Phage-antibiotic combinations to control Pseudomonas aeruginosa-Candida two-species biofilms.

Phage-antibiotic combinations to treat bacterial infections are gaining increased attention due to the synergistic effects often observed when applying both components together. Most studies however focus on a single pathogen, although in many clinical cases multiple species are present at the site of infection. The aim of this study was to investigate the anti-biofilm activity of phage-antibiotic/antifungal combinations on single- and dual-species biofilms formed by P. aeruginosa and the fungal pathogen Candida albicans. The Pseudomonas phage Motto in combination with ciprofloxacin had significant anti-biofilm activity. We then compared biofilms formed by P. aeruginosa alone with the dual-species biofilms formed by bacteria and C. albicans. Here, we found that the phage together with the antifungal fluconazole was active against 6-h-old dual-species biofilms but showed only negligible activity against 24-h-old biofilms. This study lays the first foundation for potential therapeutic approaches to treat co-infections caused by bacteria and fungi using phage-antibiotic combinations.

Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

A genetic screen identifies a role for oprF in Pseudomonas aeruginosa biofilm stimulation by subinhibitory antibiotics.

Biofilms are surface-associated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentrations (sub-MIC) of antibiotics induce biofilm formation, potentially as a defensive response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unclear. We show that treatment of Pseudomonas aeruginosa with multiple classes of sub-MIC antibiotics with distinct targets induces biofilm formation. Further, addition of exogenous eDNA or cell lysate failed to increase biofilm formation to the same extent as antibiotics, suggesting that the release of cellular contents by antibiotic-driven bacteriolysis is insufficient. Using a genetic screen for stimulation-deficient mutants, we identified the outer membrane porin OprF and the ECF sigma factor SigX as important. Similarly, loss of OmpA - the Escherichia coli OprF homolog - prevented sub-MIC antibiotic stimulation of E. coli biofilms. Our screen also identified the periplasmic disulfide bond-forming enzyme DsbA and a predicted cyclic-di-GMP phosphodiesterase encoded by PA2200 as essential for biofilm stimulation. The phosphodiesterase activity of PA2200 is likely controlled by a disulfide bond in its regulatory domain, and folding of OprF is influenced by disulfide bond formation, connecting the mutant phenotypes. Addition of reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, activation of a c-di-GMP-responsive promoter follows treatment with sub-MIC antibiotics in the wild-type but not an oprF mutant. Together, these results show that antibiotic-induced biofilm formation is likely driven by a signaling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP.

Application of cuminaldehyde and ciprofloxacin for the effective control of biofilm assembly of Pseudomonas aeruginosa: A combinatorial study.

Pseudomonas aeruginosa is widely associated with biofilm-mediated antibiotic resistant chronic and acute infections which constitute a persistent healthcare challenges. Addressing this threat requires exploration of novel therapeutic strategies involving the combination of natural compounds and conventional antibiotics. Hence, our study has focused on two compounds; cuminaldehyde and ciprofloxacin, which were strategically combined to target the biofilm challenge of P. aeruginosa. The minimum inhibitory concentration (MIC) of cuminaldehyde and ciprofloxacin was found to be 400 µg/mL and 0.4 µg/mL, respectively. Moreover, the fractional inhibitory concentration index (FICI = 0.62) indicated an additive interaction prevailed between cuminaldehyde and ciprofloxacin. Subsequently, sub-MIC doses of cuminaldehyde (25 µg/mL) and ciprofloxacin (0.05 µg/mL) were selected for an array of antibiofilm assays which confirmed their biofilm inhibitory potential without exhibiting any antimicrobial activity. Furthermore, selected doses of the mentioned compounds could manage biofilm on catheter surface by inhibiting and disintegrating existing biofilm. Additionally, the test combination of the mentioned compounds reduced virulence factors secretion, accumulated reactive oxygen species and increased cell-membrane permeability. Thus, the combination of cuminaldehyde and ciprofloxacin demonstrates potential in combating biofilm-associated Pseudomonal threats.

Quantifying the Dynamics of Bacterial Biofilm Formation on the Surface of Soft Contact Lens Materials Using Digital Holographic Tomography to Advance Biofilm Research.

The increase in bacterial resistance to antibiotics in recent years demands innovative strategies for the detection and combating of biofilms, which are notoriously resilient. Biofilms, particularly those on contact lenses, can lead to biofilm-related infections (e.g., conjunctivitis and keratitis), posing a significant risk to patients. Non-destructive and non-contact sensing techniques are essential in addressing this threat. Digital holographic tomography emerges as a promising solution. This allows for the 3D reconstruction of the refractive index distribution in biological samples, enabling label-free visualization and the quantitative analysis of biofilms. This tool provides insight into the dynamics of biofilm formation and maturation on the surface of transparent materials. Applying digital holographic tomography for biofilm examination has the potential to advance our ability to combat the antibiotic bacterial resistance crisis. A recent study focused on characterizing biofilm formation and maturation on six soft contact lens materials (three silicone hydrogels, three hydrogels), with a particular emphasis on Staphylococcus epidermis and Pseudomonas aeruginosa, both common culprits in ocular infections. The results revealed species- and time-dependent variations in the refractive indexes and volumes of biofilms, shedding light on cell dynamics, cell death, and contact lens material-related factors. The use of digital holographic tomography enables the quantitative analysis of biofilm dynamics, providing us with a better understanding and characterization of bacterial biofilms.

Discovery of Melittin as Triple-Action Agent: Broad-Spectrum Antibacterial, Anti-Biofilm, and Potential Anti-Quorum Sensing Activities.

The development of antibiotic-resistant microorganisms is a major global health concern. Recently, there has been an increasing interest in antimicrobial peptides as a therapeutic option. This study aimed to evaluate the triple-action (broad-spectrum antibacterial, anti-biofilm, and anti-quorum sensing activities) of melittin, a membrane-active peptide present in bee venom. The minimum inhibitory concentration and minimum bactericidal concentration of the melittin were determined using the microdilution method and agar plate counting. Growth curve analysis revealed that melittin showed a concentration-dependent antibacterial activity. Scanning electron microscope analysis revealed that melittin treatment altered the morphology. Confocal laser scanning microscope revealed that melittin increased the membrane permeability and intracellular ROS generation in bacteria, all of which contribute to bacterial cell death. In addition, the crystal violet (CV) assay was used to test the anti-biofilm activity. The CV assay demonstrated that melittin inhibited biofilm formation and eradicated mature biofilms. Biofilm formation mediated by quorum sensing (QS) plays a major role in this regard, so molecular docking and molecular dynamics analysis confirmed that melittin interacts with LasR receptors through hydrogen bonds, and further evaluates the anti-QS activity of melittin through the production of virulence factors (pyocyanin, elastase, and rhamnolipid), exopolysaccharides secretion, and bacterial motility, that may be the key to inhibiting the biofilm formation mechanism. The present findings highlight the promising role of melittin as a broad-spectrum antibacterial, anti-biofilm agent, and potential QS inhibitor, providing a new perspective and theoretical basis for the development of alternative antibiotics.

Immunometabolic Regulation of Bacterial Infection, Biofilms, and Antibiotic Susceptibility.

BACKGROUND: Upon infection, mucosal tissues activate a brisk inflammatory response to clear the pathogen, i.e., resistance to disease. Resistance to disease is orchestrated by tissue-resident macrophages, which undergo profound metabolic reprogramming after sensing the pathogen. These metabolically activated macrophages release many inflammatory factors, which promote their bactericidal function. However, in immunocompetent individuals, pathogens like Pseudomonas aeruginosa, Staphylococcus aureus, and Salmonella evade this type of immunity, generating communities that thrive for the long term. SUMMARY: These organisms develop features that render them less susceptible to eradication, such as biofilms and increased tolerance to antibiotics. Furthermore, after antibiotic therapy withdrawal, "persister" cells rapidly upsurge, triggering inflammatory relapses that worsen host health. How these pathogens persisted in inflamed tissues replete with activated macrophages remains poorly understood. KEY MESSAGES: In this review, we discuss recent findings indicating that the ability of P. aeruginosa, S. aureus, and Salmonella to evolve biofilms and antibiotic tolerance is promoted by the similar metabolic routes that regulate macrophage metabolic reprogramming.

Pseudomonas aeruginosa outer membrane vesicle-packed sRNAs can enter host cells and regulate innate immune responses.

Bacterial outer membrane vesicles (OMVs) can package and deliver virulence factors into host cells, which is an important mechanism mediating host-pathogen interactions. It has been reported that small RNAs (sRNAs) can be packed into OMVs with varying relative abundance, which might affect the function and/or stability of host mRNAs. In this study, we used OptiPrep density gradient ultra-high-speed centrifugation to purify OMVs from Pseudomonas aeruginosa. Next, the sequences and abundance of sRNAs were detected by using Small RNA-Seq. In particular, sRNA4518698, sRNA2316613 and sRNA809738 were the three most abundant sRNAs in OMVs, which are all fragments of P. aeruginosa non-coding RNAs. sRNAs were shielded within the interior of OMVs and remained resistant to external RNase cleavage. The miRanda and RNAhybrid analysis demonstrated that those sRNAs could target a large number of host mRNAs, which were enriched in host immune responses by the functions of GO and KEGG enrichment. Experimentally, we demonstrated that the transfection of synthetic sRNA4518698, sRNA2316613, or sRNA809738 could reduce the expression of innate immune response genes in RAW264.7 cells. Together, we demonstrated that P. aeruginosa OMVs sRNAs can regulate innate immune responses. This study uncovered a mechanism in which the OMVs regulate host responses by transferring bacterial sRNAs.

Ciprofloxacin Poly(ß-amino ester) Conjugates Enhance Antibiofilm Activity and Slow the Development of Resistance.

To tackle the emerging antibiotic resistance crisis, novel antimicrobial approaches are urgently needed. Bacterial biofilms are a particular concern in this context as they are responsible for over 80% of bacterial infections and are inherently more recalcitrant toward antimicrobial treatments. The high tolerance of biofilms to conventional antibiotics has been attributed to several factors, including reduced drug diffusion through the dense exopolymeric matrix and the upregulation of antimicrobial resistance machinery with successful biofilm eradication requiring prolonged high doses of multidrug treatments. A promising approach to tackle bacterial infections involves the use of polymer drug conjugates, shown to improve upon free drug toxicity and bioavailability, enhance drug penetration through the thick biofilm matrix, and evade common resistance mechanisms. In the following study, we conjugated the antibiotic ciprofloxacin (CIP) to a small library of biodegradable and biocompatible poly(ß-amino ester) (PBAE) polymers with varying central amine functionality. The suitability of the polymers as antibiotic conjugates was then verified in a series of assays including testing of efficacy and resistance response in planktonic Gram-positive and Gram-negative bacteria and the reduction of viability in mono- and multispecies biofilm models. The most active polymer within the prepared PBAE-CIP library was shown to achieve an over 2-fold increase in the reduction of biofilm viability in a Pseudomonas aeruginosa monospecies biofilm and superior elimination of all the species present within the multispecies biofilm model. Hence, we demonstrate that CIP conjugation to PBAEs can be employed to achieve improved antibiotic efficacy against clinically relevant biofilm models.

The Inovirus Pf4 Triggers Antiviral Responses and Disrupts the Proliferation of Airway Basal Epithelial Cells.

BACKGROUND: The inovirus Pf4 is a lysogenic bacteriophage of Pseudomonas aeruginosa (Pa). People with Cystic Fibrosis (pwCF) experience chronic airway infection with Pa and a significant proportion have high numbers of Pf4 in their airway secretions. Given the known severe damage in the airways of Pa-infected pwCF, we hypothesized a high Pf4 burden can affect airway healing and inflammatory responses. In the airway, basal epithelial cells (BCs) are a multipotent stem cell population critical to epithelium homeostasis and repair. We sought to investigate the transcriptional responses of BCs under conditions that emulate infection with Pa and exposure to high Pf4 burden. METHODS: Primary BCs isolated from pwCF and wild-type (WT) donors were cultured in vitro and exposed to Pf4 or bacterial Lipopolysaccharide (LPS) followed by transcriptomic and functional assays. RESULTS: We found that BCs internalized Pf4 and this elicits a strong antiviral response as well as neutrophil chemokine production. Further, we found that BCs that take up Pf4 demonstrate defective migration and proliferation. CONCLUSIONS: Our findings are highly suggestive of Pf4 playing a role in the pathogenicity of Pa in the airways. These findings provide additional evidence for the ability of inoviruses to interact with mammalian cells and disrupt cell function.

In vitro prevention and inactivation of biofilms using controlled-release iodine foam dressings for wound healing.

Microbial biofilms are a major hindrance in the wound healing process, prolonging the inflammatory response phase, thus making them a target in treatment. The aim of this study is to assess the antibacterial properties of commercially available wound dressings, of various material composition and antibacterial agents, towards multiple in vitro microbial and biofilm models. A variety of in vitro microbial and biofilm models were utilised to evaluate the ability of wound dressing materials to sequester microbes, prevent dissemination and manage bioburden. Sequestering and dissemination models were used to evaluate the ability of wound dressing materials to prevent the biofilm-forming bacterium, Pseudomonas aeruginosa, from migrating through dressing materials over a 24-72 h challenge period. Additionally, Centre for Disease Control (CDC) Bioreactor and Drip Flow models were used to evaluate antibacterial killing efficacy towards established P. aeruginosa and Staphylococcus aureus biofilms using more challenging, wound-like models. Controlled-release iodine foam and silver-impregnated carboxymethylcellulose (CMC) wound dressing materials demonstrated potent biofilm management properties in comparison to a methylene blue and gentian violet-containing foam dressing. Both the iodine-containing foam and silver-impregnated CMC materials effectively prevented viable P. aeruginosa dissemination for up to 72 h. In addition, the controlled-release iodine foam and silver-impregnated CMC materials reduced P. aeruginosa bioburden in the Drip Flow model. The controlled-release iodine foam demonstrated superiority in the CDC Bioreactor model, as both the silver- and iodine-containing materials reduced S. aureus to the limit of detection, but P. aeruginosa growth was only completely reduced by controlled-release iodine foam dressing materials. The data generated within the in vitro biofilm models supports the clinical data available in the public domain for the implementation of iodine foam dressings for effective biofilm management and control in wound care.

Bacterial synergies and antagonisms affecting Pseudomonas aeruginosa virulence in the human lung, skin and intestine.

Pseudomonas aeruginosa requires a significant breach in the host defense to cause an infection. While its virulence factors are well studied, its tropism cannot be explained only by studying its interaction with the host. Why are P. aeruginosa infections so rare in the intestine compared with the lung and skin? There is not enough evidence to claim specificity in virulence factors deployed by P. aeruginosa in each anatomical site, and host physiology differences between the lung and the intestine cannot easily explain the observed differences in virulence. This perspective highlights a relatively overlooked parameter in P. aeruginosa virulence, namely, potential synergies with bacteria found in the human skin and lung, as well as antagonisms with bacteria of the human intestine.

Pseudomonas aeruginosa is human bacterial pathogen that requires a significant weakness in the body's defenses to trigger an infection. The reasons for its preference in targeting certain body parts (e.g., the lungs and skin) over others (e.g., the intestine) remain unclear. This article considers why P. aeruginosa infections are less common in the intestine. We suggest that the differences in host physiology alone cannot explain this difference and that the synergy of P. aeruginosa with other bacteria that live on the skin and in the lungs, and its antagonism with other bacteria in the intestine, play a role in its infection patterns. In essence, bacterial interactions can shed light to our understanding P. aeruginosa's ability to cause infection.

Extracellular vesicles secreted by primary human bronchial epithelial cells reduce Pseudomonas aeruginosa burden and inflammation in cystic fibrosis mouse lung.

Cystic fibrosis (CF) results in a reduction in the volume of airway surface liquid, increased accumulation of viscous mucus, persistent antibiotic-resistant lung infections that cause chronic inflammation, and a decline in lung function. More than 50% of adults with CF are chronically colonized by Pseudomonas aeruginosa (P. aeruginosa), the primary reason for morbidity and mortality in people with CF (pwCF). Although highly effective modulator therapy (HEMT) is an important part of disease management in CF, HEMT does not eliminate P. aeruginosa or lung inflammation. Thus, new treatments are required to reduce lung infection and inflammation in CF. In a previous in vitro study, we demonstrated that primary human bronchial epithelial cells (HBECs) secrete extracellular vesicles (EVs) that block the ability of P. aeruginosa to form biofilms by reducing the abundance of several proteins necessary for biofilm formation as well as enhancing the sensitivity of P. aeruginosa to ß-lactam antibiotics. In this study, using a CF mouse model of P. aeruginosa infection, we demonstrate that intratracheal administration of EVs secreted by HBEC reduced P. aeruginosa lung burden and several proinflammatory cytokines including IFN-γ, TNF-α, and MIP-1ß in bronchoalveolar lavage fluid (BALF), even in the absence of antibiotics. Moreover, EVs decreased neutrophils in BALF. Thus, EVs secreted by HBEC reduce the lung burden of P. aeruginosa, decrease inflammation, and reduce neutrophils in a CF mouse model. These results suggest that HBEC via the secretion of EVs may play an important role in the immune response to P. aeruginosa lung infection.NEW & NOTEWORTHY Our findings show that extracellular vesicles secreted by primary human bronchial epithelial cells significantly reduce Pseudomonas aeruginosa burden, inflammation, and weight loss in a cystic fibrosis mouse model of infection.

Genetic and Phenotypic Analysis of Phage-Resistant Mutant Fitness Triggered by Phage-Host Interactions.

The emergence of phage-resistant bacterial strains is one of the biggest challenges for phage therapy. However, the emerging phage-resistant bacteria are often accompanied by adaptive trade-offs, which supports a therapeutic strategy called "phage steering". The key to phage steering is to guide the bacterial population toward an evolutionary direction that is favorable for treatment. Thus, it is important to systematically investigate the impacts of phages targeting different bacterial receptors on the fitness of the bacterial population. Herein, we employed 20 different phages to impose strong evolutionary pressure on the host Pseudomonas aeruginosa PAO1 and examined the genetic and phenotypic responses of their phage-resistant mutants. Among these strains with impaired adsorptions, four types of mutations associated with bacterial receptors were identified, namely, lipopolysaccharides (LPSs), type IV pili (T4Ps), outer membrane proteins (OMPs), and exopolysaccharides (EPSs). PAO1, responding to LPS- and EPS-dependent phage infections, mostly showed significant growth impairment and virulence attenuation. Most mutants with T4P-related mutations exhibited a significant decrease in motility and biofilm formation ability, while the mutants with OMP-related mutations required the lowest fitness cost out of the bacterial populations. Apart from fitness costs, PAO1 strains might lose their resistance to antibiotics when counteracting with phages, such as the presence of large-fragment mutants in this study, which may inspire the usage of phage-antibiotic combination strategies. This work provides methods that leverage the merits of phage resistance relative to obtaining therapeutically beneficial outcomes with respect to phage-steering strategies.

Biofilm heterogeneity-adaptive photoredox catalysis enables red light-triggered nitric oxide release for combating drug-resistant infections.

The formation of biofilms is closely associated with persistent and chronic infections, and physiological heterogeneity such as pH and oxygen gradients renders biofilms highly resistant to conventional antibiotics. To date, effectively treating biofilm infections remains a significant challenge. Herein, we report the fabrication of micellar nanoparticles adapted to heterogeneous biofilm microenvironments, enabling nitric oxide (NO) release through two distinct photoredox catalysis mechanisms. The key design feature involves the use of tertiary amine (TA) moieties, which function as sacrificial agents to avoid the quenching of photocatalysts under normoxic and neutral pH conditions and proton acceptors at acidic pH to allow deep biofilm penetration. This biofilm-adaptive NO-releasing platform shows excellent antibiofilm activity against ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) biofilms both in vitro and in a mouse skin infection model, providing a strategy for combating biofilm heterogeneity and biofilm-related infections.

Human Complement Inhibits Myophages against Pseudomonas aeruginosa.

Therapeutic bacteriophages (phages) are primarily chosen based on their in vitro bacteriolytic activity. Although anti-phage antibodies are known to inhibit phage infection, the influence of other immune system components is less well known. An important anti-bacterial and anti-viral innate immune system that may interact with phages is the complement system, a cascade of proteases that recognizes and targets invading microorganisms. In this research, we aimed to study the effects of serum components such as complement on the infectivity of different phages targeting Pseudomonas aeruginosa. We used a fluorescence-based assay to monitor the killing of P. aeruginosa by phages of different morphotypes in the presence of human serum. Our results reveal that several myophages are inhibited by serum in a concentration-dependent way, while the activity of four podophages and one siphophage tested in this study is not affected by serum. By using specific nanobodies blocking different components of the complement cascade, we showed that activation of the classical complement pathway is a driver of phage inhibition. To determine the mechanism of inhibition, we produced bioorthogonally labeled fluorescent phages to study their binding by means of microscopy and flow cytometry. We show that phage adsorption is hampered in the presence of active complement. Our results indicate that interactions with complement may affect the in vivo activity of therapeutically administered phages. A better understanding of this phenomenon is essential to optimize the design and application of therapeutic phage cocktails.

Cell-Based Assay to Determine Type 3 Secretion System Translocon Assembly in Pseudomonas aeruginosa Using Split Luciferase.

Multi-drug-resistant Pseudomonas aeruginosa poses a serious threat to hospitalized patients. This organism expresses an arsenal of virulence factors that enables it to readily establish infections and disseminate in the host. The Type 3 secretion system (T3SS) and its associated effectors play a crucial role in the pathogenesis of P. aeruginosa, making them attractive targets for the development of novel therapeutic agents. The T3SS translocon, composed of PopD and PopB, is an essential component of the T3SS secretion apparatus. In the properly assembled translocon, the N-terminus of PopD protrudes into the cytoplasm of the target mammalian cell, which can be exploited as a molecular indicator of functional translocon assembly. In this article, we describe a novel whole-cell-based assay that employs the split NanoLuc luciferase detection system to provide a readout for translocon assembly. The assay demonstrates a favorable signal/noise ratio (13.6) and robustness (Z' = 0.67), making it highly suitable for high-throughput screening of small-molecule inhibitors targeting T3SS translocon assembly.

Mechanism Insights of Antibacterial Surfaces Coated with Dead Probiotics.

To understand the antimicrobial effect of surfaces fabricated with dead probiotics, we prepared surfaces decorated with dead probiotics Lactobacillus rhamnosus GG (LGG) with varied inactivation methods and explored their inhibitory interactions with Pseudomonas aeruginosa (PAO1). By combining several techniques, i.e., digital holographic microscopy (DHM), atomic force microscopy (AFM), RNA sequencing, and metabolomic analysis, we studied the three-dimensional (3D) swimming behaviors, surface adhesion, biofilm formation, and adaptive responses of PAO1 near such surfaces. The results show that planktonic PAO1 decreases their flick and reverse motions by downregulating the chemotaxis pathway and accelerates with less accumulation near dead LGG surfaces by upregulating the flagellar assembly pathway and decreasing cyclic adenosine monophosphate. Distinct from live siblings, the surfaces decorated with dead LGG show a significant reduction in adhesion strength with PAO1 and inhibit biofilm formation with more downregulated genes in the Pseudomonas quinolone signal and biofilm formation pathway. We demonstrate that the antibacterial ability of such surfaces stems from the gradually released lysate from the dead LGG that is unfavorable to PAO1 in close proximity. The releasing rate and order depend on the cell membrane integrity, which closely relates to the inactivation methods.

Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

Phenylalanine-Arginine-ß-Naphthylamide Enhances the Photobactericidal Effect of Methylene Blue on Pseudomonas aeruginosa.

Objective: To investigate the effectiveness, dosing sequence, concentration, and mechanism of antimicrobial photodynamic inactivation (aPDI) using methylene blue (MB) plus phenylalanine-arginine-ß-naphthylamide (PAßN) against Pseudomonas aeruginosa. Methods: P. aeruginosa bacterial suspension was incubated with MB for different times (5-240 min), and then, 10 J/cm2 red light was irradiated. The efflux pump inhibitor (EPI) PAßN (10-100 µg/mL) was combined with MB (1-20 µM) in different sequences (PAßN-first, PAßN+MB, PAßN-after). Colony-forming units were then determined by serial dilution. Results: Using MB 10 µM plus 10 J/cm2, the killing effect of MB-aPDI on P. aeruginosa increased first and then decreased with longer incubation time. The killing effect of MB+PAßN-aPDI on P. aeruginosa was better than that of MB-aPDI (p < 0.05) by up to 2 logs. PAßN-first had the best killing effect, whereas PAßN-after had the worst killing effect. The killing effect increased with PAßN concentration and at 100 µg/mL reached 5.1 logs. Conclusions: The EPI PAßN enhanced the bactericidal effect of MB-aPDI on P. aeruginosa, especially when added before MB. It is proposed that MB is a substrate of the resistance-nodulation-division family efflux pump.