enzyme sites, were pulled together and amplified using Pfu DNA polymerase to produce the expected DNA products with sizes of ~500bp and ~600bp. The PCR products were ligated into pGEM-T plasmid vector to facilitate DNA sequencing process of the constructed huEPO gene and downstream cloning manipulation. DNA sequence analysis showed correctly assembled oligonucleotide sets, representing the huEPO gene sequence albeit with minor base mutations. Hence, oligonucleotides assembly and PCR amplification provide a convenient and speedy method for the synthesis of huEPO gene without depending on mRNA isolation and reverse transcription or the need to have a genomic library.
