ANTIBIOTICS AND SURFACE DISINFECTANT INDUCE VBNC AND EMERGENCE OF RESISTANCE IN HOSPITAL-ASSOCIATED PATHOGENS

Abstract

Intro: In an unfavorable environment, bacteria improvise living strategy by ceasing growth and reducing metabolism to attain Viable But Non-Culturable (VBNC) state. Once these bacteria find a favorable environment, they resume growth process. In the last few decades, researchers have demonstrated that several human pathogens can attain VBNC state after exposure to several physical and chemical conditions. Due to low metabolism and non-culturability, VBNC escapes culture-based detection. Pieces of evidence are there to indicate emergence of new mutations and AMR phenotypes in VBNC and resuscitated states. Commonly used antibiotics, ciprofloxacin, amoxicillin and FDA-approved surface disinfectant Glutaraldehyde have not been tested for VBNC induction. We hypothesized that these xenobiotics can induce VBNC in bacterial species commonly found in hospital environments putatively driving emergence of AMR. This knowledge will help in improving pathogen detection, and infection and AMR control. Methods: Ciprofloxacin, Amoxicillin and Glutaraldehyde were demonstrated to induce VBNC in Klebsiella pneumoniae, Acinetobacter baumannii and Escherichia coli. VBNC induction was validated by CFU count, energy production, membrane integrity and basal metabolism. Confocal microscope for morphology visualizaion, disk diffusion for AST phynotype. Findings: All three xenobiotics induced VBNC state in ∼ 85% of cells in the bacterial population, leaving the rest 15% non-viable, and successfully resuscitated by removing the xenobiotic. The resuscitation started after 24 hours to 48 hours in various conditions and showed increased AMR phenotype. The microscopic visualization showed shrunken but elongated morphology in VBNC as well as resuscitated cells. Conclusion: Our study demonstrated that commonly used antibiotics and surface disinfectant can induce VBNC, which in turn poses a highly probable risk of missing surface microbial contamination in hospital settings. Also, the emergence of new AMR traits due to exposure to these xenobiotics indicates the necessity of further insights into the mechanism of VBNC induction and resuscitation at molecular level and development of safer alternative antimicrobial agents