Detection of Autoinducer-ii (Ai-2) Regulatory Genes in Multi-drug Resistance (Mdr) Zoonotic Bacterial Pathogens and Their Inhibitory Phytochemicals

Abstract

The worldwide utilization and abuse of antibiotics has led to the progression and outspread of bacterial resistance against all regularly used antibacterials. In order to successfully tackle broaden of antimicrobial resistance (AMR) through development of novel antimicrobial compounds or antimicrobial stewardship, interpretating how bacteria transform and develop to live on antibiotic treatments is critical. Keeping these aspects in mind present study was designed to isolate and identify multi drug resistance (MDR) zoonotic bacterial pathogens mainly focusing on E. coli and pseudomonas. In total of 100 samples received in two groups i.e. 50 samples from milk and 50 from faecal. The study revealed 23 isolates based on cultural, morphological, biochemical and molecular characterization. Out of 50 milk samples 9 isolate was confirmed as E.coli and 6 as Pseudomonas and in another 50 samples of faecal, 4 isolates was confirmed as E.coli and 4 as Pseudomonas. All the isolates of E. coli were confirmed by the amplification species specific of 556 base pair uidA genes and isolates of pseudomonas were confirmed by PseudF/R- 16Sr RNA primers of 618 base pair. drug resistance was performed by phenotypic and molecular screening of multi drug resistance isolates. All the bacterial isolates were subjected to antibiotic sensitivity test against 9 antibacterial drugs like: Amoxyclav, Amikacin, Cefoperazone, Cefalexin, Celtriaxene, Levofloxacin, Gentamicin, Streptomycin and PenicillinG. PenicillinG was the only drug against which 100% resistance was recorded by all the isolates irrespective of species of isolates and also the origin of isolates. When all the isolates were classified for antibiotyping, they revealed 13 biotypes of E. coli and 5 biotypes in Pseudomonas. For molecular screening of the multi drug resistance isolates two different group of antimicrobial resistance were selected for the screening i.e. ESBL resistance and carbapenem resistance. All E. coli isolates were subjected for the amplification of two different resistant gene i.e. blaSHV and blaNDM. Amplicons were subjected to Sanger sequencing and sequences of all these genes were submitted in NCBI database to obtain the accession number. Out of 13 E. coli isolates 4 each confirmed the presence of blaSHV and blaNDM genes. Discovery of microbial quorum sensing (QS) has administered new hope for studying the regulatory mechanism of drug resistance and overcoming drug resistance QS system. Numerous bacteria can communicate by contributing and responding to a small diffusible molecule that act as signals. These molecules can be described as auto inducers (AIs). Keeping these aspects study was designed for its second objective for the designing of Autoinducer-II (AI-2) gene primers for isolated major zoonotic bacterial pathogens. These primers were used to fullfil third objective of molecular detection of AI-2 gene in multi drug resistance (MDR) in zoonotic bacterial pathogens. Out of 13 confirmed isolates 12 isolates of E. coli revealed an amplification product of 290bp and accordingly confirmed to be mqsr- AI-2. Whereas. out of 10 confirmed isolates only 6 isolates of the pseudomonas revealed an amplification product of 345bp, resulting in the confirmation of rhIR AI-2 gene. Amplicons were subjected to Sanger sequencing and received data was analyzed. The forth objective was in silico docking based assessment of phytochemicals for inhibition of quorum sensing AI-2 gene. The ten phytoconstituents of Ocimum sanctum showed different positive docking scores against autoinducer-2 translational protein of E. coli and Pseudomonas aeruginosa. However, Germacrene and Rosmarinic acid suggested good docking scores for autoinducer-2 translational protein of Pseudomonas aeruginosa. Out of these Rosmarinic acid full fill all the basec requirements to be a potential drug moleculae.