Ivacaftor ameliorates mucus burden, bacterial load, and inflammation in acute but not chronic P. aeruginosa infection in hG551D rats.

BACKGROUND: Newly approved highly effective modulation therapies (HEMT) have been life-changing for people with CF. Although these drugs have resulted in significant improvements in lung function and exacerbation rate, bacterial populations in the lung have not been eradicated. The mechanisms behind the continued colonization are not completely clear. METHODS: We used a humanized rat to assess the effects of ivacaftor therapy on infection outcomes. Rats harbor an insert expressing humanized CFTR cDNA, including the G551D mutation. hG551D rats were treated with ivacaftor either during or before infection with P. aeruginosa. The response to infection was assessed by bacterial burden in the lung and mucus burden in the lung. RESULTS: We found that hG551D rats treated with ivacaftor had reduced bacteria present in the lung in the acute phase of the infection but were not different than vehicle control in the chronic phase of the infection. Similarly, the percentage of neutrophils in the airways were reduced at the acute, but not chronic, timepoints. Overall weight data indicated that the hG551D rats had significantly better weight recovery during the course of infection when treated with ivacaftor. Potentiation of the G551D mutation with ivacaftor resultant in short-circuit current measurements equal to WT, even during the chronic phase of the infection. Despite the persistent infection, hG551D rats treated with ivacaftor had fewer airways with mucus plugs during the chronic infection. CONCLUSIONS: The data indicate that the hG551D rats have better outcomes during infection when treated with ivacaftor compared to the vehicle group. Rats have increased weight gain, increased CFTR protein function, and decreased mucus accumulation, despite the persistence of infection and inflammation. These data suggest that ivacaftor improves tolerance of infection, rather than eradication, in this rat model.

H2S scavenger as a broad-spectrum strategy to deplete bacteria-derived H2S for antibacterial sensitization.

Bacteria-derived H2S plays multifunctional protective roles against antibiotics insult, and the H2S biogenesis pathway is emerging as a viable target for the antibacterial adjuvant design. However, the development of a pan-inhibitor against H2S-synthesizing enzymes is challenging and underdeveloped. Herein, we propose an alternative strategy to downregulate the H2S levels in H2S-producing bacteria, which depletes the bacteria-derived H2S chemically by H2S scavengers without acting on the synthesizing enzymes. After the screening of chemically diversified scaffolds and a structural optimization campaign, a potent and specific H2S scavenger is successfully identified, which displays efficient H2S depletion in several H2S-producing bacteria, potentiates both bactericidal agents and photodynamic therapy, enhances the bacterial clearance of macrophages and polymorphonuclear neutrophils, disrupts the formation of bacterial biofilm and increases the sensitivity of bacterial persister cells to antibiotics. Most importantly, such an H2S scavenger exhibits sensitizing effects with gentamicin in Pseudomonas aeruginosa -infected pneumonia and skin wound female mouse models. In aggregate, our results not only provide an effective strategy to deplete bacteria-derived H2S and establish the H2S biogenesis pathway as a viable target for persisters and drug-resistant bacteria, but also deliver a promising antibacterial adjuvant for potential clinical translation.

Novel management of pseudomonas biofilm-like structure in a post-pneumonectomy empyema.

We present a patient with a post-pneumonectomy empyema refractory to surgical debridement and systemic antibiotics. The patient initially presented with a bronchopleural fistula and pneumothorax secondary to tuberculosis (TB) destroyed lung, which required a pneumonectomy with Eloesser flap. Ongoing pleural infection delayed the closure of the Eloesser flap, and thoracoscopic inspection of his chest cavity revealed a green, mucous biofilm-like structure lining the postpneumonectomy pleural cavity. Cultures identified pan-susceptible Pseudomonas aeruginosa. Despite debriding this biofilm-like structure and administering systemic antibiotics, the patient continued to show persistent signs of infection and regrowth of the film. We employed a novel approach to dissolve the biofilm-like structure using intrapleural dornase alfa followed by intrapleural antibiotic washes. After 3 weeks of daily washes, repeat inspection demonstrated the biofilm-like structure had completely resolved. Resolving the pseudomonas biofilm-like structure allowed permanent closure of his chest without further need for systemic antibiotics. At follow up 3 months later, he showed no sequalae. This treatment option can be an important adjunct to improve likelihood of chest closure in patients with post-pneumonectomy empyema that resists standard treatment options due to biofilm formation.

Antibiotic susceptibility of Pseudomonas aeruginosa in Saudi Arabia: a national antimicrobial resistance surveillance study.

Background: Pseudomonas aeruginosa is a common pathogen causing healthcare-associated infections. Most surveillance studies from Saudi Arabia that assessed the resistance by P. aeruginosa were conducted in single centers or did not use broth microdilution (BMD), the gold standard test. This is the first national multicenter study to assess the resistance profiles of P. aeruginosa isolates in Saudi Arabia using BMD. Methods: Between 2022 and 2023, isolates from various infection sites were collected from seven hospitals in seven different regions of Saudi Arabia. The isolates were shipped to an academic microbiology lab, where their susceptibility was tested by BMD following Clinical Laboratory Standards Institute guidelines using Sensititre GNX3F plates. %Susceptibility to each antibiotic, and MIC50 and MIC90 were determined. Results: In total, 185 P. aeruginosa isolates were collected. Most isolates came from respiratory specimens (34.1%), followed by urine (21.1%) and skin/soft tissue (17.8%). The highest susceptibility was to amikacin (76.8%). Concurrently, susceptibility to meropenem was 52%, but it was 43.8% to colistin. While all P. aeruginosa isolates met the definition of multidrug-resistance, 41 (22.2%) were difficult-to-treat and 10 (5.4%) were pandrug-resistant. Difficult-to-treat isolates made up 30.3% of skin and soft tissue isolates, 25.4% of respiratory isolates, 21.7% of blood isolates, and 17.9% of urine isolates. Conclusion: Pseudomonas aeruginosa demonstrated an unexpectedly high level of resistance to several commonly used antibiotics. Therefore, antimicrobial stewardship and infection control policies should be strictly enforced by hospitals across the country to optimize treatment, prevent the emergence and spread of resistant strains, and track resistance trends with local antibiograms.

Galleria mellonella as an Antimicrobial Screening Model.

To combat the rising global issue of antibiotic resistance, the accelerated development of novel antibiotics is essential. Current preclinical antimicrobial development yields a significant number of leads that prove unsuitable either prior to or during clinical trials. To increase the efficiency of preclinical development, relevant, standardized, accessible, and cost-effective models must be developed. Galleria mellonella (greater wax moth) larvae are widely used as an infection model to assess microbial virulence, conduct drug toxicity testing, and serve as a preliminary means of evaluating the in vivo efficacy of novel antimicrobial compounds. These infection models have greater biological relevance than many in vitro screens of comparable throughput and decrease reliance on mammalian models when used as a pre-screen for antimicrobial testing. This protocol describes a standardized methodology for the optimization of G. mellonella infection models, which can be applied to bacterial species and antimicrobial therapeutics of choice. Using the WHO priority pathogen Pseudomonas aeruginosa as an exemplar, we outline steps that can be undertaken to develop a reproducible model of infection and therapeutic testing. This includes recommendations on experimental setup, sample preparation, and infection and treatment protocols. Integration of this model within preclinical antimicrobial development pipelines would decrease reliance on mammalian models, reduce the number of ineffective compounds reaching clinical trials, and ultimately increase the efficiency of preclinical antimicrobial development.

QTL Mapping-Based Identification of Visceral White-Nodules Disease Resistance Genes in Larimichthys polyactis.

Disease outbreaks in aquaculture have recently intensified. In particular, visceral white-nodules disease, caused by Pseudomonas plecoglossicida, has severely hindered the small yellow croaker (Larimichthys polyactis) aquaculture industry. However, research on this disease is limited. To address this gap, the present study employed a 100K SNP chip to genotype individuals from an F1 full-sib family, identify single nucleotide polymorphisms (SNPs), and construct a genetic linkage map for this species. A high-density genetic linkage map spanning a total length of 1395.72 cM with an average interval of 0.08 cM distributed across 24 linkage groups was obtained. Employing post-infection survival time as an indicator of disease resistance, 13 disease resistance-related quantitative trait loci (QTLs) were detected, and these regions included 169 genes. Functional enrichment analyses pinpointed 11 candidate disease resistance-related genes. RT-qPCR analysis revealed that the genes of chmp1a and arg1 are significantly differentially expressed in response to P. plecoglossicida infection in spleen and liver tissues, indicating their pivotal functions in disease resistance. In summary, in addition to successfully constructing a high-density genetic linkage map, this study reports the first QTL mapping for visceral white-nodules disease resistance. These results provide insight into the intricate molecular mechanisms underlying disease resistance in the small yellow croaker.

Resistance patterns, virulence determinants, and biofilm genes of multidrug-resistant Pseudomonas aeruginosa isolated from fish and fish handlers.

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterium that is widely distributed in aquatic environments and causes major economic losses in fish and public health hazards.This study aimed to identify the occurrence of P. aeruginosa in samples collected from fish and fish handlers, and to investigate the antimicrobial susceptibility, virulence determinants, and biofilm genes of P. aeruginosa isolates. A total of 276 samples were cross-sectionally collected from Nile tilapia (53), Golden grey mullet (52), Mediterranean horse mackerel (50), Striped red mullet (71), and fish handlers (50) at five different retail fish markets in Damietta Governorate, Egypt. Pseudomonas species (spp.) were biochemically identified in 57.9% of the total examined samples. Peudomonas aeruginosa were the most prevalent species isolated from the fish and human samples via PCR technique. Peudomonas aeruginosa isolates exhibited full resistance (100%) to tobramycin (TOB), gentamicin (CN), and colistin (CL), with a high level of susceptibility (88.5%) to imipenem (IPM) using the disk diffusion method. Most P. aeruginosa isolates (84.6%) exhibited drug resistance, with 61.5% were multidrug resistance (MDR) and 23.1% were extensive drug resistance (XDR). Most isolates had at least four virulence-associated genes (lasB, toxA, exoU, and oprL) and three biofilm genes (psIA, peIA, and lasR) by using uniplex PCR. The lasI, and rhlR Quorum Sensing (QS) genes were identified in 84.6% and 61.5% in the examined P. aeruginosa isolates, respectively. The highest mortality rate in Nile tilapia experimentally infected with P. aeruginosa isolate encoding most of virulent genes. Multivariate analyses revealed high heterogeneity among the examined isolates. This study revealed the emergence of virulent and drug resistant P. aeruginosa isolates in fish, poses high risks to consumers and food. Thus, strict hygienic measures should be considered when catching, handling, and storing fish, in addition to the routine application of antimicrobial susceptibility testing.

Activity of polymyxin B combined with cefepime-avibactam against the biofilms of polymyxin B-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae in in vitro and in vivo models.

Bacterial biofilms, often forming on medical devices, can lead to treatment failure due to their increased antimicrobial resistance. Cefepime-avibactam (CFP-AVI) exhibits potent activities against Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) when used with polymyxin B (PMB). However, its efficacy in biofilm-related infections is unknown. The present study aimed to evaluate the activity of PMB combined with CFP-AVI against the biofilms of PMB-resistant Gram-negative bacteria. Five K. pneumoniae strains and three P. aeruginosa strains known to be PMB-resistant and prone to biofilm formation were selected and evaluated. Antimicrobial susceptibility assays demonstrated that the minimal biofilm inhibitory and eradication concentrations of PMB and CFP-AVI for biofilms formed by the eight strains were significantly higher than the minimal inhibitory concentrations of the antibiotics for planktonic cells. The biofilm formation inhibition and eradication assays showed that PMB combined with CFP-AVI cannot only suppress the formation of biofilm but also effectively eradicate the preformed mature biofilms. In a modified in vitro pharmacokinetic/pharmacodynamic biofilm model, CFP-AVI monotherapy exhibited a bacteriostatic or effective activity against the biofilms of seven strains, whereas PMB monotherapy did not have any activity at 72 h. However, PMB combined with CFP-AVI demonstrated bactericidal activity against the biofilms of all strains at 72 h. In an in vivo Galleria mellonella infection model, the 7-day survival rates of larvae infected with biofilm implants of K. pneumoniae or P. aeruginosa were 0-6.7%, 40.0-63.3%, and 46.7-90.0%, respectively, for PMB alone, CFP-AVI alone, and PMB combined with CFP-AVI; the combination therapy increased the rate by 6.7-33.3% (P < 0.05, n = 6), compared to CFP-AVI monotherapy. It is concluded that PMB combined with CFP-AVI exhibits effective anti-biofilm activities against PMB-resistant K. pneumoniae and P. aeruginosa both in vitro and in vivo, and thus may be a promising therapeutic strategy to treat biofilm-related infections.

Molecular investigation of LasA, LasB, and PIV genes in clinical isolates of Pseudomonas aeruginosa in Mazandaran Province, North Iran.

AIMS: Pseudomonasaeruginosa plays an important role in hospital infections caused by several virulence factors, such as elastase and proteases. This study aimed to evaluate the prevalence of LasA, LasB, and PIV genes, encoding these enzymes, in clinical isolates of P.aeruginosa.

Isovanillin decreases the virulence regulated by the quorum sensing system of Pseudomonas aeruginosa.

The quorum-sensing (QS) system of Pseudomonas aeruginosa dominates the pathogenicity of the acute or chronic infection process. Hence, curbing the pathogenicity of P. aeruginosa by targeting QS system is an ideal strategy. This study aims to identify potential anti-virulence compounds that can effectively disrupt the QS system of P. aeruginosa using a combination of virtual screening and experimental validation techniques. We explored inhibitory effect of isovanillin obtained by virtual screening on P. aeruginosa QS regulated virulence factors extracellular protease, biofilm, and pyocyanin. Results displayed that isovanillin could inhibit the virulence phenotypes regulated by the las- and pqs-QS systems of P. aeruginosa. The synthesis of extracellular proteases, pyocyanin, and biofilm formation by P. aeruginosa were dramatically inhibited by sub-MICs doses of isovanillin. The results of RNA sequencing and quantitative PCR revealed that the QS-activated genes down-regulated by subinhibitory isovanillin in the transcriptional evels. Furthermore, the presence of isovanillin increased the susceptibility of drug-resistant P. aeruginosa to kanamycin, meropenem, and polymyxin B. Treatment of isovanillin as a monotherapy significantly decreased the mortality of C. elegans in P. aeruginosa PAO1 or UCBPP-PA14 (PA14) infection. Our study reported the anti-virulence activity of isovanillin against P. aeruginosa, and provided a structural foundation for developing anti-virulence drugs targeting the QS system of P. aeruginosa.

Macrolide resistance through uL4 and uL22 ribosomal mutations in Pseudomonas aeruginosa.

Macrolides are widely used antibiotics for the treatment of bacterial airway infections. Due to its elevated minimum inhibitory concentration in standardized culture media, Pseudomonas aeruginosa is considered intrinsically resistant and, therefore, antibiotic susceptibility testing against macrolides is not performed. Nevertheless, due to macrolides' immunomodulatory effect and suppression of virulence factors, they are used for the treatment of persistent P. aeruginosa infections. Here, we demonstrate that macrolides are, instead, effective antibiotics against P. aeruginosa airway infections in an Air-Liquid Interface (ALI) infection model system resembling the human airways. Importantly, macrolide treatment in both people with cystic fibrosis and primary ciliary dyskinesia patients leads to the accumulation of uL4 and uL22 ribosomal protein mutations in P. aeruginosa which causes antibiotic resistance. Consequently, higher concentrations of antibiotics are needed to modulate the macrolide-dependent suppression of virulence. Surprisingly, even in the absence of antibiotics, these mutations also lead to a collateral reduction in growth rate, virulence and pathogenicity in airway ALI infections which are pivotal for the establishment of a persistent infection. Altogether, these results lend further support to the consideration of macrolides as de facto antibiotics against P. aeruginosa and the need for resistance monitoring upon prolonged macrolide treatment.

Tracing the path of Quorum sensing molecules in cystic fibrosis mucus in a biomimetic in vitro permeability platform.

P. aeruginosa employs specific quorum sensing (QS) mechanisms to orchestrate biofilm formation, enhancing resistance to host defences. In physiological conditions, QS molecules permeate the lung environment and cellular membrane to reach the cytoplasmic Aryl Hydrocarbon Receptor (AhR) that is pivotal for activating the immune response against infection. In pathological conditions like cystic fibrosis (CF) this interkingdom communication is altered, favouring P. aeruginosa persistence and chronic infection. Here, we aim to investigate the molecular journey of QS molecules from CF-like environments to the cytoplasm by quantifying via HPLC-MS the permeability of selected QS molecules (quinolones, lactones, and phenazines) through in vitro models of the two main biological lung barriers: CF-mucus and cellular membrane. While QS molecules not activating AhR exhibit intermediate permeability through the cellular membrane model (PAMPA) (1.0-4.0 × 10-6 cm/s), the AhR-activating molecule (pyocyanin) shows significantly higher permeability (8.6 ± 1.4 × 10-6 cm/s). Importantly, combining the CF mucus model with PAMPA induces a 50% decrease in pyocyanin permeability, indicating a strong mucus-shielding effect with pathological implications in infection eradication. This study underscores the importance of quantitatively describing the AhR-active bacterial molecules, even in vitro, to offer new perspectives for understanding P. aeruginosa virulence mechanisms and for proposing new antibacterial therapeutic approaches.

Critical resistance to carbapenem and aminoglycosides in Pseudomonas aeruginosa: spread of blaNDM/16S methylase armA harboring isolates with intrinsic resistance mechanisms in Kerman, Iran.

BACKGROUND: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the main Gram-negative bacterium causes of infections in hospital settings, and the spread of them is a significant challenge to public health. METHODS: A total of 30 non-duplicate isolates of CRPA were collected. Antibacterial susceptibility of isolates to antibiotic agents, AmpC ß-lactamase production, and biofilm formation were determined. Minimum biofilm inhibitory concentrations (MBIC) of isolates to cefepime (FEP), imipenem (IPM), ceftazidime (CAZ), and meropenem (MEM) were evaluated with/without cloxacillin (CLX). The carbapenemase and 16 S rRNA methylase genes were identified by PCR, and the transcription levels of oprD, ampC, and mexA genes were determined by quantitative real-time PCR (qPCR). ERIC-PCR was used to detect genetic relationships among isolates. RESULTS: All isolates were multidrug resistant (MDR) and strong biofilm producers. The resistance genes including blaNDM, blaIMP, blaVIM, blaSIM, blaGES, and armA were detected in 21 (70%), 6 (20%), 3 (10%), 2 (6.6%), 1 (3.3%), and 17 (56.6%) of the isolates, respectively. CLX at 500 and 1000 µg/mL significantly reduced the level of MIC to MEM, IPM, CAZ, and FEP, also at 2000 µg/mL significantly reduced the level of MBIC to MEM, IPM, CAZ, and FEP. In all isolates, the transcription levels of oprD were significantly downregulated as well as significantly increased for ampC and mexA. ERIC-PCR typing results divided 30 isolates into four clusters A to D. CONCLUSION: In this study, we reported the spread of different clones of CRPA harboring co-existence of various carbapenemase genes with armA 16 S rRNA methylase for the first time in Kerman, Iran. Also, our isolates had several mechanisms of resistance to carbapenems as well as ability biofilm formation along with resistance to aminoglycosides, the further spread of which could cause serious challenges in our hospital settings. Therefore, serious monitoring is necessary to reduce their prevalence.

Targeting Pseudomonas aeruginosa biofilm with an evolutionary trained bacteriophage cocktail exploiting phage resistance trade-offs.

Spread of multidrug-resistant Pseudomonas aeruginosa strains threatens to render currently available antibiotics obsolete, with limited prospects for the development of new antibiotics. Lytic bacteriophages, the viruses of bacteria, represent a path to combat this threat. In vitro-directed evolution is traditionally applied to expand the bacteriophage host range or increase bacterial suppression in planktonic cultures. However, while up to 80% of human microbial infections are biofilm-associated, research towards targeted improvement of bacteriophages' ability to combat biofilms remains scarce. This study aims at an in vitro biofilm evolution assay to improve multiple bacteriophage parameters in parallel and the optimisation of bacteriophage cocktail design by exploiting a bacterial bacteriophage resistance trade-off. The evolved bacteriophages show an expanded host spectrum, improved antimicrobial efficacy and enhanced antibiofilm performance, as assessed by isothermal microcalorimetry and quantitative polymerase chain reaction, respectively. Our two-phage cocktail reveals further improved antimicrobial efficacy without incurring dual-bacteriophage-resistance in treated bacteria. We anticipate this assay will allow a better understanding of phenotypic-genomic relationships in bacteriophages and enable the training of bacteriophages against other desired pathogens. This, in turn, will strengthen bacteriophage therapy as a treatment adjunct to improve clinical outcomes of multidrug-resistant bacterial infections.

Metallo-ß-lactamase-producing Pseudomonas aeruginosa Isolates from Two Tertiary Care Centres in a District of Nepal: A Descriptive Cross-sectional Study.

INTRODUCTION: Pseudomonas aeruginosa isolates producing metallo-ß-lactamase have caused nosocomial outbreaks, severe infections, and ineffective carbapenem therapy worldwide since 1991. Due to their prevalence, hospital infection control techniques are difficult. This study aimed to find out the prevalence of metallo-ß-lactamase among P. aeruginosa isolates from two tertiary care hospitals in Kathmandu. METHODS: A descriptive cross-sectional study was conducted at the Department of Microbiology and Department of Pathology of two tertiary care centres in Kathmandu from 7 December 2021 to 6 April 2023, after receiving ethical approval from the Ethical Review Board. Isolated strains were identified and tested for antibiotic susceptibility by modified Kirby-Bauer Methods. Metallo-ß-lactamase presence was confirmed using an imipenem-imipenem/ ethylenediaminetetraacetic acid disc. A convenience sampling method was used. The point estimate was calculated at 95% Confidence Interval. RESULTS: Among 255, Pseudomanas aeruginosa isolates, the distribution of metallo-ß-lactamase-producing Pseudomanas aeruginosa was 103 (40.39%) (34.32-46.69 at 95% Confidence Interval). Multidrug resistance categories included multidrug resistance 74 (71.80%), extensively drug resistance 32 (31.10%), P. aeruginosa difficult-to-treat 16 (15.53%) and carbapenem-resistant P. aeruginosa was determined to be 82 (79.60%). CONCLUSIONS: The study found a high prevalence of metallo-ß-lactamase-producing Pseudomanas aeruginosa isolates, requiring early identification, infection control measures, and an all-inclusive antimicrobial therapy protocol to reduce their spread in medical settings.

Tn4661-mediated transfer of bla CTX-M-15 from Klebsiella michiganensis to an outbreak clone of Pseudomonas aeruginosa.

Carriage of CTX-M-type extended-spectrum ß-lactamase (ESBL) is rare in Pseudomonas aeruginosa. During routine surveillance of an endemic ST-621 P. aeruginosa at a large hospital, isolate MRSN 100690 carrying bla CTX-M-15 was cultured from a patient (P2). This was the first detection of this ESBL in the endemic ST-621 lineage. All 1 488 bacterial isolates collected from the same facility in the 12 months prior to the incidence of 100 690 were screened for the presence of bla CTX-M-15. A set of 183 isolates was identified, in which corresponding patient metadata was evaluated for spatiotemporal overlaps with P2. The resulting three isolates, along with 100 690, were long-read sequenced using the Oxford Nanopore MinION platform to determine a potential donor of bla CTX-M-15. The screen revealed a single Klebsiella michiganensis isolate, MRSN 895358, which carried an IncA/C2 plasmid harbouring bla CTX-M-15. Notably, the patient harbouring 895358, P1, occupied the same hospital room as P2 9 months prior. Genomic alignment revealed that both isolates shared an identical 80.8 kb region containing the IncA/C2 plasmid replicon and bla CTX-M-15. This region was plasmid bound in 895 358, but chromosomally bound in 100 690 due to Tn4661-mediated transposition. ESBL bla CTX-M-15 was acquired and subsequently integrated into the chromosome of a ST-621 P. aeruginosa, likely initiated by plasmid transfer from a K. michiganensis strain.

Purified L-glutaminase effects against multidrug-resistant Pseudomonas aeruginosa in experimental vaginosis model: An immunological and histopathological observation.

The antimicrobial activity of crude and purified L-glutaminase (EC 3.5.1.2), obtained from Lactobacillus gasseri, was evaluated against multidrug-resistant Pseudomonas aeruginosa in the in vivo vaginosis condition. The L-glutaminase possessed significant antimicrobial and anti-biofilm formation activity against multi-drug resistance P. aeruginosa, which were confirmed in the BALBc rat vaginosis model, together with its effects on the immunological and histopathological aspects. The untreated animals showed heavy vaginitis, characterized by sub-epithelial edema and infiltration of mononuclear leukocytes, perivascular heavy inflammatory cells infiltration in the vaginal tissue, and moderate stromal edema. However, the L-glutaminase treatment exhibited no changes in vaginal tissue structure with normal appearance of the epithelium and lamina propria with marked repair of the vaginal section when compared with normal, uninfected, control group A. The immunomodulatory actions of the L-glutaminase were confirmed by observance of higher concentrations of tumor necrosis factor-γ (TNF-γ), and interleukin -12 (IL-12) in treated animals, while the interleukin-10 (IL-10) was higher in the infected, untreated animals' sera samples. Therefore, the L-glutaminase showed corrective and healing actions, which were observed through histopathological observations of the vaginal tissue. The investigations led to imply that L-glutaminase may have the potential to be an effective antimicrobial agent for preventing and inhibiting bacterial growth, as well as inhibiting the biofilm formation in the P. aeruginosa-originated vaginosis. The observations may be of promising value for future clinical use.

Global genomic diversity of Pseudomonas aeruginosa in bronchiectasis.

OBJECTIVES: Pseudomonas aeruginosa is the most common pathogen in the bronchiectasis lung, associated with worsened outcomes. P. aeruginosa genomic studies in this context have been limited to single-country, European studies. We aimed to determine strain diversity, adaptation mechanisms, and AMR features to better inform treatment. METHODS: P. aeruginosa from 180 bronchiectasis patients in 15 countries, obtained prior to a phase 3, randomised clinical trial (ORBIT-3), were analysed by whole-genome sequencing. Phylogenetic groups and sequence types were determined, and between versus within patient genetic diversity compared using Analysis of Molecular Variance (AMOVA). The frequency of AMR-associated genes and mutations was also determined. RESULTS: A total of 2854 P. aeruginosa isolates were analysed, predominantly belonging to phylogenetic group 1 (83%, n = 2359). Genetic diversity was far greater between than within patients, responsible for >99.9% of total diversity (AMOVA: phylogroup 1: df = 145, P < 0.01). Numerous pathways were under selection, some shared with CF (e.g., motility, iron acquisition), some unique to bronchiectasis (e.g., novel efflux pump PA1874). Multidrug resistance features were also frequent. CONCLUSIONS: We present a 10-fold increase in the availability of genomic data for P. aeruginosa in bronchiectasis, highlighting key distinctions with cystic fibrosis and potential targets for future treatments.

Monitoring of Pseudomonas aeruginosa mutational resistome dynamics using an enrichment panel for direct sequencing of clinical samples.

BACKGROUND: Pseudomonas aeruginosa is a major cause of hospital-acquired and chronic infections, characterised by an extraordinary capacity to develop antimicrobial resistance through the selection of chromosomal mutations, leading to treatment failure. Here, we designed and tested a hybridisation-based capture system for the enrichment of genes of interest before sequencing to monitor resistant populations genomics directly from clinical samples. METHODS: A panel for enrichment before sequencing of close to 200 genes related to P. aeruginosa antimicrobial resistance, multilocus sequence typing, mutability or virulence was designed, synthesised (KAPA HyperCap, Roche) and initially validated in vitro using a multidrug-resistant ST175 isolate and representative isolates from major P. aeruginosa clades. In vivo testing included ventilator associated pneumonia by MDR P. aeruginosa in ICU (3-10 sequential samples from 3 patients) and chronic respiratory infection by hypermutable P. aeruginosa in cystic fibrosis (8 sequential samples from a single patient covering a 4-year period). Results from direct sequencing with the enrichment panel were compared with those of whole genome sequencing (WGS) and phenotypic profiling of 10 isolated colonies per sample. FINDINGS: In vitro assays confirmed the selectivity of the enrichment panel and the correct identification of the vast mutational resistome of ST175, including specific mutations even when introduced in a 1:100 proportion. In vivo performance was at least equivalent to sequencing 10 colonies per sample, including the accurate identification of the sequence types and the basal and acquired mutational resistome. To note, specific resistance mutations, such as those in ampC leading to resistance to novel ß-lactams, could be traced even at frequencies of 1%. Moreover, the coselection of mutator populations and antibiotic resistance mutations, predicted in theoretical and in vitro studies, was evidenced in vivo. INTERPRETATION: This proof-of-concept study demonstrates that resistance genomics of P. aeruginosa can be analysed directly from clinical samples, determining not only a considerable reduction in turnaround time and cost from a diagnostics perspective, but also an unprecedented potency for accurate monitoring of in vivo population dynamics in bacterial infections. FUNDING: Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea-NextGenerationEU.

Evaluation and Molecular Characterization of Colistin-Resistant Isolates of Pseudomonas aeruginosa and Klebsiella pneumoniae from the Infected Wounds of Hospitalized Patients.

This study was planned to determine the colistin-resistant (CR) gene distribution among two species of gram-negative bacteria, Pseudomonas aeruginosa and Klebsiella pneumoniae. In total, 50 isolates of K. pneumoniae (14 isolates, 28%) and P. aeruginosa (36 isolates, 72%) were isolated between August 2023 and October 2023 from clinical wound samples at Jinnah Hospital and Lahore General Hospital Lahore, Pakistan. To determine the resistance genes linked to CR and assess antimicrobial susceptibility, all isolates were kept at -80°C in 15% glycerol broth. Using the right primer sets, a polymerase chain reaction (PCR) was utilized to identify the CR-associated mcr-1 gene of the gram-negative isolates. Out of 50, 40 isolates (80%) showed resistance against colistin with MICs of 8 and 128 µg/ml. The majority (97%) of P. aeruginosa CR strains were considered multidrug resistant (MDR). All K. pneumoniae isolates were resistant to cefepime, cotrimoxazole, ceftriaxone, and imipenem. The clinical CR isolates of P. aeruginosa were highly resistant to ceftriaxone, imipenem, cefepime, cotrimoxazole, ciprofloxacin, amikacin, and piperacillin/tazobactum. The antibiotic resistance pattern was terrifyingly high among both bacterial species. According to the PCR results, CR was prevalent among the gram-negative samples, and the mcr-1 gene was positive in 6/40 (15%) of the CR isolates, including four P. aeruginosa and two K. pneumoniae strains. The high CR (80%) reported in this research is cause for concern and underscores an urgent need to use colistin in a limited and logical manner, similar to other antibiotics.