Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry

Abstract

Pseudomonas aeruginosa is a Gram‐negative bacterium that is abundant in the environment and water systems, with strains that cause serious infections, especially in patients with compromised immune systems. In times of stress or as part of its natural life cycle, P. aeruginosa can adopt a viable but not culturable (VBNC) state, which renders it undetectable by current conventional food and water testing methods and makes it highly resistant to antibiotic treatment. Specific conditions can resuscitate these coccoid VBNC P. aeruginosa cells, which returns them to their active, virulent rod‐shaped form. Underreporting the VBNC cells of P. aeruginosa by standard culture‐based methods in water distribution systems may therefore pose serious risks to public health. As such, being able to accurately detect and quantify the presence of VBNC P. aeruginosa, especially in a hospital setting, is of critical importance. Herein, we describe a method to analyze VBNC P. aeruginosa using imaging flow cytometry. With this technique, we can accurately distinguish between active and VBNC forms. We also show here that association of VBNC P. aeruginosa with Acanthamoeba polyphaga results in resuscitation of P. aeruginosa to an active form within 2 h. Our approach could provide an alternative, reliable detection method of VBNC P. aeruginosa when coupled with species‐specific staining. Most importantly, our experiments demonstrate that the coculture with amoebae can lead to a resuscitation of P. aeruginosa of culturable morphology after only 2 h, indicating that VBNC P. aeruginosa could potentially resuscitate in piped water (healthcare) environments colonized with amoebae. © 2019 International Society for Advancement of Cytometry

Role of amoebae for survival and recovery of ‘non-culturable’ Helicobacter pylori cells in aquatic environments

“… 2007 ; Dey et al . 2019 ). We thus investigated VBNC- H. pylori reactivation in co-culture with amoebae using culture and imaging flow cytometry.…”

“…Intracellular locations of GFP- H. pylori in W. magna coculture was performed using ImageStream ® cytometry as previously described (Dey et al . 2019 ). Briefly, FLA were infected for 2 h with viable spiral GFP- H. pylori at a MOI of 100, washed and re-suspended in PBS prior to processing through the ImageStream ®X Mark II (Millipore Sigma).…”

“…The bacteria gating strategy as previously described (Dey et al . 2019 ) and briefly provided in ( Supplementary data SD. 2 ).…”

“…To investigate this further, we analyzed the morphological changes of H. pylori in co-culture with FLA using an imaging flow cytometric approach, that we already have developed (Dey et al . 2019 ). In addition, we investigated growth and survival strategies of H. pylori in co-culture with FLA by culture, molecular and imaging techniques.…”

In Flow Cytometry, Image Is Everything

“…Specifically, the first part of the Special Issue consists of nine papers (while the second part follows in a subsequent issue). Dey et al (15) report the use of IFC to accurately differentiate between active and viable but not-culturable forms of Psedomonas aeruginosa (a Gram-negative bacterium abundant in the environment and water systems). Kim et al (16) present their investigation of the physical and magnetic properties of monocytes in human blood together with plasma platelets, oxyhemoglobin red blood cells, and methemoglobin red blood cells by using IFC.…”