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Oxygen Restriction Generates Difficult-to-Culture P. aeruginosa.

ABSTRACT: Induction of a non-culturable state has been demonstrated for many bacteria, e.g., Escherichia coli and various Vibrio spp. In a clinical perspective, the lack of growth due to these non-culturable bacteria can have major consequences for the treatment of patients. Here, we show how anoxic conditioning (restriction of molecular oxygen, O2) generates difficult-to-culture (DTC) bacteria during biofilm growth. A significant subpopulation of Pseudomonas aeruginosa entered a DTC state after anoxic conditioning, ranging from 5 to 90% of the total culturable population, in both planktonic and biofilm models. Anoxic conditioning also generated DTC subpopulations of Staphylococcus aureus and Staphylococcus epidermidis (89 and 42% of the total culturable population, respectively). Growth of the DTC populations were achieved by substituting O2 with 10 mM NO3 - as an alternative electron acceptor for anaerobic respiration or, in the case of P. aeruginosa, by adding sodium pyruvate or catalase as scavengers against reactive oxygen species (ROS) during aerobic respiration. An increase in normoxic plating due to addition of catalase suggests the molecule hydrogen peroxide as a possible mechanism for induction of DTC P. aeruginosa. Anoxic conditioning also generated a true viable but non-culturable (VBNC) population of P. aeruginosa that was not resurrected by substituting O2 with NO3 - during anaerobic respiration. These results demonstrate that habituation to an anoxic micro-environment could complicate diagnostic culturing of bacteria, especially in the case of chronic infections where oxygen is restricted due to the host immune response.

SUBMITTER: Kvich L

PROVIDER: S-EPMC6727857 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Oxygen Restriction Generates Difficult-to-Culture P. aeruginosa.

Kvich Lasse L Fritz Blaine B Crone Stephanie S Kragh Kasper N KN Kolpen Mette M Sønderholm Majken M Andersson Mikael M Koch Anders A Jensen Peter Ø PØ Bjarnsholt Thomas T

Frontiers in microbiology 20190829

Induction of a non-culturable state has been demonstrated for many bacteria, e.g., Escherichia coli and various Vibrio spp. In a clinical perspective, the lack of growth due to these non-culturable bacteria can have major consequences for the treatment of patients. Here, we show how anoxic conditioning (restriction of molecular oxygen, O2) generates difficult-to-culture (DTC) bacteria during biofilm growth. A significant subpopulation of Pseudomonas aeruginosa ent ...[more]

PMID: 31555231

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Project description:The aim of this work was to develop, characterize and test a novel 3D bioscaffold matrix which can accommodate pancreatic islets and provide them with a continuous, controlled and steady source of oxygen to prevent hypoxia-induced damage following transplantation. Hence, we made a collagen based cryogel bioscaffold which incorporated calcium peroxide (CPO) into its matrix. The optimal concentration of CPO integrated into bioscaffolds was 0.25wt.% and this generated oxygen at 0.21±0.02mM/day (day 1), 0.19±0.01mM/day (day 6), 0.13±0.03mM/day (day 14), and 0.14±0.02mM/day (day 21). Accordingly, islets seeded into cryogel-CPO bioscaffolds had a significantly higher viability and function compared to islets seeded into cryogel alone bioscaffolds or islets cultured alone on traditional cell culture plates; these findings were supported by data from quantitative computational modelling. When syngeneic islets were transplanted into the epididymal fat pad (EFP) of diabetic mice, our cryogel-0.25wt.%CPO bioscaffold improved islet function with diabetic animals re-establishing glycemic control. Mice transplanted with cryogel-0.25wt.%CPO bioscaffolds showed faster responses to intraperitoneal glucose injections and had a higher level of insulin content in their EFP compared to those transplanted with islets alone (P<0.05). Biodegradability studies predicted that our cryogel-CPO bioscaffolds will have long-lasting biostability for approximately 5 years (biodegradation rate: 16.00±0.65%/year). Long term implantation studies (i.e. 6 months) showed that our cryogel-CPO bioscaffold is biocompatible and integrated into the surrounding fat tissue with minimal adverse tissue reaction; this was further supported by no change in blood parameters (i.e. electrolyte, metabolic, chemistry and liver panels). Our novel oxygen-generating bioscaffold (i.e. cryogel-0.25wt.%CPO) therefore provides a biostable and biocompatible 3D microenvironment for islets which can facilitate islet survival and function at extra-hepatic sites of transplantation.

Project description:BACKGROUND:Rhamnolipids are the most extensively studied biosurfactants and has been successfully used in various areas from bioremediation to industrial fields. Rhamnolipids structural composition decide their physicochemical properties. Different physicochemical properties influence their application potential. Rhamnolipids can be produced at both aerobic conditions and anaerobic conditions by Pseudomonas aeruginosa. This study aims to evaluate the oxygen effects on the rhamnolipids yield, structural composition, physicochemical properties and the rhl-genes expression in P. aeruginosa SG. Results will guide researchers to regulate microbial cells to synthesize rhamnolipids with different activity according to diverse application requirements. RESULTS:Quantitative real-time PCR analysis revealed that rhlAB genes were down-regulated under anaerobic conditions. Therefore, strain P. aeruginosa SG anaerobically produced less rhamnolipids (0.68 g/L) than that (11.65 g/L) under aerobic conditions when grown in media containing glycerol and nitrate. HPLC-MS analysis showed that aerobically produced rhamnolipids mainly contained Rha-C8-C10, Rha-Rha-C10-C12:1 and Rha-Rha-C8-C10; anaerobically produced rhamnolipids mainly contained Rha-C10-C12 and Rha-C10-C10. Anaerobically produced rhamnolipids contained more mono-rhamnolipids (94.7%) than that (54.8%) in aerobically produced rhamnolipids. rhlC gene was also down-regulated under anaerobic conditions, catalyzing less mono-rhamnolipids to form di-rhamnolipids. Aerobically produced rhamnolipids decreased air-water surface tension (ST) from 72.2 to 27.9 mN/m with critical micelle concentration (CMC) of 60 mg/L; anaerobically produced rhamnolipids reduced ST to 33.1 mN/m with CMC of 80 mg/L. Anaerobically produced rhamnolipids emulsified crude oil with EI24 = 80.3%, and aerobically produced rhamnolipids emulsified crude oil with EI24 = 62.3%. Both two rhamnolipids products retained surface activity (ST < 35.0 mN/m) and emulsifying activity (EI24 > 60.0%) under temperatures (4-121 °C), pH values (4-10) and NaCl concentrations less than 90 g/L. CONCLUSIONS:Oxygen affected the rhl-genes expression in P. aeruginosa, thus altering the rhamnolipids yield, structural composition and physicochemical properties. Rhamnolipids produced at aerobic or anaerobic conditions was structurally distinct. Two rhamnolipids products had different application potential in diverse biotechnologies. Although both rhamnolipids products were thermo-stable and halo-tolerant, aerobically produced rhamnolipids possessed better surface activity, implying its well wetting activity and desorption property; anaerobically produced rhamnolipids exhibited better emulsifying activity, indicating its applicability for enhanced oil recovery and bioremediation of petroleum pollution.

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Oxygen Restriction Generates Difficult-to-Culture P. aeruginosa.

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Oxygen Restriction Generates Difficult-to-Culture <i>P. aeruginosa</i>.

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