Antimicrobial Tolerance of Pseudomonas aeruginosa Biofilms Is Activated during an Early Developmental Stage and Requires the Two-Component Hybrid SagS

Abstract

A hallmark characteristic of biofilms is their extraordinary tolerance to antimicrobial agents. While multiple factors are thought to contribute to the high level of antimicrobial tolerance of biofilms, little is known about the timing of induction of biofilm tolerance. Here, we asked when over the course of their development do biofilms gain their tolerance to antimicrobial agents? We demonstrate that in Pseudomonas aeruginosa, biofilm tolerance is linked to biofilm development, with transition to the irreversible attachment stage regulated by the two-component hybrid SagS, marking the timing when biofilms switch to the high-level tolerance phenotype. Inactivation of sagS rendered biofilms but not planktonic cells more susceptible to tobramycin, norfloxacin, and hydrogen peroxide. Moreover, inactivation of sagS also eliminated the recalcitrance of biofilms to killing by bactericidal antimicrobial agents, a phenotype comparable to that observed upon inactivation of brlR, which encodes a MerR-like transcriptional regulator required for biofilm tolerance. Multicopy expression of brlR in a ⌬sagS mutant restored biofilm resistance and recalcitrance to killing by bactericidal antibiotics to wild-type levels. In contrast, expression of sagS did not restore the susceptibility phenotype of ⌬brlR mutant biofilms to wild-type levels, indicating that BrlR functions downstream of SagS. Inactivation of sagS correlated with reduced BrlR levels in biofilms, with the produced BrlR being impaired in binding to the previously described BrlR-activated promoters of the two multidrug efflux pump operons mexAB-oprM and mexEF-oprN. Our findings demonstrate that biofilm tolerance is linked to early biofilm development and SagS, with SagS contributing indirectly to BrlR activation.

Using Phage’s to Exterminate Biofilms

“…Although they are capable of blocking antibiotics effect within their beginning stages of formation. This finding will follow that of Gupta et al [57], who also stated that the antibiotic resistance begins to appear in the first stages of biofilm formation. Thus, bacteria can be destroyed by phages in cases where antibiotics did have no effect on them.…”

The Diguanylate Cyclase GcbA Facilitates Pseudomonas aeruginosa Biofilm Dispersion by Activating BdlA

“…This is based on our finding that BdlA, a central modulator of these types of dispersion events, is activated via posttranslational processing soon after initial attachment and, thus, prior to initiation of biofilm maturation, significant biomass accumulation, or microcolony formation. Evidence for this is also provided by previous microscopic observations of nutrient-induced dispersion occurring within and outside microcolonies, as well as by findings of nutrient-induced dispersion not being affected in certain biofilms (such as those formed by ⌬flgB or ⌬pilA mutant strains or the dipA-overexpressing PAO1) that were characterized by significantly reduced biomass accumulation and thicknesses comparable to those of wildtype cells at the initial attachment stages of biofilm development (12,37,54,57,60). Together with these findings, the present results suggest that attached cells are likely able to respond to changing environmental conditions in a manner independent of biomass accumulation and microcolony size, with GcbA contributing to the activation of BdlA and mechanisms for sensing and relaying dispersion-inducing cues soon after the initial surface attachment.…”

“…Cells dispersing from biofilms following the induction of dispersion as described above were collected directly into RNAprotect (Qiagen). Isolation of mRNA and cDNA synthesis were carried out as previously described (54)(55)(56)(57). qRT-PCR was performed using the Bio-Rad CFX Connect real-time PCR detection system and SsoAdvanced SYBR green supermix (Bio-Rad) with oligonucleotide pairs PA4843RTf/PA4843RTr (39) and bdlA-RT-for (CTACGCGCAATCGGA AGAC)/bdlA-RT-rev (GGACATTGCCGTCGAGGTC).…”

Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics

“…On the other hand, brlR overexpression increased P. aeruginosa tolerance toward antimicrobials. The same group identified BrlR as an activator of mexAB-oprM and mexEF-oprN (79) and the two-component hybrid protein SagS as a possible upstream regulator of BrlR (80). Recently, SagS was shown to contribute to BrlR activation and tolerance toward antibiotics through an increase of the level of the second messenger cyclic di-GMP (c-di-GMP) (81).…”