Induction of the viable but non-culturable state in bacterial pathogens by household cleaners and inorganic salts

Abstract

Effective monitoring of microbial pathogens is essential for a successful preventive food safety and hygiene strategy. However, as most monitoring strategies are growth-based, these tests fail to detect pathogenic bacteria that have entered the viable but non-culturable (VBNC) state. The present study reports the induction of the VBNC state in five human pathogens by commercially available household cleaners in combination with inorganic salts. We determined that non-ionic surfactants, a common ingredient in household cleaners, can induce the VBNC state, when combined with salts. A screening study with 630 surfactant/salt combinations indicates a correlation between the hydrophobicity of the surfactant and VBNC induction in L. monocytogenes, E. coli, S. enterica serovar Typhimurium, S. aureus and toxin-producing enteropathogenic E. coli. Cells that were exposed to combinations of surfactants and salts for 5 min and up to 1 h lost their culturability on standard growth media while retaining their ATP production, fermentation of sugars and membrane integrity, which suggests intact and active metabolism. Screening also revealed major differences between Gram-negative and Gram-positive bacteria; the latter being more susceptible to VBNC induction. Combinations of such detergents and salts are found in many different environments and reflect realistic conditions in industrial and domestic surroundings. VBNC cells present in industrial environments, food-processing plants and even our daily routine represent a serious health risk due to possible resuscitation, unknown spreading, production of toxins and especially their invisibility to routine detection methods, which rely on culturability of cells and fail to detect VBNC pathogens.

A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens

“…Induction and confirmation of the VBNC state was performed according to Robben et al (2018). One milliliter of early log phase bacteria (OD 600 : 0.6) was centrifuged for 5 min at 8,000 × g .…”

“…However, research has shown that large populations of VBNC cells also exist in unstressed environments, which appear to be a stochastic physiological transformation to protect a subpopulation of cells from unexpected stresses (Ayrapetyan et al, 2015; Gonçalves and de Carvalho, 2016). It has also been demonstrated that VBNC bacteria are able to resist a wide range of unfavorable conditions including starvation, temperature shifts, high or low pH and salinity, oxidative stress, cleaners, antimicrobial compounds, and antibiotics (Nowakowska and Oliver, 2013; Li et al, 2014; Zhao et al, 2017; Ayrapetyan et al, 2018; Robben et al, 2018). In addition, the accumulation of several non-toxic stresses has been shown to switch pathogens into the VBNC state (Overney et al, 2017).…”

A Novel Method for Sampling and Long-Term Monitoring of Microbes That Uses Stickers of Plain Paper

“…Therefore, the method thus detects living, dead, and viable but nonculturable cells (VBNCs). While detection of nongrowing cells in the past was often discussed as a disadvantage (1), increasing interest and awareness of VBNCs today highlight that nongrowing cells are also a potential threat (30). Further, the detection of nongrowing cells advantageously attests to badly cleansed areas, as most chemical disinfectants alone cannot remove DNA.…”

Functionalized Inorganic Nanoparticles for the Detection of Food and Waterborne Bacterial Pathogens

“…In some cases, the futility of normal culture plate technique often requires pre-enrichment, selective enrichment, selective plating and identifications. The sequence of the method is stereotyped, lengthy, time-consuming, limited by its low sensitivity -giving false-negative results even with the presence of viable but non-culturable (VBNC) cells [23,24]. Immunology-based methods present varying disadvantages such as the use of expensive test kits, false result due to cross-reactivity with closely related antigens, the need for pre-enrichment to attain detectable antigen level in samples.…”