Survival strategies of imipenem-resistant P. aeruginosa: a comparative analysis of surface-bound biofilms and unbound aggregates

Abstract

This study investigated imipenem-resistant Pseudomonas aeruginosa (P. aeruginosa) isolates recovered from various ready-to-eat food items. Isolates were identified as P. aeruginosa based on growth on selective P-Pseudomonas media and confirmed by PCR amplification of the oprI and oprL genes. These isolates formed biofilms under laboratory conditions at 35 °C in Tryptic Soy Broth (TSB). The biofilms were induced by a sub-inhibitory dose of imipenem. Two types of biofilm aggregates were observed: surface-bound biofilms and unbound cell aggregates. Surface-bound biofilms appeared after 48 h of incubation and reached maximum biomass after 96 h. Unbound aggregates were observed after 72 h of incubation. The biomass of aggregates was measured using a crystal violet binding assay. Further characterization revealed two types of unbound or floating aggregates: aggregates detached from surface-bound biofilms and spontaneously formed cell clusters. Both aggregate types exhibited similar imipenem resistance profiles. Comparative analysis of surface-bound and unbound biofilm cells revealed that surface-bound cells were more hydrophobic and relatively more resistant to high temperatures. Both types of aggregates survived at 80 °C for 12 h. Atomic force microscopy showed that surface-bound P. aeruginosa cells were stiffer, with an average force constant of 56.36 ± 5.21 pN nm^-1, compared to cells from unbound aggregates [44.55 ± 4.87 pN nm^-1]. Similarly, surface-bound cells exhibited greater adhesiveness, with an average adhesion force of 553.25 ± 62.18 pN, whereas cells from floating aggregates demonstrated lower adhesion force values, averaging 451.81 ± 58.32 pN.