Long-term survival and resistance of submerged pseudomonad cultures in the exopolymer mass

Abstract

An issue on the cellular forms that ensure survival of pseudomonads is important due to wide occurrence of these bacteria in the environment and their role for clinical microbiology. The present work demonstrates the high survival potential of Pseudomonas aurantiaca and P. аeruginosa in the mass of exopolymers produced by cells. Exopolymer formation occurred only during incubation of the post-stationary phase cultures of P. aurantiaca (at 4°C) and P. aeruginosa (at 4 and 20°C). After storage for 1.5–12 months, the number of colony-forming units in the exopolymer was 30 to 68% of the viable cell titer in stationary-phase cultures. Antibiotic-tolerant persister cells that were revealed in the exopolymer cultures after treatment with ciprofloxacin (2.5–100 μg/mL) were more resistant to the antibiotic than persisters in suspension cultures, with the threshold doses of 25 and 2.5 μg/mL, respectively. The cells embedded in the exopolymer were found to be more resistant to 5-min heating at 60–70°C than the vegetative cells of suspension cultures, which did not survive such heat treatment conditions. Electron microscopic investigation revealed morphological heterogeneity of exopolymer-embedded pseudomonads, including the presence of the cells similar to cystlike dormant forms. The populations developing on solid media inoculated with the exopolymer mass with cells were found to contain 1.5 to 2 orders of magnitude more persisters tolerant to high ciprofloxacin doses (25 μg/mL for P. aurantiaca and 100 μg/mL for P. aeruginosa) than the populations developing after inoculation with second-transfer vegetative cells of the cells of planktonic cultures. The results obtained improve our understanding of pseudomonad survival in the environment.