Sub-lethal antimicrobial photodynamic inactivation affects Pseudomonas aeruginosa PAO1 quorum sensing and cyclic di-GMP regulatory systems.

BACKGROUND: Antimicrobial photodynamic inactivation (APDI) is a new therapeutic modality which needs more precision during application due to the possibility of exposure of bacteria to sub-lethal doses (sAPDI). In this study, we aimed to evaluate the effect of sAPDI on Pseudomonas aeruginosa quorum sensing (QS) and c-di-GMP signaling which are important virulence factor regulatory systems. METHODS: Biofilm formation, pyoverdine, pyocyanin and protease production of P. aeruginosa was evaluated before and after a single sAPDI treatment with 0.8 mM methylene blue (MB) plus 1, 2, and 5-min irradiation with red laser light. Fluorescent lasB, rhlA, pqsA, and cdrA reporters of P. aeruginosa PAO1 and P. aeruginosa ΔmexAB-oprM were treated individually with sAPDI and the regulatory signals were detected. The gene expressions were also assessed after sAPDI using quantitative real-time PCR analysis. RESULTS: Morphological observations and molecular assessments indicated that sAPDI with 0.8 mM MB along with 2- and 5-min irradiation led to an increase in the expression of the Las QS system and c-di-GMP signaling, while 1 min irradiation revealed dissimilar results (increase in lasB expression and decrease in c-di-GMP levels). Expression of rhlA and pqsA did not change in response to sAPDI. Further, a severe lethal effect of sAPDI was observed in P. aeruginosa ΔmexAB-oprM as compared with the wild type strain, whilst there was no difference in QS and c-di-GMP levels as detected by reporters between treated and untreated samples. CONCLUSION: The results suggest that sAPDI affects QS and c-di-GMP signaling inP. aeruginosa in a time-dependent manner.

Quorum-sensing-regulated virulence factors in Pseudomonas aeruginosa are affected by sub-lethal photodynamic inactivation.

BACKGROUND: Photodynamic inactivation (PDI) is recognized as a new antimicrobial approach. It is likely that in human hosts receiving this therapy, pathogens may encounter sub-lethal doses of PDI (sPDI), which may affect microbial virulence. This study was aimed to evaluate the effect of sPDI using methylene blue (MB) on the expression of genes belonging to two quorum sensing (QS) operons (rhl and las systems) and two genes necessary for pyocyanin and rhamnolipid production (phzM and rhlA) under QS control in Pseudomonas aeruginosa. METHODS: Ability of pyocyanin and rhamnolipid production of P. aeruginosa ATCC 27853 and clinical isolates exposed to sPDI (MB at 0.012 mM and light dose of 23 J/cm2 was evaluated. The effect of sPDI on expression of rhlI, rhlR, lasI, lasR, phzM and rhlA were also evaluated by quantitative real time polymerase chain reaction. RESULTS: sPDI led to the down-regulation of the expression of all four QS genes (lasI, lasR, rhlI and rhlR) and rhamnolipid gene (rhlA). However, up-regulation of pyocyanin gene (phzM) was observed after sPDI. These results were consistent with phenotypic changes. CONCLUSION: This study suggests that oxidative stress induced by sPDI can affect QS-regulated virulence factors of P. aeruginosa such as pyocyanin and rhamnolipids in different ways.

Sub-lethal antimicrobial photodynamic inactivation: an in vitro study on quorum sensing-controlled gene expression of Pseudomonas aeruginosa biofilm formation.

During antimicrobial photodynamic inactivation (APDI) in the treatment of an infection, it is likely that microorganisms would be exposed to sub-lethal doses of APDI (sAPDI). Although sAPDI cannot kill microorganisms, it can significantly affect microbial virulence. In this study, we evaluated the effect of sAPDI using methylene blue (MB) on the expression of genes belonging to two quorum sensing (QS) operons (rhl and las systems) and two genes necessary for biofilm formation (pelF and pslA) under QS control in Pseudomonas aeruginosa. Biofilm formation ability of P. aeruginosa ATCC 27853 exposed to sAPDI (MB at 0.012 mM and light dose of 23 J/cm2) was evaluated using triphenyl tetrazolium chloride (TTC) assay and scanning electron microscopy (SEM). The effect of sAPDI on expression of rhlI, rhlR, lasI, lasR, pelF, and pslA were also evaluated by quantitative real-time polymerase chain reaction. Quantitative assay (TTC) results and morphological observations (SEM) indicated that a single sAPDI treatment resulted in a significant decrease in biofilm formation ability of P. aeruginosa ATCC 27853 compared to their non-treated controls (P = 0.012). These results were consistent with the expression of genes belonging to rhl and las systems and pelF and pslA genes. The results suggested that the transcriptional decreases caused by MB-sAPDI did lead to phenotypic changes.

High levels of cAMP inhibit Pseudomonas aeruginosa biofilm formation through reduction of the c-di-GMP content.

The human pathogen Pseudomonas aeruginosa can cause both acute infections and chronic biofilm-based infections. Expression of acute virulence factors is positively regulated by cAMP, whereas biofilm formation is positively regulated by c-di-GMP. We provide evidence that increased levels of cAMP, caused by either a lack of degradation or increased production, inhibit P. aeruginosa biofilm formation. cAMP-mediated inhibition of P. aeruginosa biofilm formation required Vfr, and involved a reduction of the level of c-di-GMP, as well as reduced production of biofilm matrix components. A mutant screen and characterization of defined knockout mutants suggested that a subset of c-di-GMP-degrading phosphodiesterases is involved in cAMP-Vfr-mediated biofilm inhibition in P. aeruginosa.

Assessment of Biofilm Formation and Resistance to Imipenem and Ciprofloxacin among Clinical Isolates of Acinetobacter baumannii in Tehran.

BACKGROUND: Biofilms are communities of bacteria attached to the surfaces in an extracellular polymeric matrix which are associated with many chronic infections in humans. Acinetobacter spp. are emerging as a major cause of nosocomial infections and Acinetobacter baumannii is the predominant species associated with this kind of infections. OBJECTIVES: In the present study, the potential of biofilm formation of clinical isolates, A. baumannii, was assessed by using crystal violet method. Furthermore, susceptibility pattern of these strains to ciprofloxacin and imipenem was determined. METHODS AND MATERIALS: Biofilm formation by 75 A. baumannii isolates was evaluated by using microtiter plate and tube methods and crystal violet staining. Tube method was carried out under static and shaking conditions. Then, the susceptibility of isolates to ciprofloxacin and imipenem was determined. RESULTS: Results showed that in tube method under shaking, 22% of clinical isolates were strong biofilm producers while 23% of them were not able to form biofilms. In this experiment, 18% and 42% of isolates were considered as moderate and weak biofilm-forming strains, respectively. In microtiter plate tests, 18% of strains were strong-biofilm producers and 25% of them were notable biofilm producers. In this assessment, 10% and 47% were considered as moderate and weak biofilm-forming isolates, respectively. The susceptibility tests, using microdilution method, confirmed that 92% of these isolates were resistant and 6.6% were susceptible to ciprofloxacin, although these results for imipenem were 68% and 24%, respectively. CONCLUSIONS: It can be concluded that most of A. baumannii isolates can form biofilm in microtiter plate and tube. The results also verified that most of these isolates were resistant to ciprofloxacin and imipenem.