A Method of Universal Primer Design for The Detection of Diverged CTX-M Beta-Lactamases in Multi-Drug Resistant Superbugs

Asit Kumar Chakraborty; Sourav Kumar Nandi

Abstract

Complexity of many diverged β-lactamases in common multi-drug resistant bacteria has reached very high level. We found >40% of common bacteria in Kolkata water bodies were ampicillin resistant and reported clinical isolates were >95% ampicillin resistant worldwide. More than twenty unique sequence classes of β-lactamases were known to cause MDR inactivating penicillin, cephalosporin and carbapenem drugs. Further, there are other diverged MDR genes like drug efflux genes (acrAB, mexAB-EF, tetA), drug modifying genes (aacA1/C1, strA/B, cat3B, sul1/2) and drug transporter genes (mexAB, MdtABC) were present in single bacterial plasmid suggesting too many PCR reactions to be performed to understand the drug resistomes. Thus, we devised a method of reduction in PCR assays using seq-2 and forced multi-align analysis of ~195 blaCTX-M protein sequences for universal primer design. The universal primer set recognized major type-1, type-2 and type-9 blaCTX-M mutants including clinically relevant blaCTX-M-15. However, such universal primers were detected mostly CTX-M-1 type β-lactamase gene and a TEM-SHV another universal primer were identified blaTEM-1 type gene in Kolkata superbugs whereas SHV-isomers were infrequent. BlaTEM-1 β-lactamase 132 alanine amino acid polymorphism (GCT-GCG) was detected signifying the amp gene of pBR322 plasmid origin. This study was supported the WHO recommendations to adopt a uniform policy for AMR detection and antibiotic therapy worldwide.Keywords: Universal primers, Diverged CTX-M β-lactamases, PCR diagnosis, MDR conjugative plasmidsCite this ArticleAsit Kumar Chakraborty, Sourav Kumar Nandi. A Method of Universal Primer Design for The Detection of Diverged CTX-M Beta-Lactamases in Multi-Drug Resistant Superbugs. Research & Reviews: A Journal of Biotechnology. 2019; 9(2): 1–10p.