Heavy Metal and Imidazole Rich Pharmaceutical Effluent Increases AMR Traits and Induces Microbial Persistence Through VBNC Formation

Abstract

Purpose: To explore the effect of pharmaceutical industry effluent on viability and persistence of microbial population (environmental and clinical) with regard to abundance of AMR genes and induction of Viable but non-Culturable (VBNC) state. Methods & Materials: Settings: Effluent from common effluent treatment plants (CETP),Sample collection: Samples were collected from industrial effluent inlet and river discharge pointSample Definition: CETP receives effluent from different manufacturing units and pharmaceutical industries dealing with small molecule drugs.Chemical and Phenotypical analysis: Chemical analysis of effluent, detection of AMR genes, VBNC induction by effluent and resuscitation, morphological identification of microbial species Results: Chemical analysis- Chemical analysis of samples, showed high level of lead, mercury, some hydrolyzed imidazole and other smaller hydrolyzed fragments.AMR gene detection- AMR genes for the fluoroquinolone, Aminoglycoside, beta-lactamase Class A/B/C/D, Erythromycin, Lincosamide Streptogramin_b, and vancomycin were present in test samples, with highest copy numbers of Macrolide and tetracycline. Sample from river discharge point had multidrug resistant efflux pump genes.VBNC induction and resuscitation- Based on chemical analysis and presence of AMR genes, further analysis of effect of heavy metals in test sample was explored on microbial population. Effluent sample upon ultra-centrifugation followed by ultra-filtration, did not show any microbial growth under normal culture conditions, but showed growth upon co-culture with E. coli. This indicates the presence of bacterial population in the shrunken form in effluent reflecting the VBNC state.Microbial identification- Different bacterial colonies that was observed during resuscitation were analyzed by gram staining. All 44 colonies were categorized into four groups, gram positive cocci (30), gram positive rod (8), gram negative cocci (4) and gram-negative rod (2), while from unfiltered culture it was 46, 12, 11 and 7 respectively. Conclusion: Presence of heavy metals in the industrial effluent generate selection pressure resulting in emergence of AMR traits. Also, heavy metal and imidazole rich effluent can induce VBNC state and persistence of microbial population, which upon attaining favorable condition, can be resuscitated. This work has significant implication in devising future strategies for effluent treatment to minimize environmental pressure and in turn reduce emergence of AMR.