Oxygen restriction generates difficult-to-culture pathogens

Abstract

34 35 Keywords: 36 P. aeruginosa biofilm / anoxia / biofilm / reactive oxygen species / difficult-to-culture / viable but non-culturable 37 38 Abstract: 233 words 39 Document: 4604 words 40 Abstract 41Induction of a non-culturable state has been demonstrated for many bacteria. In a clinical 42 perspective, the lack of growth due to these non-culturable bacteria can have major consequences 43 for the diagnosis and treatment of patients. Here we show how anoxic conditioning (restriction of 44 molecular oxygen, O2) generates difficult-to-culture (DTC) bacteria during biofilm growth. 45A significant subpopulation of Pseudomonas aeruginosa entered a DTC state after anoxic 46 conditioning, ranging from five to 90 % of the total culturable population, in both planktonic and 47 biofilm models. Anoxic conditioning also generated DTC subpopulations of Staphylococcus aureus 48 and Staphylococcus epidermidis (89 and 42 % of the total culturable population, respectively). 49Growth of the DTC populations were achieved by substituting O2 with 10 mM NO3as an 50 alternative electron acceptor for anaerobic respiration or, in the case of P. aeruginosa, by adding 51 sodium pyruvate or catalase as scavengers against reactive oxygen species (ROS) during aerobic 52 respiration. An increase in normoxic plating due to addition of catalase suggests the molecule 53 hydrogen peroxide as a possible mechanism for induction of DTC P. aeruginosa. Anoxic 54 conditioning also generated a true viable but non-culturable (VBNC) population of P. aeruginosa 55 that was not resurrected by substituting O2 with NO3during anaerobic respiration. 56 These results demonstrate that habituation to an anoxic micro-environment could complicate 57 diagnostic culturing of bacteria, especially in the case of chronic infections where oxygen is 58 restricted due to the host immune response. 59Oxygen restriction generates DTC pathogens 3 Importance 60 Diagnostics of bacteria from chronic infections by standard culture-based methods is challenging. 61Bacteria in a non-culturable state may contribute to the lack of culturing from these infections. 62Many stressors are known to induce a non-culturable state, among others the absence of molecular 63 oxygen, which is evident in chronic infections due to high rates of oxygen consumption by the host 64 response. In this study, we have shown that Pseudomonas aeruginosa, Staphylococcus aureus and 65 Staphylococcus epidermidis can enter a non-culturable state after oxygen restriction. Regrowth was 66 not possible using conventional normoxic plating where oxygen served as electron acceptor. 67 Instead, regrowth was enabled during anoxic conditions with added nitrate as alternative electron 68 acceptor. In the case of P. aeruginosa we were able to counteract this non-culturable state by adding 69 scavengers for reactive oxygen species during normoxic incubation. This suggest a link between 70 reactive oxygen species and a non-culturable state and it also renders a solution to circumvent this 71 problem in a clinical diag...

Metabolic flux fingerprinting differentiates planktonic and biofilm states ofPseudomonas aeruginosaandStaphylococcus aureus

“…The numerical model suggested that P. aeruginosa grown in alginate beads was associated with higher death rates than the other models. For simplicity, we used the term death rate, but it might better be explained by a switch to an inactive state, as it has previously been shown that bacteria can resume growth after prolonged starvation and electron acceptor depletion (Kvich et al, 2019).…”

“…The increased tolerance of biofilms toward antibiotics is frequently attributed to a lower metabolic rate (Kolpen et al, 2017), upregulated efflux pumps (Frimodt-Møller et al, 2018;Bartell et al, 2019), protection by matrix components (Tseng et al, 2013;Cao et al, 2016) or SOS responses (Nguyen et al, 2011;Bernier et al, 2013), and is determined by direct exposure to antibiotics and measure of killing either by plating and enumeration of surviving colony forming units (CFU) or live/dead staining. The CFU method, however, can be slow and prone to biases, such as counting aggregates as single CFUs or false negatives due to the induction of viable but non-culturable state of the bacteria (Kvich et al, 2019). Similarly, live/dead staining may overestimate the proportion of dead cells, due to the binding of propidium iodide to eDNA (Rosenberg et al, 2019).…”