Development of qPCR-PMAxx™ and ddPCR-PMAxx™ methods for quantitative detection of viable but not culturable Cronobacter sakazakii on stainless steel surfaces after desiccation and exposure to commercial disinfectant.

Abstract

Cronobacter spp. is an opportunistic foodborne pathogen responsible for life-threatening infections such as meningitis and necrotizing enterocolitis in neonates, but also for various complications in elderly and immunocompromised people. Environmental monitoring in powdered milk manufacturing plants shows that Cronobacter can persist in production environments for long periods of time. The aim of this study is to develop a method to assess the viability of Cronobacter cells after implementation of stress encountered in industries to determine whether this bacterium can persist in processing environments in a dormant state known as Viable But Non-Cultivable (VBNC) state, making it undetectable by conventional enumeration methods. We developed two detection systems specific to the genus Cronobacter based on quantitative PCR (qPCR) and Droplet Digital PCR (ddPCR) in combination with a PMAxx™ (Propidium Monoazide) treatment and agar enumeration to assess the viability of detected cells. Despite a better sensitivity for ddPCR, qPCR-PMAxx™ was more suitable for VBNC detection, as it effectively differentiates viable from dead cells. Desiccation at 58 °C and 20 % relative humidity led to a significant reduction in culturable bacteria, with a drop from 6.82 to 2.58 log copies of genome per coupon over 48 h, while qPCR-PMAxx™ revealed that the proportion of VBNC cells represents approximately 1/100 of the total population of viable bacteria (V). Similarly, treatment of Cronobacter biofilms with a peracetic acid containing disinfectant induced the VBNC state in Cronobacter. The ability of this bacterium to enter the VBNC state may explain its long-term survival in processing plants and highlights the need for appropriate detection methods for effective environmental monitoring plans.