Comparison between Flow Cytometry and Traditional Culture Methods for Efficacy Assessment of Six Disinfectant Agents against Nosocomial Bacterial Species.

Richard Massicotte,Akier A Mafu,Gilbert Pichette,Darakhshan Ahmad,Pierre Belhumeur,Francis Deshaies

doi:10.3389/fmicb.2017.00112

Abstract

The present study was undertaken to compare the use of flow cytometry (FCM) and traditional culture methods for efficacy assessment of six disinfectants used in Quebec hospitals including: two quaternary ammonium-based, two activated hydrogen peroxide-based, one phenol-based, and one sodium hypochlorite-based. Four nosocomial bacterial species, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Vancomycin-resistant Enterococci faecalis, were exposed to minimum lethal concentrations (MLCs) and sublethal concentrations (1/2 MLCs) of disinfectants under study. The results showed a strong correlation between the two techniques for the presence of dead and live cell populations, as well as, evidence of injured populations with the FCM. The only exception was observed with sodium hypochlorite at higher concentrations where fluorescence was diminished and underestimating dead cell population. The results also showed that FCM can replace traditional microbiological methods to study disinfectant efficacy on bacteria. Furthermore, FCM profiles for E. coli and E. faecalis cells exposed to sublethal concentrations exhibited distinct populations of injured cells, opening a new aspect for future research and investigation to elucidate the role of injured, cultural/noncuturable/resuscitable cell populations in infection control.

Highlights

Surface disinfectants play a major role in controlling the environmental transmission and spread of nosocomial infections by contact with hitherto “noncritical” inanimate/environmental surfaces in the hospital and healthcare facilities (Mafu et al, 2015)
Validation of Viability Profiling Method by Flow Cytometry (FCM) Compared to Fluorescent Microscopy and Classical Spot-Plating Using E. coli Cells Exposed to quaternary ammoniums (QA)-Based Disinfectant
Gating and analysis strategy for front scatter counts (FSC) vs. side scatter counts (SSC) dot plots, presented in Figure 1, shows that all cells in the sample treated with lethal concentration of the disinfectant contained only one population of cells stained with propidium iodine (PI) mapping on the left upper quadrant region (Red zone representing dead cell area)

Summary

Introduction

Surface disinfectants play a major role in controlling the environmental transmission and spread of nosocomial infections by contact with hitherto “noncritical” inanimate/environmental surfaces in the hospital and healthcare facilities (Mafu et al, 2015). It was concluded that this could be better managed if a proper assessment of the risk associated with the use of disinfectants at off-recommended strengths conditions is considered to provide guidance toward and seeking satisfactory solutions (Deshaies et al, 2012). In such in vitro efficacy studies targeted to develop guidelines for on-site/in vivo practices, the action to be taken is by nature delayed to get the results by traditional/ classical culture based methods. Flow cytometry (FCM) combined with viability staining using propidium iodine (PI) marker to assess the integrity of the cell membrane is a suitable technique for this kind of study (Davey and Kell, 1996; Fugère et al, 1996; Nebevon-Caron et al, 1999; Wozniak-Kosek and Kawiak, 2005; Allegra et al, 2008; Cronin and Wilkinson, 2010; Hwang et al, 2012)

Methods

Results

Discussion

Conclusion