Complete photodynamic inactivation of Pseudomonas aeruginosa biofilm with use of potassium iodide and its comparison with enzymatic pretreatment.

Pseudomonas aeruginosa, a gram-negative bacterium, accounts for 7% of all hospital-acquired infections. Despite advances in medicine and antibiotic therapy, P. aeruginosa infection still results in high mortality rates of up to 62% in certain patient groups. This bacteria is also known to form biofilms, that are 10 to 1000 times more resistant to antibiotics compared to their free-floating counterparts. Photodynamic Inactivation (PDI) has been proved to be an effective antimicrobial technique for microbial control. This method involves the incubation of the pathogen with a photosensitizer (PS), then, a light at appropriated wavelength is applied, leading to the production of reactive oxygen species that are toxic to the microbial cells. Studies have focused on strategies to enhance the PDI efficacy, such as a pre-treatment with enzymes to degrade the biofilm matrix and/or an addition of inorganic salts to the PS. The aim of the present study is to evaluate the effectiveness of PDI against P. aeruginosa biofilm in association with the application of the enzymes prior to PDI (enzymatic pre-treatment) or the addition of potassium iodide (KI) to the photosensitizer solution, to increase the inactivation effectiveness of the treatment. First, a range of enzymes and PSs were tested, and the best protocols for combined treatments were selected. The results showed that the use of enzymes as a pre-treatment was effective to reduce the total biomass, however, when associated with PDI, mild bacterial reductions were obtained. Then, the use of KI in association with the PS was evaluated and the results showed that, PDI mediated by methylene blue (MB) in the presence of KI was able to completely eradicate the biofilm. However, when the PDI was performed with curcumin and KI, no additive reduction was observed. In conclusion, out of all strategies evaluated in the present study, the most promising strategy to improve PDI against P. aeruginosa biofilm was the use of KI in association with MB, resulting in eradication with 108 log bacterial inactivation.

Effect of Xylitol on Inhibition and Eradication of Pseudomonas aeruginosa PAO1 and Methicillin-Resistant Staphylococcus aureus Biofilms in an Alginate Bead Model.

Biofilms formed by Pseudomonas aeruginosa and Staphylococcus aureus, along with their antibiotic tolerance have posed challenges to treatment strategies for lung, wound, and other infections, particularly when co-infecting. In the present study, the inhibitory effect of xylitol on biofilm formation, as well as its eradication potential on pre-established biofilms formed by P. aeruginosa strain PAO1, methicillin-resistant S. aureus, and a mix of both species in an alginate bead model were tested. Xylitol concentrations of 2, 1, and 0.5 M reduced biofilm formation by P. aeruginosa strain PAO1, methicillin-resistant S. aureus, and the mixed-species biofilm in a concentration-dependent manner. Additionally, biofilms formed by these species were subjected to treatment with xylitol. Xylitol was also capable of eradicating biofilms established by P. aeruginosa strain PAO1, methicillin-resistant S. aureus, and the mixed-species biofilm by at least 20%, with the most effective eradication observed for P. aeruginosa strain PAO1. The present study indicates the effectiveness of xylitol as both an inhibitory and eradicating agent against biofilms formed by P. aeruginosa strain PAO1, methicillin-resistant S. aureus, and a mix of both species in an alginate bead model, which mimics the in vivo characteristics of P. aeruginosa aggregates.

Effect of L-HSL on biofilm and motility of Pseudomonas aeruginosa and its mechanism.

Pseudomonas aeruginosa (P. aeruginosa) biofilm formation is a crucial cause of enhanced antibiotic resistance. Quorum sensing (QS) is involved in regulating biofilm formation; QS inhibitors block the QS signaling pathway as a new strategy to address bacterial resistance. This study investigated the potential and mechanism of L-HSL (N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide) as a QS inhibitor for P. aeruginosa. The results showed that L-HSL effectively inhibited the biofilm formation and dispersed the pre-formed biofilm of P. aeruginosa. The production of extracellular polysaccharides and the motility ability of P. aeruginosa were suppressed by L-HSL. C. elegans infection experiment showed that L-HSL was non-toxic and provided protection to C. elegans against P. aeruginosa infection. Transcriptomic analysis revealed that L-HSL downregulated genes related to QS pathways and biofilm formation. L-HSL exhibits a promising potential as a therapeutic drug for P. aeruginosa infection. KEY POINTS: • Chemical synthesis of N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide, named L-HSL. • L-HSL does not generate survival pressure on the growth of P. aeruginosa and can inhibit the QS system. • KEGG enrichment analysis found that after L-HSL treatment, QS-related genes were downregulated.

Exploring rose absolute and phenylethyl alcohol as novel quorum sensing inhibitors in Pseudomonas aeruginosa and Chromobacterium violaceum.

Inter-cellular signaling, referred to as quorum sensing (QS), regulates the production of virulence factors in numerous gram-negative bacteria, such as the human pathogens Pseudomonas aeruginosa and Chromobacterium violaceum. QS inhibition may provide an opportunity for the treatment of bacterial infections. This represents the initial study to examine the antibiofilm and antivirulence capabilities of rose absolute and its primary component, phenylethyl alcohol. QS inhibition was assessed by examining extracellular exopolysaccharide synthesis, biofilm development, and swarming motility in P. aeruginosa PAO1, along with violacein production in C. violaceum ATCC 12472. Molecular docking analysis was conducted to explore the mechanism by which PEA inhibits QS. Our results indicate that rose absolute and PEA caused decrease in EPS production (60.5-33.5%), swarming motility (94.7-64.5%), and biofilm formation (98.53-55.5%) in the human pathogen P. aeruginosa PAO1. Violacein production decreased by 98.1% and 62.5% with an absolute (0.5 v/v %) and PEA (2 mM). Moreover, the molecular docking analysis revealed a promising competitive interaction between PEA and AHLs. Consequently, this study offers valuable insights into the potential of rose absolute and PEA as inhibitors of QS in P. aeruginosa and C. violaceum.

Bacteria are a major determinant of Orsay virus transmission and infection in Caenorhabditis elegans.

The microbiota is a key determinant of the physiology and immunity of animal hosts. The factors governing the transmissibility of viruses between susceptible hosts are incompletely understood. Bacteria serve as food for Caenorhabditis elegans and represent an integral part of the natural environment of C. elegans. We determined the effects of bacteria isolated with C. elegans from its natural environment on the transmission of Orsay virus in C. elegans using quantitative virus transmission and host susceptibility assays. We observed that Ochrobactrum species promoted Orsay virus transmission, whereas Pseudomonas lurida MYb11 attenuated virus transmission relative to the standard laboratory bacterial food Escherichia coli OP50. We found that pathogenic Pseudomonas aeruginosa strains PA01 and PA14 further attenuated virus transmission. We determined that the amount of Orsay virus required to infect 50% of a C. elegans population on P. lurida MYb11 compared with Ochrobactrum vermis MYb71 was dramatically increased, over three orders of magnitude. Host susceptibility was attenuated even further in the presence of P. aeruginosa PA14. Genetic analysis of the determinants of P. aeruginosa required for attenuation of C. elegans susceptibility to Orsay virus infection revealed a role for regulators of quorum sensing. Our data suggest that distinct constituents of the C. elegans microbiota and potential pathogens can have widely divergent effects on Orsay virus transmission, such that associated bacteria can effectively determine host susceptibility versus resistance to viral infection. Our study provides quantitative evidence for a critical role for tripartite host-virus-bacteria interactions in determining the transmissibility of viruses among susceptible hosts.

Tyrosol blocks E. coli anaerobic biofilm formation via YbfA and FNR to increase antibiotic susceptibility.

Bacteria within mature biofilms are highly resistant to antibiotics than planktonic cells. Oxygen limitation contributes to antibiotic resistance in mature biofilms. Nitric oxide (NO) induces biofilm dispersal; however, low NO levels stimulate biofilm formation, an underexplored process. Here, we introduce a mechanism of anaerobic biofilm formation by investigating the antibiofilm activity of tyrosol, a component in wine. Tyrosol inhibits E. coli and Pseudomonas aeruginosa biofilm formation by enhancing NO production. YbfA is identified as a target of tyrosol and its downstream targets are sequentially determined. YbfA activates YfeR, which then suppresses the anaerobic regulator FNR. This suppression leads to decreased NO production, elevated bis-(3'-5')-cyclic dimeric GMP levels, and finally stimulates anaerobic biofilm formation in the mature stage. Blocking YbfA with tyrosol treatment renders biofilm cells as susceptible to antibiotics as planktonic cells. Thus, this study presents YbfA as a promising antibiofilm target to address antibiotic resistance posed by biofilm-forming bacteria, with tyrosol acting as an inhibitor.

Collagen concentration regulates neutrophil extravasation and migration in response to infection in an endothelium dependent manner.

Introduction: As the body's first line of defense against disease and infection, neutrophils must efficiently navigate to sites of inflammation; however, neutrophil dysregulation contributes to the pathogenesis of numerous diseases that leave people susceptible to infections. Many of these diseases are also associated with changes to the protein composition of the extracellular matrix. While it is known that neutrophils and endothelial cells, which play a key role in neutrophil activation, are sensitive to the mechanical and structural properties of the extracellular matrix, our understanding of how protein composition in the matrix affects the neutrophil response to infection is incomplete. Methods: To investigate the effects of extracellular matrix composition on the neutrophil response to infection, we used an infection-on-a-chip microfluidic device that replicates a portion of a blood vessel endothelium surrounded by a model extracellular matrix. Model blood vessels were fabricated by seeding human umbilical vein endothelial cells on 2, 4, or 6 mg/mL type I collagen hydrogels. Primary human neutrophils were loaded into the endothelial lumens and stimulated by adding the bacterial pathogen Pseudomonas aeruginosa to the surrounding matrix. Results: Collagen concentration did not affect the cell density or barrier function of the endothelial lumens. Upon infectious challenge, we found greater neutrophil extravasation into the 4 mg/mL collagen gels compared to the 6 mg/mL collagen gels. We further found that extravasated neutrophils had the highest migration speed and distance in 2mg/mL gels and that these values decreased with increasing collagen concentration. However, these phenomena were not observed in the absence of an endothelial lumen. Lastly, no differences in the percent of extravasated neutrophils producing reactive oxygen species were observed across the various collagen concentrations. Discussion: Our study suggests that neutrophil extravasation and migration in response to an infectious challenge are regulated by collagen concentration in an endothelial cell-dependent manner. The results demonstrate how the mechanical and structural aspects of the tissue microenvironment affect the neutrophil response to infection. Additionally, these findings underscore the importance of developing and using microphysiological systems for studying the regulatory factors that govern the neutrophil response.

Adaptive Escape of Pseudomonas aeruginosa by Application of Low-Amplitude Electric Pulses.

Marine antibiofouling using low-amplitude electric pulses (EP) is an energy-efficient and eco-friendly approach, but potential mechanisms for preventing biofouling remain unclear. In the present study, the 3D adhesion dynamics of a model microorganism─Pseudomonas aeruginosa (PAO1)─under low-amplitude cathodic EP were examined as a function of applying voltage and its duration (td). The results demonstrated that adhered bacteria escaped from the electrode surface even when EP was removed. The escaped bacteria ratio, induction period of escape, and duration of the detachment were influenced profoundly by EP amplitude but slightly by td when td ≥ 5 min. The acceleration of escaped PAO1 from the surface indicated that their flagellar motor was powered by EP. Particularly, EP enabled swimming bacteria to have adaptive motions that were sustainable and regulated by the gene rsmA. As a result, they had less accumulation near the surface. The propulsion of adhered bacteria and adaptive escape of swimming bacteria were enhanced in response to low-amplitude EP. Hence, low-amplitude and short-duration EP is promising for sustainable antibiofouling applications.

Single-cell level LasR-mediated quorum sensing response of Pseudomonas aeruginosa to pulses of signal molecules.

Quorum sensing (QS) is a communication form between bacteria via small signal molecules that enables global gene regulation as a function of cell density. We applied a microfluidic mother machine to study the kinetics of the QS response of Pseudomonas aeruginosa bacteria to additions and withdrawals of signal molecules. We traced the fast buildup and the subsequent considerably slower decay of a population-level and single-cell-level QS response. We applied a mathematical model to explain the results quantitatively. We found significant heterogeneity in QS on the single-cell level, which may result from variations in quorum-controlled gene expression and protein degradation. Heterogeneity correlates with cell lineage history, too. We used single-cell data to define and quantitatively characterize the population-level quorum state. We found that the population-level QS response is well-defined. The buildup of the quorum is fast upon signal molecule addition. At the same time, its decay is much slower following signal withdrawal, and the quorum may be maintained for several hours in the absence of the signal. Furthermore, the quorum sensing response of the population was largely repeatable in subsequent pulses of signal molecules.

Vertical confinement enhances surface exploration in bacterial twitching motility.

Bacteria are often found in environments where space is limited, and they attach themselves to surfaces. One common form of movement on these surfaces is bacterial twitching motility, which is powered by the extension and retraction of type IV pili. Although twitching motility in unrestricted conditions has been extensively studied, the effects of spatial confinement on this behaviour are not well understood. In this study, we explored the diffusive properties of individual twitching Pseudomonas aeruginosa cells in spatially confined conditions. We achieved this by placing the bacteria between layers of agarose and glass, and then tracking the long-term twitching motility of individual cells. Interestingly, we found that while confinement reduced the immediate speed of twitching, it paradoxically increased diffusion. Through a combination of mechanical and geometrical analysis, as well as numerical simulations, we showed that this increase in diffusion could be attributed to mechanical factors. The constraint imposed by the agarose altered the diffusion pattern of the bacteria from normal to superdiffusion. These findings provide valuable insights into the motile behaviour of bacteria in confined environments.

Bacteriophages and Green Synthesized Zinc Oxide Nanoparticles in Combination Are Efficient against Biofilm Formation of Pseudomonas aeruginosa.

Bacteriophages (phages) are viruses that infect the bacteria within which their reproduction cycle takes place, a process that ends in the lysis and death of the bacterial cell. Some phages are also able to destroy bacterial biofilms. Due to increased antibiotics resistance, Pseudomonas aeruginosa, another biofilm-forming pathogen, is a problem in many parts of the world. Zinc oxide (ZnO) and other metal nanoparticles (NPs) are biologically active and also possess anti-biofilm properties. ZnO-NPs were prepared by the green synthesis method using orange peels. The vibrational peaks of the ZnO-NPs were analyzed using FTIR analysis, and their size and morphological properties were determined using scanning electron microscopy (SEM). The ability of the ZnO-NPs to reduce or eliminate P. aeruginosa biofilm alone or in combination with phages PB10 and PA19 was investigated. The P. aeruginosa cells were effectively killed in the preformed 48 h biofilms during a 24 h incubation with the ZnO-NP-phage combination, in comparison with the control or ZnO-NPs alone. The treatments on growing biofilms were most efficient in the final stages of biofilm development. All five treatment groups showed a significant biofilm reduction compared to the control group (p < 0.0001) at 48 h of incubation. The influence of the ZnO-NPs and phages on the quorum sensing system of P. aeruginosa was monitored by quantitative real-time PCR (qRT-PCR) of the autoinducer biosynthesis gene lasI. While the ZnO-NPs repressed the lasI gene transcription, the phages slightly activated it at 24 and 48 h of incubation. Also, the effect of the ZnO-NPs and phage PA19 on the viability of HFF2 cells was investigated and the results showed that the combination of NPs with PA19 reduced the toxic effect of ZnO-NPs and also stimulated the growth in normal cells.

Pseudomonas aeruginosa breaches respiratory epithelia through goblet cell invasion in a microtissue model.

Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.

Extracellular vesicles released by host epithelial cells during Pseudomonas aeruginosa infection function as homing beacons for neutrophils.

BACKGROUND: Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can cause sight threatening infections in the eye and fatal infections in the cystic fibrosis airway. Extracellular vesicles (EVs) are released by host cells during infection and by the bacteria themselves; however, there are no studies on the composition and functional role of host-derived EVs during PA infection of the eye or lung. Here we investigated the composition and capacity of EVs released by PA infected epithelial cells to modulate innate immune responses in host cells. METHODS: Human telomerase immortalized corneal epithelial cells (hTCEpi) cells and human telomerase immortalized bronchial epithelial cells (HBECs) were treated with a standard invasive test strain of Pseudomonas aeruginosa, PAO1, for 6 h. Host derived EVs were isolated by qEV size exclusion chromatography. EV proteomic profiles during infection were compared using mass spectrometry and functional studies were carried out using hTCEpi cells, HBECs, differentiated neutrophil-like HL-60 cells, and primary human neutrophils isolated from peripheral blood. RESULTS: EVs released from PA infected corneal epithelial cells increased pro-inflammatory cytokine production in naïve corneal epithelial cells and induced neutrophil chemotaxis independent of cytokine production. The EVs released from PA infected bronchial epithelial cells were also chemotactic although they failed to induce cytokine secretion from naïve HBECs. At the proteomic level, EVs derived from PA infected corneal epithelial cells exhibited lower complexity compared to bronchial epithelial cells, with the latter having reduced protein expression compared to the non-infected control. CONCLUSIONS: This is the first study to comprehensively profile EVs released by corneal and bronchial epithelial cells during Pseudomonas infection. Together, these findings show that EVs released by PA infected corneal and bronchial epithelial cells function as potent mediators of neutrophil migration, contributing to the exuberant neutrophil response that occurs during infection in these tissues.

Citral and triclosan synergistically silence quorum sensing and potentiate antivirulence response in Pseudomonas aeruginosa.

Among the ESKAPE pathogens, Pseudomonas aeruginosa is an extensively notorious superbug that causes difficult-to-treat infections. Since quorum sensing (QS) directly promotes pseudomonal virulence, targeting QS circuits is a promising approach for disarming phenotypic virulence. Hence, this study scrutinizes the anti-QS, antivirulence, and anti-biofilm potential of citral (CiT; phytochemical) and triclosan (TcN; disinfectant), alone and in combination, against P. aeruginosa PAO1/PA14. The findings confirmed synergism between CiT and TcN and revealed their quorum quenching (QQ) potential. At sub-inhibitory levels, CiT-TcN combination significantly impeded pyocyanin, total bacterial protease, hemolysin, and pyochelin production alongside inhibiting biofilm formation in P. aeruginosa. Moreover, the QQ and antivirulence potential of CiT and TcN was positively correlated by molecular docking studies that predicted strong associations of the drugs with QS receptors of P. aeruginosa. Collectively, the study identifies CiT-TcN as an effective drug combination that harbors QQ, antivirulence, and anti-biofilm prospects against P. aeruginosa.

Olfaction regulates peripheral mitophagy and mitochondrial function.

The central nervous system coordinates peripheral cellular stress responses, including the unfolded protein response of the mitochondria (UPRMT); however, the contexts for which this regulatory capability evolved are unknown. UPRMT is up-regulated upon pathogenic infection and in metabolic flux, and the olfactory nervous system has been shown to regulate pathogen resistance and peripheral metabolic activity. Therefore, we asked whether the olfactory nervous system in Caenorhabditis elegans controls the UPRMT cell nonautonomously. We found that silencing a single inhibitory olfactory neuron pair, AWC, led to robust induction of UPRMT and reduction of oxidative phosphorylation dependent on serotonin signaling and parkin-mediated mitophagy. Further, AWC ablation confers resistance to the pathogenic bacteria Pseudomonas aeruginosa partially dependent on the UPRMT transcription factor atfs-1 and fully dependent on mitophagy machinery. These data illustrate a role for the olfactory nervous system in regulating whole-organism mitochondrial dynamics, perhaps in preparation for postprandial metabolic stress or pathogenic infection.

Skeletal muscle mitochondrial dysfunction mediated by Pseudomonas aeruginosa quorum-sensing transcription factor MvfR: reversing effects with anti-MvfR and mitochondrial-targeted compounds.

Sepsis and chronic infections with Pseudomonas aeruginosa, a leading "ESKAPE" bacterial pathogen, are associated with increased morbidity and mortality and skeletal muscle atrophy. The actions of this pathogen on skeletal muscle remain poorly understood. In skeletal muscle, mitochondria serve as a crucial energy source, which may be perturbed by infection. Here, using the well-established backburn and infection model of murine P. aeruginosa infection, we deciphered the systemic impact of the quorum-sensing transcription factor MvfR (multiple virulence factor regulator) by interrogating, 5 days post-infection, its effect on mitochondrial-related functions in the gastrocnemius skeletal muscle and the outcome of the pharmacological inhibition of MvfR function and that of the mitochondrial-targeted peptide, Szeto-Schiller 31 (SS-31). Our findings show that the MvfR perturbs adenosine triphosphate generation, oxidative phosphorylation, and antioxidant response, elevates the production of reactive oxygen species, and promotes oxidative damage of mitochondrial DNA in the gastrocnemius muscle of infected mice. These impairments in mitochondrial-related functions were corroborated by the alteration of key mitochondrial proteins involved in electron transport, mitochondrial biogenesis, dynamics and quality control, and mitochondrial uncoupling. Pharmacological inhibition of MvfR using the potent anti-MvfR lead, D88, we developed, or the mitochondrial-targeted peptide SS-31 rescued the MvfR-mediated alterations observed in mice infected with the wild-type strain PA14. Our study provides insights into the actions of MvfR in orchestrating mitochondrial dysfunction in the skeletal murine muscle, and it presents novel therapeutic approaches for optimizing clinical outcomes in affected patients. IMPORTANCE: Skeletal muscle, pivotal for many functions in the human body, including breathing and protecting internal organs, contains abundant mitochondria essential for maintaining cellular homeostasis during infection. The effect of Pseudomonas aeruginosa (PA) infections on skeletal muscle remains poorly understood. Our study delves into the role of a central quorum-sensing transcription factor, multiple virulence factor regulator (MvfR), that controls the expression of multiple acute and chronic virulence functions that contribute to the pathogenicity of PA. The significance of our study lies in the role of MvfR in the metabolic perturbances linked to mitochondrial functions in skeletal muscle and the effectiveness of the novel MvfR inhibitor and the mitochondrial-targeted peptide SS-31 in alleviating the mitochondrial disturbances caused by PA in skeletal muscle. Inhibiting MvfR or interfering with its effects can be a potential therapeutic strategy to curb PA virulence.

Atomic force microscopy analysis of Pel polysaccharide- and type IV pili-mediated adhesion of Pseudomonas aeruginosa PA14 to an abiotic surface.

Type IV pili (TFP) contribute to the ability of microbes such as Pseudomonas aeruginosa to engage with and move across surfaces. We reported previously that P. aeruginosa TFP generate retractive forces of ∼30 pN and provided indirect evidence that TFP-mediated surface attachment was enhanced in the presence of the Pel polysaccharide. Here, we use different mutants defective in flagellar, Pel production or TFP production - alone or in combination - to decipher the relative contribution of these biofilm-promoting factors for P. aeruginosa adhesion. By means of atomic force microscopy (AFM), we show that mutating the flagellum (ΔflgK mutant) results in an increase in Pel polysaccharide production, but this increase in Pel does not result in an increase in surface adhesive properties compared to those previously described for the WT strain. By blocking Pel production in the ΔflgK mutant (ΔflgKΔpel), we directly show that TFP play a major role in the adhesion of the bacteria to hydrophobic AFM tips, but that the adhesion force is only slightly impaired by the absence of Pel. Inversely, performing single-cell force spectroscopy measurements with the mutant lacking TFP (ΔflgKΔpilA) reveals that the Pel can modulate the attachment of the bacteria to a hydrophobic substrate in a time-dependent manner. Finally, little adhesion was detected for the ΔflgKΔpilAΔpelA triple mutant, suggesting that both TFP and Pel polysaccharide make a substantial contribution to bacteria-substratum interaction events. Altogether, our data allow us to decipher the relative contribution of Pel and TFP in the early attachment by P. aeruginosa.

Evaluation of biofilm formation and expression of psl, pel, alg genes of Pseudomonas aeruginosa in exposure to detergents.

Pseudomonas aeruginosa has been in the center of attention for several years as an opportunistic human pathogen implicated in many severe acute and chronic infections particularly in immunocompromised patients. Its high persistence and resistance against many antimicrobial agents are mostly attributed to biofilm formation. Biofilms are microbial communities mainly consisting of extracellular polymeric substances that encapsulate bacteria together and protect them from extracellular stresses. This cell aggregation is a stress response that P. aeruginosa employes as a survival strategy during growth with the toxic detergents. This process has shown to involve several operons such as psl, pel, and alg. Here we used P. aeruginosa strain PAO1 in control group, 40 P. aeruginosa strains from sink and 40 strains from surface of public places. Biofilm formation and gene expression were measured before and after exposure to sub minimum inhibitory concentration (sub-MIC) of biocides chlorhexidine diacetate and benzalkonium chloride. The qRT-PCR and biofilm formation results demonstrated an increase in biofilm formation ability and gene expression of pslA/B and pelA/B in two groups collected from sink and surface in contrast to the control group. A remarkable increase was observed in the biofilm formation and expression of pslA in the bacterial strain collected from the sink after exposure to biocides chlorhexidine diacetate. Both Pel and Psl appeared to have redundant functions as structural scaffolds in biofilms. Sub-MIC levels of detergents can improve biofilm formation ability of P. aeruginosa and therefore trigger resistance.

Effects of quorum sensing-interfering agents, including macrolides and furanone C-30, and an efflux pump inhibitor on nitrosative stress sensitivity in Pseudomonas aeruginosa.

Long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infection, despite how limited the clinically achievable concentrations are, being far below their MICs. An increase in the sub-MIC of macrolide exposure-dependent sensitivity to nitrosative stress is a typical characteristic of P. aeruginosa. However, a few P. aeruginosa clinical isolates do not respond to sub-MIC of macrolide treatment. Therefore, we examined the effects of sub-MIC of erythromycin (EM) on the sensitivity to nitrosative stress together with an efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN). The sensitivity to nitrosative stress increased, suggesting that the efflux pump was involved in inhibiting the sub-MIC of macrolide effect. Analysis using efflux pump-mutant P. aeruginosa revealed that MexAB-OprM, MexXY-OprM, and MexCD-OprJ are factors in reducing the sub-MIC of macrolide effect. Since macrolides interfere with quorum sensing (QS), we demonstrated that the QS-interfering agent furanone C-30 (C-30) producing greater sensitivity to nitric oxide (NO) stress than EM. The effect of C-30 was decreased by overproduction of MexAB-OprM. To investigate whether the increase in the QS-interfering agent exposure-dependent sensitivity to nitrosative stress is characteristic of P. aeruginosa clinical isolates, we examined the viability of P. aeruginosa treated with NO. Although treatment with EM could reduce cell viability, a high variability in EM effects was observed. Conversely, C-30 was highly effective at reducing cell viability. Treatment with both C-30 and PAßN was sufficiently effective against the remaining isolates. Therefore, the combination of a QS-interfering agent and an EPI could be effective in treating P. aeruginosa infections.

Characterization of biofilm formation and multi-drug resistance among Pseudomonas aeruginosa isolated from hospital wastewater in Dhaka, Bangladesh.

Hospital wastewater has been identified as a hotspot for the emergence and transmission of multidrug-resistant (MDR) pathogens that present a serious threat to public health. Therefore, we investigated the current status of antibiotic resistance as well as the phenotypic and genotypic basis of biofilm formation in Pseudomonas aeruginosa from hospital wastewater in Dhaka, Bangladesh. The disc diffusion method and the crystal violet assay were performed to characterize antimicrobial resistance and biofilm formation, respectively. Biofilm and integron-associated genes were amplified by the polymerase chain reaction. Isolates exhibited varying degrees of resistance to different antibiotics, in which >80% of isolates showed sensitivity to meropenem, amikacin, and gentamicin. The results indicated that 93.82% of isolates were MDR and 71 out of 76 MDR isolates showed biofilm formation activities. We observed the high prevalence of biofilm-related genes, in which algD+pelF+pslD+ (82.7%) was found to be the prevalent biofilm genotypic pattern. Sixteen isolates (19.75%) possessed class 1 integron (int1) genes. However, statistical analysis revealed no significant association between biofilm formation and multidrug resistance (χ2 = 0.35, P = 0.55). Taken together, hospital wastewater in Dhaka city may act as a reservoir for MDR and biofilm-forming P. aeruginosa, and therefore, the adequate treatment of wastewater is recommended to reduce the occurrence of outbreaks.