Pseudomonas Aeruginosa Research Articles

Molecular Detection of Virulence Genes Associated With Multi- Drug Resistant Pseudomonas Aeruginosa Isolated from Clinical and Environmental Samples within Maiduguri

Pseudomonas aeruginosa is an ubiquitous opportunistic pathogen having numerous virulence factors and the ability to acquire multidrug resistant traits. This study aimed to determine the presence of the virulence genes algD and lasB in multidrug resistant Pseudomonas aeruginosa isolated from clinical and environmental samples within Maiduguri. A total of 200 samples were collected from four sources namely; Farm soil at root region of groundnut, abattoir waste water, commercial boreholes and urine of urinary tract infected patients. All positive growths on cetrimide agar were further identified biochemically. Antibiotic profile of confirmed Pseudomonas aeruginosa was determined using Kirby-Baeur disc diffusion method. The genes algD and lasB were detected in the multidrug resistant isolates. The result also showed that 90.90%, 81.81% and 81.81% of the urine isolates were resistant to nalidixic acid, chloramphenicol and tetracycline respectively while highest sensitivity was observed against amikacin at 81.81%. Highest resistance among environmental samples was observed in abattoir wastewater with 95.24% resistance to chloramphenicol while least resistance was observed in borehole isolates with 0.00% resistance to Gentamicin, Amikaicin, Meropenem and Aztreonam. According to the findings of this study also, there is no significant difference in habouring virulence genes among the isolates of the different sources. All the multidrug resistant isolates were found to habour both algD and LasB genes. In conclusion, P. aeruginosa was found in all of the studied sources and widespread of algD and LasB genes in the multidrug resistant isolates from all the sources.

In response to bacteria, neutrophils release extracellular vesicles capable of initiating thrombin generation through DNA-dependent and independent pathways.

Neutrophils release extracellular vesicles (EVs) and some subsets of neutrophil-derived EVs are procoagulant. In response to S. aureus, neutrophils produce EVs that associate electrostatically with neutrophil extracellular traps (NETs). DNA in NETs is procoagulant, but whether neutrophil EVs produced during bacterial challenge have similar activity is unknown. Given that EV activity is agonist- and cell-type dependent and coagulation contributes to sepsis, we hypothesized that sepsis-causing bacteria increase production of neutrophil-derived EVs, as well as EV-associated DNA, and intact EVs and DNA cause coagulation. We recovered EVs from neutrophils challenged with S. aureus (SA), S. epidermidis (SE), E. coli (EC), and P. aeruginosa (PA), and measured associated DNA and procoagulant activity. EVs from SA-challenged neutrophils (SA-EVs), which were previously characterized, displayed dose-dependent procoagulant activity as measured by thrombin generation (TG) in platelet-poor plasma. EV lysis and DNase treatment reduced TG by 90% and 37%, respectively. SE, EC, and PA also increased EV production and EV-associated extracellular DNA, and these EVs were also procoagulant. Compared to spontaneously released EVs, which demonstrated some ability to amplify Factor XII-dependent coagulation in the presence of an activator, only EVs produced in response to bacteria could initiate the pathway. SA-EVs and SE-EVs had more surface-associated DNA than EC-EVs and PA-EVs, and SA-EVs and SE-EVs contributed to initiation and amplification of TG in a DNA-dependent manner. However, DNA on EC- or PA-EVs played no role, suggesting that neutrophils release procoagulant EVs which can activate the coagulation cascade through both DNA-dependent and independent mechanisms.

Pseudomonas aeruginosa Activates Quorum Sensing, Antioxidant Enzymes and Type VI Secretion in Response to Oxidative Stress to Initiate Biofilm Formation and Wound Chronicity.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen frequently isolated from cutaneous chronic wounds. How PA, in the presence of oxidative stress (OS), colonizes chronic wounds and forms a biofilm is still unknown. The purpose of this study is to investigate the changes in gene expression seen when PA is challenged with the high levels of OS present in chronic wounds. We used a biofilm-forming PA strain isolated from the chronic wounds of our murine model (RPA) and performed a qPCR to obtain gene expression patterns as RPA developed a biofilm in vitro in the presence of high levels of OS, and then compared the findings in vivo, in our mouse model of chronic wounds. We found that the planktonic bacteria under OS conditions overexpressed quorum sensing genes that are important for the bacteria to communicate with each other, antioxidant stress genes important to reduce OS in the microenvironment for survival, biofilm formation genes and virulence genes. Additionally, we performed RNAseq in vivo and identified the activation of novel genes/pathways of the Type VI Secretion System (T6SS) involved in RPA pathogenicity. In conclusion, RPA appears to survive the high OS microenvironment in chronic wounds and colonizes these wounds by turning on virulence, biofilm-forming and survival genes. These findings reveal pathways that may be promising targets for new therapies aimed at disrupting PA-containing biofilms immediately after debridement to facilitate the treatment of chronic human wounds.

Caspase-11 drives macrophage hyperinflammation in models of Polg-related mitochondrial disease.

Mitochondrial diseases (MtD) represent a significant public health challenge due to their heterogenous clinical presentation, often severe and progressive symptoms, and the lack of effective therapies. Environmental exposures, such bacterial and viral infection, can further compromise mitochondrial function and exacerbate the progression of MtD. Infections in MtD patients more frequently progress to sepsis, pneumonia, and other detrimental inflammatory endpoints. However, the underlying immune alterations that enhance immunopathology in MtD remain unclear, constituting a key gap in knowledge that complicates treatment and increases mortality in this population. Here we employ in vitro and in vivo approaches to clarify the molecular and cellular basis for innate immune hyperactivity in models of polymerase gamma (Polg)-related MtD. We reveal that type I interferon (IFN-I)-mediated upregulation of caspase-11 and guanylate-binding proteins (GBPs) increase macrophage sensing of the opportunistic microbe Pseudomonas aeruginosa (PA) in Polg mutant mice. Furthermore, we show that excessive macrophage cytokine secretion and pyroptotic cell death contribute to lung inflammation and morbidity after infection with PA. Our work sheds new light on innate immune dysregulation in MtD and reveals potential targets for limiting infection- and inflammation-related complications in Polg-related MtD.

#3055 Replacement of tunneled cuffed hemodialysis catheters on coated guidewire: a strategic approach to treat CVC-related infections

Abstract Background and Aims Infections of tunneled hemodialysis central venous catheters (CVCs) are a serious problem, and the outcome of dialysis patients strongly depends on their clinical management. Several specialists are involved in choosing the appropriate technical and clinical strategy but often there is no consensus among them on the proper management. We believe that the role of nephrologists must be pivotal in this setting throughout the entire process, from the choice of vascular access to the management of complications. Here, we present our experience using an approach to replace CVCs that was conceived to minimize the rate of infections. Method We propose a CVC replacement technique to minimize the risk of contamination by using a guidewire coating that allow no contact between the old and new CVC. We carried out a retrospective observational study of tunneled CVCs replacement with this technique performed at the Nephrology Unit of the "A. Gemelli Polyclinic" between January 2020 and June 2023. The following parameters were analyzed: age, sex, infection-supporting pathogen, CVC age, and dialysis age. Results During the reporting period, we carried out a total of 87 CVC guidewire replacement procedures for HD tunneled catheters defined “access to be preserved” using our technique. Among the patients (median age 70,6) undergoing this procedure, n=5 (5.75%) presented with endocarditis (n=4 by Staphylococcus aureus, n=1 by Pseudomonas aeruginosa). The follow-up ranged from 6 months to 3 years. During this time only one patient, with endocarditis, had a recurrent infection caused by Pseudomonas aeruginosa after 3 months from the CVC replacement whereas 12 patients presented a de novo CVC infection caused by a different pathogen (n=4 by Staphylococcus aureus, n=1 by Pseudomonas Aeruginosa. Conclusion Overall, our data are encouraging as the lower rate of recurrent infections in our cohort suggests that our CVC replacement technique can be potentially used even in case of dangerous pathogens.

Advances in Bioreceptor Layer Engineering in Nanomaterial-based Sensing of Pseudomonas Aeruginosa and its Metabolites.

Pseudomonas aeruginosa is a pathogen that infects wounds and burns and causes severe infections in immunocompromised humans. The high virulence, the rise of antibiotic-resistant strains, and the easy transmissibility of P.aeruginosa necessitate its fast detection and control. The gold standard for detecting P.aeruginosa, the plate culture method, though reliable, takes several days to complete. Therefore, developing accurate, rapid, and easy-to-use diagnostic tools for P.aeruginosa is highly desirable. Nanomaterial-based biosensors are at the forefront of detecting P.aeruginosa and its secondary metabolites. This review summarises the biorecognition elements, biomarkers, immobilisation strategies, and current state-of-the-art biosensors for P.aeruginosa. The review highlights the underlying principles of bioreceptor layer engineering and the design of optical, electrochemical, mass-based, and thermal biosensors based on nanomaterials. The advantages and disadvantages of these biosensors and their future point-of-care applications are also discussed. This review outlines significant advancements in biosensors and sensors for detecting P.aeruginosa and its metabolites. Research efforts have identified biorecognition elements specific and selective towards P.aeruginosa. The stability, ease of preparation, cost-effectiveness, and integration of these biorecognition elements onto transducers are pivotal for their application in biosensors and sensors. At the same time, when developing sensors for clinically significant analytes such as P.aeruginosa, virulence factors need to be addressed, such as the sensor's sensitivity, reliability, and response time in samples obtained from patients. The point-of-care applicability of the developed sensor may be an added advantage since it enables onsite determination. In this context, optical methods developed for P.aeruginosa offer promising potential.

Evaluation of Antimicrobial and Antifungal Effects of Ag and Mg Dual-Doped Zinc Oxide Nanoparticles against Staphylococcus Epidermidis, Pseudomonas Aeruginosa, and Candida Albicans

Background: Microbial infectious diseases are one of the serious health problems that have attracted the attention of the public as a threat to human health all over the world. Zinc oxide nanoparticles due to their various properties such as antibacterial, antifungal, and anticancer are of great interest. Methods: The antimicrobial activity of synthesized un-doped and silver and magnesium-doped zinc oxide nanoparticles with Salvadora persica extract against different strains of Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans was investigated through two methods of broth diffusion and the minimum microbial inhibitory concentration (MIC). Results: According to results, MIC of un-doped and silver and magnesium-doped zinc oxide nanoparticles showed 160, 80, 40, and 40 µg/mL on Staphylococcus epidermidis, and 80, 40, 40, and 40 µg/mL on Staphylococcus aureus. These values on Pseudomonas aeruginosa were obtained ˃5120, 640, 320, and 320 µg/mL. Conclusion: The results of the broth dilution method on Staphylococcus epidermidis and Staphylococcus aureus showed the presence of silver has a very good inhibition effect on these bacteria. In Pseudomonas aeruginosa and Candida albicans fungus, it has been observed that by increasing the amount of silver and magnesium doping, the inhibitory rate of nanoparticles on Gram-negative bacteria and fungi has increased.

Antibiotic-Induced Changes in Efflux Transporter Expression: A Key Factor in Pseudomonas aeruginosa Biofilm Resistance

Listed by WHO as an antibiotic-resistant priority pathogen, Pseudomonas aeruginosa (P.A.) is a serious threat in nosocomial infections. Its high antibiotic resistance is attributed to major mechanisms that can be categorized into intrinsic, acquired, and adaptive resistance. This study tests the ability of three commonly used antibiotics to inhibit new biofilm formation and eradicate mature biofilm growth, as well as investigate changes in the expression levels of selected genes coding for multidrug efflux pumps in P.A. planktonic cells and biofilms before and after treatment with antibiotics to provide a conceptual estimate of the activity of the efflux transporters that work to extrude antibiotics leading to a reduction in their effectiveness. Antimicrobial susceptibility testing was conducted with Ofloxacin (OFLX), Tobramycin (TOB), and Ceftazidime (CAZ) to determine Mean Inhibitory Concentration (MIC) and Mean Bactericidal Concentration (MBC) using microtiter plate-based biofilm assay and spectrophotometric quantification. Extraction of total RNA was performed from planktonic cultures, inhibition phase, and eradication phase P.A. biofilms. Real-time quantitative reverse transcriptase PCR was utilized to analyze the changes in expression of the mexAB, mexXY, and oprM genes. Three (3) antibiotics that have proven to show less resistance are OFLX, TOB, and CAZ when tested against overnight cultures of P.A. strain PA01. Results showed that OFLX is best for bactericidal properties, which is also supported by the viability assay data obtained from Propidium Iodide staining. Our study showed that the PAO1 strain is susceptible to OFLX for both inhibition and eradication of mature biofilms. TOB was most effective at higher concentrations in the eradication phase.

Exendin-4 blockade of T1R2/T1R3 activation improves Pseudomonas aeruginosa-related pneumonia in an animal model of chemically induced diabetes

ObjectivePoorly controlled diabetes frequently exacerbates lung infection, thereby complicating treatment strategies. Recent studies have shown that exendin-4 exhibits not only hypoglycemic but also anti-inflammatory properties. This study aimed to explore the role of exendin-4 in lung infection with diabetes, as well as its association with NOD1/NF-κB and the T1R2/T1R3 sweet taste receptor.Methods16HBE human bronchial epithelial cells cultured with 20 mM glucose were stimulated with lipopolysaccharide (LPS) isolated from Pseudomonas aeruginosa (PA). Furthermore, Sprague‒Dawley rats were fed a high-fat diet, followed by intraperitoneal injection of streptozotocin and intratracheal instillation of PA. The levels of TNF-α, IL-1β and IL-6 were evaluated using ELISAs and RT‒qPCR. The expression of T1R2, T1R3, NOD1 and NF-κB p65 was assayed using western blotting and immunofluorescence staining. Pathological changes in the lungs of the rats were observed using hematoxylin and eosin (H&E) staining.ResultsAt the same dose of LPS, the 20 mM glucose group produced more proinflammatory cytokines (TNF-α, IL-1β and IL-6) and had higher levels of T1R2, T1R3, NOD1 and NF-κB p65 than the normal control group (with 5.6 mM glucose). However, preintervention with exendin-4 significantly reduced the levels of the aforementioned proinflammatory cytokines and signaling molecules. Similarly, diabetic rats infected with PA exhibited increased levels of proinflammatory cytokines in their lungs and increased expression of T1R2, T1R3, NOD1 and NF-κB p65, and these effects were reversed by exendin-4.ConclusionsDiabetic hyperglycemia can exacerbate inflammation during lung infection, promote the increase in NOD1/NF-κB, and promote T1R2/T1R3. Exendin-4 can ameliorate PA-related pneumonia with diabetes and overexpression of NOD1/NF-κB. Additionally, exendin-4 suppresses T1R2/T1R3, potentially through its hypoglycemic effect or through a direct mechanism. The correlation between heightened expression of T1R2/T1R3 and an intensified inflammatory response in lung infection with diabetes requires further investigation.

High-Intensity Blue Light (450-460 nm) Phototherapy for Pseudomonas aeruginosa-Infected Wounds.

Background: Nosocomial wound infection with Pseudomonas aeruginosa (PA) is a serious complication often responsible for septic mortality of burn patients. High-intensity antimicrobial blue light (aBL) treatment may represent an alternative therapy for PA infections. Methods: Antibacterial effects of an light-emitting diode (LED) array (450-460 nm; 300 mW/cm2; 15/30 min; 270/540J/cm2) against PA were determined by suspension assay, biofilm assay, and a human skin wound model and compared with 15-min topically applied 3% citric acid (CA) and wound irrigation solution (Prontosan®; PRT). Results: The aBL reduced the bacterial number (2.51-3.56 log10 CFU/mL), whereas PRT or CA treatment achieved a 4.64 or 6.60 log10 CFU/mL reduction in suspension assays. The aBL reduced biofilm formation by 60%-66%. PRT or CA treatment showed reductions by 25% or 13%. In this study, aBL reduced bacterial number in biofilms (1.30-1.64 log10 CFU), but to a lower extent than PRT (2.41 log10 CFU) or CA (2.48 log10 CFU). In the wound skin model, aBL (2.21-2.33 log10 CFU) showed a bacterial reduction of the same magnitude as PRT (2.26 log10 CFU) and CA (2.30 log10 CFU). Conclusions: The aBL showed a significant antibacterial efficacy against PA and biofilm formation in a short time. However, a clinical application of aBL in wound therapy requires effective active skin cooling and eye protection, which in turn may limit clinical implementation.

Design, Synthesis, and Biological Evaluation of the Quorum-Sensing Inhibitors of Pseudomonas aeruginosa PAO1.

Due to the resistance of Gram-negative bacteria Pseudomonas aeruginosa PAO1 to most clinically relevant antimicrobials, the use of traditional antibiotic treatments in hospitals is challenging. The formation of biofilms, which is regulated by the quorum-sensing (QS) system of Pseudomonas aeruginosa (PA), is an important cause of drug resistance. There are three main QS systems in P. aeruginosa: the las system, the rhl system, and the pqs system. The inhibitors of the las system are the most studied. Previously, the compound AOZ-1 was found to have a certain inhibitory effect on the las system when screened. In this study, twenty-four compounds were designed and synthesized by modifying the Linker and Rings of AOZ-1. Using C. violaceum CV026 as a reporter strain, this study first assessed the inhibitory effects of new compounds against QS, and their SAR was investigated. Then, based on the SAR analysis of compound AOZ-1 derivatives, the parent core of AOZ-1 was replaced to explore the structural diversity. Then, nine new compounds were designed and synthesized with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one. The compound Y-31 (IC50 = 91.55 ± 3.35 µM) was found to inhibit the QS of C. violaceum CV026. Its inhibitory effect on C. violaceum CV026 was better than that of compound AOZ-1 (IC50 > 200 µM). Furthermore, biofilm formation is one of the important causes of Pseudomonas aeruginosa PAO1 resistance. In this study, it was found that compound Y-31, with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one, had the highest biofilm inhibition rate (40.44%). The compound Y-31 has a certain inhibitory effect on the production of PAO1 virulence factors (pyocyanin, rhamnolipid, and elastase) and swarming. When the concentration of compound Y-31 was 162.5 µM, the inhibition rates of pyocyanin, rhamnolipid, and elastase were 22.48%, 6.13%, and 22.67%, respectively. In vivo, the lifetime of wildtype Caenorhabditis elegans N2 infected with P. aeruginosa PAO1 was markedly extended by the new parent nucleus Y-31. This study also performed cytotoxicity experiments and in vivo pharmacokinetics experiments on the compound Y-31. In conclusion, this study identified a compound, Y-31, with a new nucleus core component of 3-amino-tetrahydro-l,3-oxazin-2-one, which is a potential agent for treating P. aeruginosa PAO1 that is resistant to antibiotics and offers a way to discover novel antibacterial medications.

Dry powder inhalation containing muco-inert ciprofloxacin and colistin co-loaded liposomes for pulmonary P. Aeruginosa biofilm eradication

Pseudomonas aeruginosa (PA), a predominant pathogen in lung infections, poses significant challenges due to its biofilm formation, which is the primary cause of chronic and recalcitrant pulmonary infections. Bacteria within these biofilms exhibit heightened resistance to antibiotics compared to their planktonic counterparts, and their secreted toxins exacerbate lung infections. Diverging from traditional antibacterial therapy for biofilm eradication, this study introduces a novel dry powder inhalation containing muco-inert ciprofloxacin and colistin co-encapsulated liposomes (Cipro-Col-Lips) prepared using ultrasonic spray freeze drying (USFD) technique. This USFD dry powder is designed to efficiently deliver muco-inert Cipro-Col-Lips to the lungs. Once deposited, the liposomes rapidly diffuse into the airway mucus, reaching the biofilm sites. The muco-inert Cipro-Col-Lips neutralize the biofilm-secreted toxins and simultaneously trigger the release of their therapeutic payload, exerting a synergistic antibiofilm effect. Our results demonstrated that the optimal USFD liposomal dry powder formulation exhibited satisfactory in vitro aerosol performance in terms of fine particle fraction (FPF) of 44.44 ± 0.78 %, mass median aerodynamic diameter (MMAD) of 4.27 ± 0.21 μm, and emitted dose (ED) of 99.31 ± 3.31 %. The muco-inert Cipro-Col-Lips effectively penetrate the airway mucus and accumulate at the biofilm site, neutralizing toxins and safeguarding lung cells. The triggered release of ciprofloxacin and colistin works synergistically to reduce the biofilm’s antibiotic resistance, impede the development of antibiotic resistance, and eliminate 99.99 % of biofilm-embedded bacteria, including persister bacteria. Using a PA-beads induced biofilm-associated lung infection mouse model, the in vivo efficacy of this liposomal dry powder aerosol was tested, and the results demonstrated that this liposomal dry powder aerosol achieved a 99.7 % reduction in bacterial colonization, and significantly mitigated inflammation and pulmonary fibrosis. The USFD dry powder inhalation containing muco-inert Cipro-Col-Lips emerges as a promising therapeutic strategy for treating PA biofilm-associated lung infections.

Residual risk of Pseudomonas aeruginosa waterborne contamination in an intensive care unit despite the presence of filters at all water points-of-use

To assess the residual risk of waterborne contamination by Pseudomonas aeruginosa from a water network colonized by a single genotype [sequence type (ST) 299] despite the presence of antimicrobial filters in a medical intensive care unit (ICU). During the first 19-month period since the ICU opened, contamination of the water network was assessed monthly by collecting water upstream of the filters. Downstream water was also sampled to assess the efficiency of the filters. P.aeruginosa isolates from patients were collected and compared with the waterborne ST299 P.aeruginosa by multiplex-rep polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. Cross-transmission events by other genotypes of P.aeruginosa were also assessed. Overall, 1.3% of 449 samples of filtered water were positive for P.aeruginosa in inoculum, varying between 1 and 104colony-forming units/100mL according to the tap. All P.aeruginosa hydric isolates belonged to ST299 and displayed fewer than two single nucleotide polymorphisms (SNPs). Among 278 clinical isolates from 122 patients, 10 isolates in five patients showed identical profiles to the hydric ST299 clone on both multiplex-rep PCR and PFGE, and differed by an average of fewer than five SNPs, confirming the water network reservoir as the source of contamination by P.aeruginosa for 4.09% of patients. Cross-transmission events by other genotypes of P.aeruginosa were responsible for the contamination of 1.75% of patients. Antimicrobial filters are not sufficient to protect patients from waterborne pathogens when the water network is highly contaminated. A microbiological survey of filtered water may be needed in units hosting patients at risk of P.aeruginosa infections, even when all water points-of-use are fitted with filters.

Prevalence and Antibiotic Susceptibility Pattern of Pseudomonas Aeruginosa from the Foot Ulcer of Diabetic Patients in Hayatabad Medical Complex Peshawar, Pakistan

Background: Diabetic foot ulcers (DFUs) are a major complication of diabetes mellitus, affecting 15% of diabetic patients and often leading to severe outcomes like amputation. These ulcers frequently become infected with bacteria such as Pseudomonas aeruginosa, which is known for its robust antibiotic resistance. Objective: To investigate the prevalence and antibiotic susceptibility patterns of Pseudomonas aeruginosa isolated from the foot ulcers of diabetic patients in Hayatabad Medical Complex, Peshawar. Methods: A total of 103 clinical samples from diabetic foot ulcers were collected and analyzed for the presence of Pseudomonas aeruginosa using culture techniques and biochemical tests. The antibiotic susceptibility was assessed using the Kirby-Bauer disk diffusion method to identify effective treatments. Results: Pseudomonas aeruginosa was identified in 48 (46.60%) of the samples. High resistance was noted against amoxicillin-clavulanic acid (100%), and notable sensitivity was observed to gentamicin (79.16%) and cefoperazone-sulbactam (87.5%). The susceptibility to other tested antibiotics varied, with moderate resistance seen in agents like cefotaxime (25%) and trimethoprim-sulfamethoxazole (33.33%). Conclusion: The study highlights a significant presence of Pseudomonas aeruginosa in diabetic foot ulcers with substantial antibiotic resistance, emphasizing the need for precise susceptibility testing to guide effective treatment strategies.

High-Intensity Blue Light (450-460 nm) Phototherapy for Pseudomonas aeruginosa-Infected Wounds.

Background: Nosocomial wound infection with Pseudomonas aeruginosa (PA) is a serious complication often responsible for the septic mortality of burn patients. Objective: High-intensity antimicrobial blue light (aBL) treatment may represent an alternative therapy for PA infections and will be investigated in this study. Methods: Antibacterial effects of a light-emitting diode array (450-460 nm; 300 mW/cm2; 15/30 min; 270/540 J/cm2) against PA were determined by suspension assay, biofilm assay, and a human skin wound model and compared with 15-min topically applied 3% citric acid (CA) and wound irrigation solution (Prontosan®; PRT). Results: aBL reduced the bacterial number [2.51-3.56 log10 colony-forming unit (CFU)/mL], whereas PRT or CA treatment achieved a 4.64 or 6.60 log10 CFU/mL reduction in suspension assays. aBL reduced biofilm formation by 60-66%. PRT or CA treatment showed reductions by 25% or 13%. Here, aBL reduced bacterial number in biofilms (1.30-1.64 log10 CFU), but to a lower extend than PRT (2.41 log10 CFU) or CA (2.48 log10 CFU). In the wound skin model, aBL (2.21-2.33 log10 CFU) showed a bacterial reduction of the same magnitude as PRT (2.26 log10 CFU) and CA (2.30 log10 CFU). Conclusions: aBL showed a significant antibacterial efficacy against PA and biofilm formation in a short time. However, a clinical application of aBL in wound therapy requires effective active skin cooling and eye protection, which in turn may limit clinical implementation.

Highly Motile Neutrophil Cytoplast ameliorate Bacterial Pneumonia

Abstract Polymorphonuclear neutrophils (PMN) function as innate immune cell through rapid mobilization at site of infection and prompt phagocytosis and bactericidal activity. We addressed in vivo in mouse lungs the function of cytoplasts (PMNcyt), the PMN generated following release of extracellular traps (NETs). We found ~50% of PMN lost their nucleus post-NETosis following pneumonia induced by Pseudomonas aeruginosa (PA), and become PMNcyt. Interestingly, , PMNcyt in lung microvessels showed >2 greater velocity and > 3 greater deformability as compared to PMN. PMNcyt were also 9x more efficient in killing airway PA as compared to PMN. Using the protein arginine deiminase 4 (PAD4) inhibitor (Cl-amidine) to prevent NET formation in mice, we prevented the generation of PMNcyt demonstrating that PMNcyt were derived post-vital NETosis. We also showed PMNcyt retained the ability to phagocytose PA. Moreover, the inhibition of PMNcyt generation by PAD4 blocker induced severe lung injury in the pneumonia model, indicating an important anti-bacterial protective role of PMNcyt. Adoptive transfer of PMNcyt in PA infected mice also significantly reduced bacterial load and prevented lung tissue injury in mice. The results suggest a crucial host-defense function of PMNcyt generated post vital NETosis and their usefulness as cell-based therapy in inflammatory diseases such as pneumonia.

Essential Roles of Mitochondrial Reactive Oxygen Species Signaling in Airway Smooth Muscle from Mice following Nicotine, Pyoverdine, and their Co-exposure Authors: Subika Khatoon, Ed Wilson Santos, Yun-Min Zheng, Yong-Xiao Wang Corresponding Author: Yong-Xiao Wang, wangy@amc.edu

Consumption of nicotine, a major active component of cigarettes or e-cigarette smoke, is a global health problem, causing a myriad of life-threatening diseases. Pseudomonas aeruginosa is a common cause of lung infections and often exacerbate nicotine-associated lung diseases. However, the underlying cellular and molecular mechanisms remain poorly understood. In this study, we aim to address these important questions by performing a series of experiments. establish a clinical threshold of Pseudomonas Aeruginosa Infection in a COPD animal model and determine the underlying molecular mechanisms underlying the mitochondrial dysfunctions in Airway Smooth Muscle Cells (ASMCs). Utilizing in vitro and in vivo models, our data revealed that mice following inhalation of nicotine showed increased lung resistance, decreased lung compliance, and elevated airway resistance in vivo. In support, nicotine caused airway hyperresponsiveness in isolated mouse tracheal and bronchial smooth muscles. We also found that reactive oxygen species (ROS) production was largely increased in isolated mitochondria from mice after nicotine inhalation. Application of pyoverdine, a primary active component of Pseudomonas aeruginosa, produced a similar effect on ROS production in isolated mouse airway smooth muscle (ASM) mitochondria. Furthermore, mice co-exposed to nicotine inhalation and pyoverdine had a higher increase in ROS production in isolated ASM mitochondria compared with mice following nicotine or pyoverdine exposure alone. In conclusion, our findings for the first time demonstrate mitochondrial ROS signaling plays essential roles in airway dysfunctions following nicotine, pyoverdine, and their co-exposure and may also offer new strategies to treat lung diseases induced by nicotine inhalation and/or Pseudomonas aeruginosa. Funding: This work was supported by the NIH (R01HL122865, R03AG070784, and R01HL108232, to Yong-Xiao Wang). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

ObjectiveRecombinase-aided polymerase chain reaction (RAP) is a sensitive, single-tube, two-stage nucleic acid amplification method. This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Acinetobacter baumannii (AB) in the bloodstream based on recombinant human mannan-binding lectin protein (M1 protein)-conjugated magnetic bead (M1 bead) enrichment of pathogens combined with RAP. MethodsRecombinant plasmids were used to evaluate the assay sensitivity. Common blood influenza bacteria were used for the specific detection. Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR (M-RAP) and quantitative PCR (qPCR) assays. Kappa analysis was used to evaluate the consistency between the two assays. ResultsThe M-RAP method had sensitivity rates of 1, 10, and 1 copies/μL for the detection of SA, PA, and AB plasmids, respectively, without cross-reaction to other bacterial species. The M-RAP assay obtained results for < 10 CFU/mL pathogens in the blood within 4 h, with higher sensitivity than qPCR. M-RAP and qPCR for SA, PA, and AB yielded Kappa values of 0.839, 0.815, and 0.856, respectively (P < 0.05). ConclusionAn M-RAP assay for SA, PA, and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

Radiographic Outcomes in Pediatric Bronchiectasis and Factors Associated with Reversibility.

Rationale: Conventionally considered irreversible, bronchiectasis has been demonstrated to be reversible in children in small studies. However, the factors associated with radiographic reversibility of bronchiectasis have yet to be defined. Objectives: In a large cohort of children with bronchiectasis, we aimed to determine: 1) if and to what extent bronchiectasis is reversible and 2) factors associated with radiographic chest high-resolution computed tomography (cHRCT) resolution. Methods: We identified children with bronchiectasis who had a repeat multidetector cHRCT scan between 2010 and 2021. We excluded those with cystic fibrosis, surgical pulmonary resection, traction bronchiectasis only, or lobar opacification. Measurements and Main Results: cHRCT scans were scored using the modified Reiff score (MRS) with a pediatric correction. Resolution was defined as an absence of abnormal bronchoarterial ratio (>0.8) on the second cHRCT scan. We included 142 children (median age, 5 years; IQR, 2.6-7.4). Inter- and intrarater agreement in MRSs was excellent (weighted κ = 0.83-0.86 and 0.95, respectively). There was radiographic resolution in 57 of 142 patients (40.1%), improvement in 56 of 142 (39.4%), and no change or worsening in 29 of 142 (20.4%). Pseudomonas aeruginosa (PsA) was absolutely associated with a lack of resolution. On multivariable regression, in those without PsA cultured, younger age at the time of diagnosis (risk ratio [RR], 0.94; 95% confidence interval [CI], 0.88-0.99), lower MRS (RR, 0.89; 95% CI, 0.82-0.97), and lower annual rate of exacerbations requiring intravenous antibiotic therapy (RR, 0.60; 95% CI, 0.37-0.98) increased the likelihood of radiographic resolution. Conclusions: This first large cohort confirms that bronchiectasis in children is often reversible with appropriate management. Younger children and those with lesser radiographic severity at diagnosis were most likely to exhibit radiographic reversibility, whereas those with PsA infection were least likely.

Impact of Pseudomonas aeruginosa on resource utilization and costs in patients with exacerbated non-cystic fibrosis bronchiectasis

Aims Non‐cystic fibrosis bronchiectasis (NCFB) is a chronic progressive respiratory disorder occurring at a rate ranging from 4.2 to 278.1 cases per 100,000 persons, depending on age, in the United States. For many patients with NCFB, the presence of Pseudomonas aeruginosa (PA) makes treatment more complicated and typically has worse outcomes. Management of NCFB can be challenging, warranting a better understanding of the burden of illness for NCFB, treatments applied, healthcare resources used, and subsequent treatment costs. Comparing patients diagnosed with exacerbated NCFB, with or without PA on antibiotic utilization, treatments, and healthcare resources utilization and costs was the purpose of this study. Materials and methods This was a retrospective cohort study of commercial claims from IQVIA’s PharMetrics Plus database (January 1,2006–December 31, 2020). Study patients with a diagnosis of NCFB were stratified into two groups based on the presence or absence of PA, then followed to identify demographic characteristics, comorbid conditions, antibiotic treatment regimen prescribed, healthcare resources utilized, and costs of care. Results The results showed that patients with exacerbated NCFB who were PA+ had significantly more oral antibiotic fills per patient per year, more inpatient admissions with a longer length of stay, and more outpatient encounters than those who were PA−. For costs, PA+ patients also had significantly greater total healthcare costs per patient when compared to those who were PA−. Conclusion Exacerbated NCFB with PA+ was associated with increased antibiotic usage, greater resource utilization, and increased costs. The major contributor to the cost differences was the use of inpatient services. Treatment strategies aimed at reducing the need for inpatient treatment could lessen the disparities observed in patients with NCFB.