Role of psl Genes in Antibiotic Tolerance of Adherent Pseudomonas aeruginosa.

Bacteria attached to a surface are generally more tolerant to antibiotics than their planktonic counterparts, even without the formation of a biofilm. The mechanism of antibiotic tolerance in biofilm communities is multifactorial, and the genetic background underlying this antibiotic tolerance has not yet been fully elucidated. Using transposon mutagenesis, we isolated a mutant with reduced tolerance to biapenem (relative to that of the wild type) from adherent cells. Sequencing analysis revealed a mutation in the pslL gene, which is part of the polysaccharide biosynthesis operon. The Pseudomonas aeruginosa PAO1ΔpslBCD mutant demonstrated a 100-fold-lower survival rate during the exposure of planktonic and biofilm cells to biapenem; a similar phenotype was observed in a mouse infection model and in clinical strains. Transcriptional analysis of adherent cells revealed increased expression of both pslA and pelA, which are directly regulated by bis-(3',5')-cyclic dimeric GMP (c-di-GMP). Inactivation of wspF resulted in significantly increased tolerance to biapenem due to increased production of c-di-GMP. The loss of pslBCD in the ΔwspF mutant background abolished the biapenem-tolerant phenotype of the ΔwspF mutant, underscoring the importance of psl in biapenem tolerance. Overexpression of PA2133, which can catalyze the degradation of c-di-GMP, led to a significant reduction in biapenem tolerance in adherent cells, indicating that c-di-GMP is essential in mediating the tolerance effect. The effect of pslBCD on antibiotic tolerance was evident, with 50- and 200-fold-lower survival in the presence of ofloxacin and tobramycin, respectively. We speculate that the psl genes, which are activated by surface adherence through elevated intracellular c-di-GMP levels, confer tolerance to antimicrobials.

Effects of an autoinducer analogue on antibiotic tolerance in Pseudomonas aeruginosa.

Objectives: Antibiotic tolerance causes chronic, refractory and persistent infections. In order to advance the development of a new type of drug for the treatment of infectious diseases, we herein investigated the effects of a newly synthesized analogue of the Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 ( a uto i nducer a nalogue) on antibiotic tolerance in P. aeruginosa . Methods: A P. aeruginosa luminescent strain derived from PAO1 was injected into neutropenic ICR mice and bioluminescence images were acquired for a period of time after treatments with antibiotics and AIA-1. In vitro susceptibility testing and killing assays for the planktonic and biofilm cells of PAO1 were performed using antibiotics and AIA-1. The expression of quorum-sensing-related genes was examined using real-time PCR. Results: In vivo and in vitro assays showed that AIA-1 alone did not exert any bactericidal effects and also did not affect the MICs of antibiotics. However, the combined use of AIA-1 and antibiotics exerted markedly stronger therapeutic effects against experimental infection than antibiotics alone. The presence of AIA-1 also enhanced the killing effects of antibiotics in planktonic and biofilm cells. Although AIA-1 did not inhibit the expression of lasB and rhlA genes, which are directly regulated by quorum sensing, it clearly suppressed expression of the rpoS gene. Conclusions: The new compound, AIA-1, did not alter the antibiotic susceptibility of P. aeruginosa by itself; however, its addition enhanced the antibacterial activity of antibiotics. AIA-1 did not inhibit quorum sensing, but reduced the antibiotic tolerance of P. aeruginosa by suppressing rpoS gene expression.

RpoN Modulates Carbapenem Tolerance in Pseudomonas aeruginosa through Pseudomonas Quinolone Signal and PqsE.

The ability of Pseudomonas aeruginosa to rapidly modulate its response to antibiotic stress and persist in the presence of antibiotics is closely associated with the process of cell-to-cell signaling. The alternative sigma factor RpoN (σ(54)) is involved in the regulation of quorum sensing (QS) and plays an important role in the survival of stationary-phase cells in the presence of carbapenems. Here, we demonstrate that a ΔrpoN mutant grown in nutrient-rich medium has increased expression of pqsA, pqsH, and pqsR throughout growth, resulting in the increased production of the Pseudomonas quinolone signal (PQS). The link between pqsA and its role in carbapenem tolerance was studied using a ΔrpoN ΔpqsA mutant, in which the carbapenem-tolerant phenotype of the ΔrpoN mutant was abolished. In addition, we demonstrate that another mechanism leading to carbapenem tolerance in the ΔrpoN mutant is mediated through pqsE Exogenously supplied PQS abolished the biapenem-sensitive phenotype of the ΔrpoN ΔpqsA mutant, and overexpression of pqsE failed to alter the susceptibility of the ΔrpoN ΔpqsA mutant to biapenem. The mutations in the ΔrpoN ΔrhlR mutant and the ΔrpoN ΔpqsH mutant led to susceptibility to biapenem. Comparison of the changes in the expression of the genes involved in QS in wild-type PAO1 with their expression in the ΔrpoN mutant and the ΔrpoN mutant-derived strains demonstrated the regulatory effect of RpoN on the transcript levels of rhlR, vqsR, and rpoS The findings of this study demonstrate that RpoN negatively regulates the expression of PQS in nutrient-rich medium and provide evidence that RpoN interacts with pqsA, pqsE, pqsH, and rhlR in response to antibiotic stress.

Role of the interplay between quorum sensing regulator VqsR and the Pseudomonas quinolone signal in mediating carbapenem tolerance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coordinates its response to environmental conditions through activation of a quorum sensing (QS) system. In this study, we investigated the regulatory interaction between the QS transcriptional regulator VqsR and the Pseudomonas quinolone signal (PQS) through integration of sigma factor RpoS, and we addressed whether one of the pathways controlling carbapenem tolerance can be attributed to VqsR. We demonstrate that vqsR expression at the transcriptional level is regulated by pqsA, pqsR, and pqsE. Assessment of the transcriptional expression of vqsR, lasI, rhlI, and qscR in ΔpqsA and ΔpqsAΔrpoS mutants provided insight into pqsA- and rpoS-dependent regulation of vqsR and vqsR-controlled genes. Exogenously supplemented PQS reversed expression of vqsR and vqsR-controlled genes in the ΔpqsA mutant to wild-type levels, but failed to increase expression levels of lasI and qscR in the ΔpqsAΔrpoS mutant to levels observed in wild-type PAO1. The ΔvqsR mutant showed reduced survival when challenged with carbapenems compared to wild-type PAO1. Introduction of a pqsA mutation into the ΔvqsR mutant completely abolished its carbapenem-sensitive phenotype. We conclude that a regulatory link between PQS and vqsR exists, and that RpoS is important in their interaction. We also demonstrate that VqsR affects carbapenem tolerance.

RpoN Promotes Pseudomonas aeruginosa Survival in the Presence of Tobramycin.

Pseudomonas aeruginosa has developed diverse strategies to respond and adapt to antibiotic stress. Among the factors that modulate survival in the presence of antibiotics, alternative sigma factors play an important role. Here, we demonstrate that the alternative sigma factor RpoN (σ54) promotes survival in the presence of tobramycin. The tobramycin-sensitive phenotype of logarithmic phase ΔrpoN mutant cells is suppressed by the loss of the alternative sigma factor RpoS. Transcriptional analysis indicated that RpoN positively regulates the expression of RsmA, an RNA-binding protein, in the P. aeruginosa stationary growth phase in a nutrient-rich medium. The loss of RpoS led to the upregulation of gacA expression in the nutrient-limited medium-grown stationary phase cells. Conversely, in the logarithmic growth phase, the ΔrpoS mutant demonstrated lower expression of gacA, underscoring a regulatory role of RpoS for GacA. Supplementation of tobramycin to stationary phase ΔrpoN mutant cells grown in nutrient-rich medium resulted in decreased expression of gacA, relA, and rpoS without altering the expression of rsmA relative to wild-type PAO1. The observed downregulation of gacA and relA in the ΔrpoN mutant in the presence of tobramycin could be reversed through the mutation of rpoS in the ΔrpoN mutant background. The tobramycin-tolerant phenotype of the ΔrpoNΔrpoS mutant logarithmic phase cells may be associated with the expression of relA, which remained unresponsive upon addition of tobramycin. The logarithmic phase ΔrpoS and ΔrpoNΔrpoS mutant cells demonstrated increased expression of gacA in response to tobramycin. Together, these results suggest that a complex regulatory interaction between RpoN, RpoS, the Gac/Rsm pathway, and RelA modulates the P. aeruginosa response to tobramycin.

A Pseudomonas aeruginosa Quorum-Sensing autoinducer analog enhances the activity of antibiotics against resistant strains.

In this study, we have investigated the effects of the newly synthesized analog of Pseudomonas aeruginosa quorum-sensing autoinducer named AIA-1 (autoinducer analog) against antibiotic-resistant bacteria. In vitro susceptibility and killing assays for P. aeruginosa PAO1ΔoprD mutant and clinical isolates were performed by using antibiotics and AIA-1. In an in vivo assay, a luminescent carbapenem-resistant strain derived from PAO1ΔoprD was injected into neutropenic ICR mice and bioluminescence images were acquired after the treatment with antibiotics and AIA-1. Additionally, we investigated the effects of the combination use against carbapenem-resistant Enterobacteriaceae (CRE). Using killing assays in P. aeruginosa, the survival rates in the presence of antibiotics and AIA-1 significantly decreased in comparison with those with antibiotics alone. Furthermore, dual treatment of biapenem and AIA-1 was more effective than biapenem alone in a mouse infection model. AIA-1 did not change the MICs in P. aeruginosa, suggesting that AIA-1 acts on the mechanism of antibiotic tolerance. Conversely, the MICs of antibiotics decreased in the presence of AIA-1 in some CRE strains, indicating that AIA-1 may require additional mechanism to act on CRE. In conclusion, AIA-1 may be a potent drug for clinical treatment of infections caused by antibiotic-resistant bacteria. J. Med. Invest. 64: 101-109, February, 2017.

Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance.

The alternative sigma factor, RpoS has been described as a central regulator of many stationary phase-inducible genes and a master stress-response regulator under various stress conditions. We constructed an rpoS mutant in Pseudomonas aeruginosa and investigated the role of rpoS gene in antibiotic tolerance. The survival of the rpoS mutant cells in stationary phase was approximately 70 times lower when compared with that of the parental strain at 37 degrees C for 2 h after the addition of biapenem. For imipenem, the survival was approximately 40 times lower. Heat stress promoted an increase in the survival of the parental strain to biapenem, but the same was not found to be the case for the rpoS mutant. Our results indicate that rpoS gene is involved in tolerance to antibiotics in P. aeruginosa during the stationary phase and heat stress. However, under osmotic stress, tolerance to biapenem was not dependent on the rpoS gene.

rpoN gene of Pseudomonas aeruginosa alters its susceptibility to quinolones and carbapenems.

The alternative sigma factor sigma54 has been implicated in diverse functions within the cells. In this study, we have constructed an rpoN mutant of Pseudomonas aeruginosa and investigated its importance as a target for antimicrobial agents, such as quinolones and carbapenems. The stationary-phase cells of the rpoN mutant displayed a survival rate approximately 15 times higher than that of the wild-type cells in the presence of quinolones and carbapenems. The stationary phase led to substantial production of pyoverdine by the P. aeruginosa rpoN mutant. Pyoverdine synthesis correlated with decreased susceptibility to antimicrobial agents. Quantitative real-time PCR revealed that stationary-phase cells of the rpoN mutant grown without an antimicrobial agent had approximately 4- to 140- and 2- to 14-fold-higher levels of transcripts of the pvdS and vqsR genes, respectively, than the wild-type strain. In the presence of an antimicrobial agent, levels of pvdS and vqsR transcripts were elevated 400- and 5-fold, respectively, in comparison to the wild-type levels. Flow cytometry assays using a green fluorescent protein reporter demonstrated increased expression of the vqsR gene in the rpoN mutant throughout growth. A pvdS mutant of P. aeruginosa, deficient in pyoverdine production, was shown to be susceptible to biapenem. These findings suggest that rpoN is involved in tolerance to antimicrobial agents in P. aeruginosa and that its tolerant effect is partly dependent on increased pyoverdine production and vqsR gene expression.

Functional analysis of spoT, relA and dksA genes on quinolone tolerance in Pseudomonas aeruginosa under nongrowing condition.

To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of the genes involved or linked with the stringent response, such as relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of 8 microg/ml of ofloxacin and 1 microg/ml of ciprofloxacin were shown to be approximately 20-180 and 10-40 times respectively, higher than the same for the wild type strain. The intracellular levels of ppGpp determined with high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa.

Effect of Varidase (streptodornase) on biofilm formed by Pseudomonas aeruginosa.

The biofilm of Pseudomonas aeruginosa could be removed by Varidase (streptodornase) that was used as defibrinating drug. After cultivating the biofilm on a silicone chip in a modified alginate-producing medium in vitro, it was treated with Varidase or DNase I. In both treatments, the biofilm was removed depending on the concentration of the reagents. Varidase was apparently effective under the condition that the biofilm was exposed to more than 625 U/ml (a quarter of the concentration of the medical use) for 3 h, twice, at 37 degrees C. The result of this experiment indicates that (a) the DNases, DNase I and Varidase, were effective in destroying the biofilm of P. aeruginosa in vitro, and (b) in a clinical field, Varidase could be useful for P. aeruginosa focal infection, such as urinary tract infection, by removing the biofilm.

Novel Pseudomonas aeruginosa gene that suppresses tolerance to carbapenems.

A biapenem-tolerant mutant of Pseudomonas aeruginosa was isolated by Tn1737KH insertion. The survival of the mutant 3 h after the addition of biapenem was about 1000 times greater than that of the wild type. The mutant was also tolerant to other biapenems, such as imipenem, panipenem, and meropenem.