Role of antibodies in neutralization of beta- lactamase enzymes from antibiotic resistant bacteria

Summary:-Antibiotic resistant bacteria impose substantial burden on the human population animal health. So society as a whole must pay for development of new antibiotic to keep pace with continually evolving pathogens so the current research aims to treat1- E. coli isolates obtained from different sources were resistant to β-lactams in addition to other drugs, the isolates contained resistant genes TEM, SHV and OXA.2- β-lactamase expressed by TEM, SHV and OXA genes were extracted as 30, 25 KD as that obtained with standard enzyme the protein content of the extracted enzymes were determined by lowery method.3- Anti β-lactamase antibodies could be produced in rabbits as polyclonal antibodies.4- Anti β-lactamase had the ability to combine with extracted β-lactamase through western blot analysis technique.5- Exposure of - β-lactams resistant E. coli to β-lactams and other drugs with anti β-lactamase.The obtained results were as follow:1. Disc diffusion susceptibility patterns out of 11 E. coli isolates 4 out of 11 E. coli isolates were sensitive to enrofoxacin by (36.4%) 7 isolates were resistant by (63.6%), 6 isolates were sensitive to cefotaxime by (54.5%), 1 isolate was intermediate2. Purification of beta lactamase by gel filtration technique using column chromatography using sephadex G75 was the ideal method that yields maximum volume of β–lactamases enzymes.3. Sodium dodecycle sulfate gel electrophoresis, extract of 2 E. coli isolates (cod No.Q1 and Q2) in addition to standard TEM enzyme were subjected to SDS-PAGE. 3 bands were observed TEM and or SHV at 30 KD, standard TEM at 30 KD and OXA at 25 KD. (Q1–TEM4. The protein concentration using was 625 and 750µg/ml of Q1 and Q2 E.coli strains respectively.5. Immunoblotting of β-lactamase enzymes extracted from strain Q1 and Q2 transferred to nitrocellulose paper and probed with serum samples from injected rabbits in addition to standard TEM revealed that formation of 3 lanes at molecular weight 30KD.6. The MIC range of amoxicillin / clavulanic acid against 11 E. coli isolates was < 4 µg/ml. The MIC range of cefotaxime against 6 E-coli isolates was < 4 µg/ ml.7. Increase in the MIC of amoxicillin /clavulanic acid value after addition of β-lactamase antigen in all tested 11 E. coli isolates.8. Increase in the MIC of cefotaxime antibiotic value after addition of β-lactamase antigen in all tested 6 E. coli isolates.9. Neutralization effect of prepared antiserum(from strain Q1) 10 of total 11 E. coli isolates show DROP in MIC of amoxicillin /clavulanic acid by one fold but strain H4 E. coli show no enhancement.10. Neutralization effect of prepared antiserum against standard TEM which showed no effect on 11 E. coli isolates.11. Decrease in the total colony count of tested E. coli isolates after addition of anti β-lactamase serum prepared against extracted β-lactamases from strain Q1 E. coli with cefotaxime antibiotic by 50-70% in all tested 6 E. coli isolates.12. Decrease in the total colony count of tested E. coli isolates after addition of anti β-lactamase serum prepared against extracted β-lactamases from strain Q1 E. coli with cefotaxime antibiotic by 40-50%.by 50% in all tested 6 E. coli isolates in addition to production of pinpoint colonies after subculture on nutrient agar.13. Disc diffusion sensitivity test of 6 E. coli isolates showed inhibition zone to gentamicin, amoxicillin, cefotaxime, streptomycin and piperacillin, two isolates showed inhibition zone to oxacillin while 4 isolates showed no inhibition zone, but 1 of 6