Assessment of bacteriocin production by clinical Pseudomonas aeruginosa isolates and their potential as therapeutic agents.

INTRODUCTION: Bacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential. METHODS: The identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml-1). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS-PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene. RESULT: The antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR. CONCLUSION: These findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries.

Assessment of the ability of Pseudomonas aeruginosa and Staphylococcus aureus to create biofilms during wound healing in a rat model treated with carboxymethyl cellulose/carboxymethyl chitosan hydrogel containing EDTA.

The primary objective of this study was to develop a carboxymethyl cellulose (CMC) and carboxymethyl chitosan (CMCS) hydrogel containing ethylene diamine tetra acetic acid (EDTA) as the materials for wound healing. CMC and CMCS solutions were prepared with a concentration of 4% (w/v). These solutions were made using normal saline serum with a concentration of 0.5% (v/v). Additionally, EDTA with the concentrations of 0.01%, 0.05%, 0.1%, 0.5%, 1%, and 2% (w/v) was included in the prepared polymer solution. The analysis of the hydrogels revealed that they possess porous structures with interconnected pores, with average in size 88.71 ± 5.93 µm. The hydrogels exhibited a swelling capacity of up to 60% of their initial weight within 24 h, as indicated by the weight loss and swelling measurements. The antibacterial experiments showed that the formulated CMC/CMCS/EDTA 0.5% hydrogel inhibited the growth of Staphylococcus aureus and Pseudomonas aeruginosa. Moreover, the produced hydrogels were haemocompatible and biocompatible. At the last stage, the evaluation of wound healing in the animal model demonstrated that the use of the produced hydrogels significantly improved the process of wound healing. Finally, the findings substantiated the effectiveness of the formulated hydrogels as the materials for promoting wound healing and antibacterial agents.

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.

Resistance, mechanism, and fitness cost of specific bacteriophages for Pseudomonas aeruginosa.

The bacteriophage is an effective adjunct to existing antibiotic therapy; however, in the course of bacteriophage therapy, host bacteria will develop resistance to bacteriophages, thus affecting the efficacy. Therefore, it is important to describe how bacteria evade bacteriophage attack and the consequences of the biological changes that accompany the development of bacteriophage resistance before the bacteriophage is applied. The specific bacteriophage vB3530 of Pseudomonas aeruginosa (P. aeruginosa) has stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. Ten bacteriophage-resistant strains (TL3780-R) were induced using the secondary infection approach, and the plaque assay showed that vB3530 was less sensitive to TL3780-R. Identification of bacteriophage adsorption receptors showed that the bacterial surface polysaccharide was probably the adsorption receptor of vB3530. In contrast to the TL3780 parental strain, TL3780-R is characterized by the absence of long lipopolysaccharide chains, which may be caused by base insertion of wzy or deletion of galU. It is also intriguing to observe that, in comparison to the parent strain, the bacteriophage-resistant strains TL3780-R mostly exhibited a large cost of fitness (growth rate, biofilm formation, motility, and ability to produce enhanced pyocyanin). In addition, TL3780-R9 showed increased susceptibility to aminoglycosides and chlorhexidine, which may be connected to the loss and down-regulation of mexX expression. Consequently, these findings fully depicted the resistance mechanism of P. aeruginosa to vB3530 and the fitness cost of bacteriophage resistance, laying a foundation for further application of bacteriophage therapy.IMPORTANCEThe bacteriophage is an effective adjunct to existing antibiotic therapy; However, bacteria also develop defensive mechanisms against bacteriophage attack. Thus, there is an urgent need to deeply understand the resistance mechanism of bacteria to bacteriophages and the fitness cost of bacteriophage resistance so as to lay the foundation for subsequent application of the phage. In this study, a specific bacteriophage vB3530 of P. aeruginosa had stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. In addition, we found that P. aeruginosa may lead to phage resistance due to the deletion of galU and the base insertion of wzy, involved in the synthesis of lipopolysaccharides. Simultaneously, we showed the association with the biological state of the bacteria after bacteria acquire bacteriophage resistance, which is extremely relevant to guide the future application of therapeutic bacteriophages.

Assessment of the Glycan-Binding Profile of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen that can establish acute and chronic infections in individuals who lack fully functional innate immunity. In particular, phagocytosis by neutrophils and macrophages is a key mechanism that modulates host control and clearance of P. aeruginosa. Individuals with neutropenia or cystic fibrosis are highly susceptible to P. aeruginosa infection, thus underscoring the importance of the host innate immune response. Cell-to-cell contact between host innate immune cells and the pathogen, a first step in phagocytic uptake, is facilitated by simple and complex glycan structures present at the host cell surface. We have previously shown that endogenous polyanionic N-linked glycans localized to the cell surface of phagocytes mediate the binding and subsequent phagocytosis of P. aeruginosa cells. However, the suite of glycans that P. aeruginosa cells bind to on host phagocytic cells remains poorly characterized. Here, we demonstrate, with the use of exogenous N-linked glycans and a glycan array, that P. aeruginosa PAO1 cells preferentially attach to a subset of glycans, including a bias toward monosaccharide versus more complex glycan structures. Consistent with these findings, we were able to competitively inhibit bacterial adherence and uptake by the addition of exogenous N-linked mono- and disaccharide glycans. We discuss our findings in the context of previous reports of P. aeruginosa glycan binding. IMPORTANCE P. aeruginosa cells bind to a variety of glycans as part of their interaction with host cells, and a number of P. aeruginosa-encoded receptors and target ligands have been described that allow this microbe to bind to such glycans. Here, we extend this work by studying the glycans used by P. aeruginosa PAO1 cells to bind to phagocytic cells and by using a glycan array to characterize the suite of such molecules that can facilitate host cell binding by this microbe. This study provides an increased understanding of the glycans bound by P. aeruginosa and furthermore provides a useful data set for future studies of P. aeruginosa-glycan interactions.

Assessment of mortality-related risk factors and effective antimicrobial regimens for treatment of bloodstream infections caused by carbapenem-resistant Pseudomonas aeruginosa in patients with hematological diseases.

Background: Infections caused by carbapenem-resistant Pseudomonas aeruginosa (CRPA) are related to higher mortality. The objective of this study was to explore clinical outcomes of CRPA bacteremia, identify risk factors and also, compare the efficacy of traditional and novel antibiotic regimens. Methods: This retrospective study was conducted at a blood diseases hospital in China. The study included hematological patients who were diagnosed with CRPA bacteremia between January 2014 and August 2022. The primary endpoint was all-cause mortality at day 30. Secondary endpoints included 7-day and 30-day clinical cure. Multivariable Cox regression analysis was employed to identify mortality-related risk factors. Results: 100 patients infected with CRPA bacteremia were included and 29 patients accepted allogenic-hematopoietic stem cell transplantation. 24 received ceftazidime-avibactam (CAZ-AVI)-based therapy and 76 received other traditional antibiotics. 30-day mortality was 21.0%. Multivariable cox regression analysis showed neutropenia >7 days after bloodstream infections (BSI) (P=0.030, HR: 4.068, 95%CI: 1.146~14.434), higher Pitt bacteremia score (P<0.001, HR:1.824, 95%CI: 1.322~2.517), higher Charlson comorbidity index (P=0.01, HR: 1.613, 95%CI: 1.124~2.315) and bacteremia due to multidrug-resistant Pseudomonas aeruginosa (MDR-PA) (P=0.024, HR:3.086, 95%CI: 1.163~8.197) were identified as independent risk factors of 30-day mortality. After controlling for confounders, an additional multivariable cox regression analysis revealed definitive regimens containing CAZ-AVI were associated with lower mortality in CRPA bacteremia (P=0.016, HR: 0.150, 95%CI: 0.032~0.702), as well as in MDR-PA bacteremia (P=0.019, HR: 0.119, 95%CI: 0.020~0.709). Conclusions: For patients with hematological diseases and CRPA bacteremia, 30-day mortality rate was 21.0% (21/100). Neutropenia >7 days after BSI, higher Pitt bacteremia score, higher Charlson comorbidity index and bacteremia due to MDR-PA increased 30-day mortality. CAZ-AVI-based regimens were effective alternatives for bacteremia due to CRPA or MDR-PA.

Superiority of ceftazidime off-label high-dose regimen in PK/PD target attainment during treatment of extensively drug-resistant Pseudomonas aeruginosa infections in cancer patients.

AIM: The objective of this study was to evaluate off-label high-dose ceftazidime population pharmacokinetics in cancer patients with suspected or proven extensively drug-resistant (XDR) Pseudomonas aeruginosa infections and then to compare the achievement of the pharmacokinetic/pharmacodynamic (PK/PD) target after standard and off-label high-dose regimens using population model-based simulations. A further aim was to clinically observe the occurrence of adverse effects during the off-label high-dose ceftazidime treatment. METHODS: In patients treated with off-label high-dose ceftazidime (3 g every 6 h), blood samples were collected and ceftazidime serum levels measured using LC-MS/MS. A pharmacokinetic population model was developed using a nonlinear mixed-effects modelling approach and Monte Carlo simulations were then used to compare standard and high-dose regimens for PK/PD target attainment. RESULTS: A total of 14 cancer patients with serious infection suspected of XDR P. aeruginosa aetiology were eligible for PK analysis. XDR P. aeruginosa was confirmed in 10 patients as the causative pathogen. Population ceftazidime volume of distribution was 13.23 L, while clearance started at the baseline of 1.48 L/h and increased by 0.0076 L/h with each 1 mL/min/1.73 m2 of eGFR. High-dose regimen showed significantly higher probability of target attainment (i.e., 86% vs. 56% at MIC of 32 mg/L). This was translated into a very low mortality rate of 20%. Only one case of reversible neurological impairment was observed. CONCLUSION: We proved the superiority of the ceftazidime off-label high-dose regimen in PK/PD target attainment with very low occurrence of adverse effects. The off-label high-dose regimen should be used to optimize treatment of XDR P. aeruginosa infections.

Role of Enzymatic Activity in the Biological Cost Associated with the Production of AmpC ß-Lactamases in Pseudomonas aeruginosa.

In the current scenario of growing antibiotic resistance, understanding the interplay between resistance mechanisms and biological costs is crucial for designing therapeutic strategies. In this regard, intrinsic AmpC ß-lactamase hyperproduction is probably the most important resistance mechanism of Pseudomonas aeruginosa, proven to entail important biological burdens that attenuate virulence mostly under peptidoglycan recycling alterations. P. aeruginosa can acquire resistance to new ß-lactam-ß-lactamase inhibitor combinations (ceftazidime-avibactam and ceftolozane-tazobactam) through mutations affecting ampC and its regulatory genes, but the impact of these mutations on the associated biological cost and the role that ß-lactamase activity plays per se in contributing to the above-mentioned virulence attenuation are unknown. The same questions remain unsolved for plasmid-encoded AmpC-type ß-lactamases such as FOX enzymes, some of which also provide resistance to new ß-lactam-ß-lactamase inhibitor combinations. Here, we assessed from different perspectives the effects of changes in the active center and, thus, in the hydrolytic spectrum resistance to inhibitors of AmpC-type ß-lactamases on the fitness and virulence of P. aeruginosa, using site-directed mutagenesis; the previously described AmpC variants T96I, G183D, and ΔG229-E247; and, finally, blaFOX-4 versus blaFOX-8. Our results indicate the essential role of AmpC activity per se in causing the reported full virulence attenuation (in terms of growth, motility, cytotoxicity, and Galleria mellonella larvae killing), although the biological cost of the above-mentioned AmpC-type variants was similar to that of the wild-type enzymes. This suggests that there is not an important biological burden that may limit the selection/spread of these variants, which could progressively compromise the future effectiveness of the above-mentioned drug combinations. IMPORTANCE The growing antibiotic resistance of the top nosocomial pathogen Pseudomonas aeruginosa pushes research to explore new therapeutic strategies, for which the resistance-versus-virulence balance is a promising source of targets. While resistance often entails significant biological costs, little is known about the bases of the virulence attenuations associated with a resistance mechanism as extraordinarily relevant as ß-lactamase production. We demonstrate that besides potential energy and cell wall alterations, the enzymatic activity of the P. aeruginosa cephalosporinase AmpC is essential for causing the full attenuation associated with its hyperproduction by affecting different features related to pathogenesis, a fact exploitable from the antivirulence perspective. Less encouraging, we also show that the production of different chromosomal/plasmid-encoded AmpC derivatives conferring resistance to some of the newest antibiotic combinations causes no significantly increased biological burdens, which suggests a free way for the selection/spread of these types of variants, potentially compromising the future effectiveness of these antipseudomonal therapies.

An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates.

Understanding quorum sensing (QS) and its role in the development of pathogenesis may provide new avenues for diagnosing, surveillance, and treatment of infectious diseases. For this purpose, the availability of reliable and efficient analytical diagnostic tools suitable to specifically detect and quantify these essential QS small molecules and QS regulated virulence factors is crucial. Here, we reported the development and evaluation of antibodies and an enzyme-linked immunosorbent assay (ELISA) for HQNO (2-heptyl-4-quinoline N-oxide), a QS product of the PqsR system, which has been found to act as a major virulence factor that interferes with the growth of other microorganisms. Despite the nonimmunogenic character of HQNO, the antibodies produced showed high avidity and the microplate-based ELISA developed could detect HQNO in the low nM range. Hence, a limit of detection (LOD) of 0.60 ± 0.13 nM had been reached in Müeller Hinton (MH) broth, which was below previously reported levels using sophisticated equipment based on liquid chromatography coupled to mass spectrometry. The HQNO profile of release of different Pseudomonas aeruginosa clinical isolates analyzed using this ELISA showed significant differences depending on whether the clinical isolates belonged to patients with acute or chronic infections. These data point to the possibility of using HQNO as a specific biomarker to diagnose P. aeruginosa infections and for patient surveillance. Considering the role of HQNO in inhibiting the growth of coinfecting bacteria, the present ELISA will allow the investigation of these complex bacterial interactions underlying infections. IMPORTANCE Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa-specific QS signaling molecule PQS (Pseudomonas quinolone signal). In this work, the development of highly specific antibodies and an immunochemical diagnostic technology (ELISA) for the detection and quantification of HQNO was reported. The ELISA allowed profiling of the release of HQNO by clinical bacterial isolates, showing its potential value for diagnosing and surveillance of P. aeruginosa infections. Moreover, the antibodies and the ELISA reported here may contribute to the knowledge of other underlying conditions related to the pathology, such as the role of the interactions with other bacteria of a particular microbiota environment.

Histological assessment, anti-quorum sensing, and anti-biofilm activities of Dioon spinulosum extract: in vitro and in vivo approach.

Pseudomonas aeruginosa is an opportunistic bacterium causing several health problems and having many virulence factors like biofilm formation on different surfaces. There is a significant need to develop new antimicrobials due to the spreading resistance to the commonly used antibiotics, partly attributed to biofilm formation. Consequently, this study aimed to investigate the anti-biofilm and anti-quorum sensing activities of Dioon spinulosum, Dyer Ex Eichler extract (DSE), against Pseudomonas aeruginosa clinical isolates. DSE exhibited a reduction in the biofilm formation by P. aeruginosa isolates both in vitro and in vivo rat models. It also resulted in a decrease in cell surface hydrophobicity and exopolysaccharide quantity of P. aeruginosa isolates. Both bright field and scanning electron microscopes provided evidence for the inhibiting ability of DSE on biofilm formation. Moreover, it reduced violacein production by Chromobacterium violaceum (ATCC 12,472). It decreased the relative expression of 4 quorum sensing genes (lasI, lasR, rhlI, rhlR) and the biofilm gene (ndvB) using qRT-PCR. Furthermore, DSE presented a cytotoxic activity with IC50 of 4.36 ± 0.52 µg/ml against human skin fibroblast cell lines. For the first time, this study reports that DSE is a promising resource of anti-biofilm and anti-quorum sensing agents.

Subpalpebral Antibiotic Lavage as Safe, Emergent, and Cost-Effective Management of Acute Infectious Keratitis Related to Contact Lens Overwear: Case Report and Literature Review.

PURPOSE: The aim of this study is to describe the technique of subpalpebral antibiotic lavage (SAL), which is a highly therapeutic, efficient, and cost-effective method for managing severe bacterial keratitis. METHODS: This case report describes a 26-year-old woman with severe bacterial keratitis in the right eye due to contact lens overwear, with progressive corneal thinning, a hypopyon, impending perforation, and marked visual loss to perception of light despite treatment with intensive topical antibiotics. This was managed with SAL that involves the insertion of a cannula transcutaneously into the upper conjunctival fornix to provide continuous antibiotic irrigation of the ocular surface. RESULTS: By 11 weeks after presentation, the cornea and anterior chamber appeared clinically quiescent, and visual acuity improved to 20/40 corrected in the right eye. CONCLUSIONS: Bacterial keratitis is a potentially blinding condition for which contact lens wear is an important risk factor. Most cases are successfully managed with topical medications; however, in cases of treatment failure, a second-line approach such as SAL can be sight-saving. SAL uses readily available equipment for the delivery of high concentrations of antibiotics to the ocular surface, thus increasing therapeutic efficacy and reducing nursing staff workload. Despite its advantages, the literature reveals apparent underutilization of this technique.

Intravenous or oral antibiotic treatment in adults and children with cystic fibrosis and Pseudomonas aeruginosa infection: the TORPEDO-CF RCT.

BACKGROUND: People with cystic fibrosis are susceptible to pulmonary infection with Pseudomonas aeruginosa. This may become chronic and lead to increased mortality and morbidity. If treatment is commenced promptly, infection may be eradicated through prolonged antibiotic treatment. OBJECTIVE: To compare the clinical effectiveness, cost-effectiveness and safety of two eradication regimens. DESIGN: This was a Phase IV, multicentre, parallel-group, randomised controlled trial. SETTING: Seventy UK and two Italian cystic fibrosis centres. PARTICIPANTS: Participants were individuals with cystic fibrosis aged > 28 days old who had never had a P. aeruginosa infection or who had been infection free for 1 year. INTERVENTIONS: Fourteen days of intravenous ceftazidime and tobramycin or 3 months of oral ciprofloxacin. Inhaled colistimethate sodium was included in both regimens over 3 months. Consenting patients were randomly allocated to either treatment arm in a 1 : 1 ratio using simple block randomisation with random variable block length. MAIN OUTCOME MEASURES: The primary outcome was eradication of P. aeruginosa at 3 months and remaining free of infection to 15 months. Secondary outcomes included time to reoccurrence, spirometry, anthropometrics, pulmonary exacerbations and hospitalisations. Primary analysis used intention to treat (powered for superiority). Safety analysis included patients who had received at least one dose of any of the study drugs. Cost-effectiveness analysis explored the cost per successful eradication and the cost per quality-adjusted life-year. RESULTS: Between 5 October 2010 and 27 January 2017, 286 patients were randomised: 137 patients to intravenous antibiotics and 149 patients to oral antibiotics. The numbers of participants achieving the primary outcome were 55 out of 125 (44%) in the intravenous group and 68 out of 130 (52%) in the oral group. Participants randomised to the intravenous group were less likely to achieve the primary outcome; although the difference between groups was not statistically significant, the clinically important difference that the trial aimed to detect was not contained within the confidence interval (relative risk 0.84, 95% confidence interval 0.65 to 1.09; p = 0.184). Significantly fewer patients in the intravenous group (40/129, 31%) than in the oral group (61/136, 44.9%) were hospitalised in the 12 months following eradication treatment (relative risk 0.69, 95% confidence interval 0.5 to 0.95; p = 0.02). There were no clinically important differences in other secondary outcomes. There were 32 serious adverse events in 24 participants [intravenous: 10/126 (7.9%); oral: 14/146 (9.6%)]. Oral therapy led to reductions in costs compared with intravenous therapy (-£5938.50, 95% confidence interval -£7190.30 to -£4686.70). Intravenous therapy usually necessitated hospital admission, which accounted for a large part of this cost. LIMITATIONS: Only 15 out of the 286 participants recruited were adults - partly because of the smaller number of adult centres participating in the trial. The possibility that the trial participants may be different from the rest of the cystic fibrosis population and may have had a better clinical status, and so be more likely to agree to the uncertainty of trial participation, cannot be ruled out. CONCLUSIONS: Intravenous antibiotics did not achieve sustained eradication of P. aeruginosa in a greater proportion of cystic fibrosis patients. Although there were fewer hospitalisations in the intravenous group during follow-up, this confers no advantage over the oral therapy group, as intravenous eradication frequently requires hospitalisation. These results do not support the use of intravenous antibiotics to eradicate P. aeruginosa in cystic fibrosis. FUTURE WORK: Future research studies should combine long-term follow-up with regimens to reduce reoccurrence after eradication. TRIAL REGISTRATION: Current Controlled Trials ISRCTN02734162 and EudraCT 2009-012575-10. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 25, No. 65. See the NIHR Journals Library website for further project information.

Cystic fibrosis is a genetic condition that affects mucous glands, causing sticky mucus in the lungs and digestive system. People with cystic fibrosis are prone to lung infection with a bacterium called Pseudomonas aeruginosa, which can lead to serious long-term complications and death. It is possible to eradicate P. aeruginosa if antibiotics are started promptly and taken for several months. The Trial of Optimal TheRapy for Pseudomonas EraDicatiOn in Cystic Fibrosis (TORPEDO-CF) was designed to find out if intravenous ceftazidime and tobramycin were better at eradicating P. aeruginosa than oral ciprofloxacin. A total of 286 children, young people and adults with cystic fibrosis joined the study from 70 UK and two Italian centres. Approximately half of the participants received treatment with intravenous antibiotics and half with oral antibiotics. All participants received inhaled colistin for 3 months and were followed up for a minimum of 15 months. We studied whether or not either treatment eradicated P. aeruginosa, and if reinfection happened during follow-up. We also collected data on lung function, other chest infections and hospital admissions, and examined whether or not one treatment was more cost-effective than the other. In total, 15 adults joined TORPEDO-CF, so the study population may not totally match the wider cystic fibrosis population; however, in TORPEDO-CF, we found that intravenous antibiotics did not achieve persistent eradication of P. aeruginosa in a greater proportion of cystic fibrosis patients. We also found that oral antibiotics were more cost-effective than intravenous antibiotics. The intravenous antibiotics group had fewer hospital admissions during follow-up, but, as they were usually admitted for their initial treatment, this was not considered an advantage over the oral antibiotics group. The TORPEDO-CF results do not support the use of intravenous antibiotics to eradicate P. aeruginosa in cystic fibrosis and, when the findings of this trial are applied in routine clinical practice in the NHS, patients will most likely receive oral treatment as an outpatient, avoiding the need for hospital admission.

The Epidemiology and Pathogenesis and Treatment of Pseudomonas aeruginosa Infections: An Update.

Pseudomonas aeruginosa is a Gram-negative bacterial pathogen that is a common cause of nosocomial infections, particularly pneumonia, infection in immunocompromised hosts, and in those with structural lung disease such as cystic fibrosis. Epidemiological studies have identified increasing trends of antimicrobial resistance, including multi-drug resistant (MDR) isolates in recent years. P. aeruginosa has several virulence mechanisms that increase its ability to cause severe infections, such as secreted toxins, quorum sensing and biofilm formation. Management of P. aeruginosa infections focuses on prevention when possible, obtaining cultures, and prompt initiation of antimicrobial therapy, occasionally with combination therapy depending on the clinical scenario to ensure activity against P. aeruginosa. Newer anti-pseudomonal antibiotics are available and are increasingly being used in the management of MDR P. aeruginosa.

Assessment of cefepime toxicodynamics: comprehensive examination of pharmacokinetic/pharmacodynamic targets for cefepime-induced neurotoxicity and evaluation of current dosing guidelines.

BACKGROUND: Cefepime-induced neurotoxicity (CIN) is an increasingly reported adverse event; however, the toxicity threshold remains unclear. This study was conducted to provide a comprehensive examination of the most appropriate threshold for CIN, and evaluate the ability of current dosing regimens to attain therapeutic targets. METHODS: Data of the incidence of CIN and cefepime plasma concentrations were collected retrospectively from patients administered cefepime. Population pharmacokinetic modelling was used to determine daily cefepime trough concentration (Cmin), maximum serum concentration and area under the concentration-time curve. The ability of each pharmacokinetic parameter to predict CIN was evaluated using receiver operating characteristic (ROC) curves, from which optimal toxicity thresholds were determined. Pharmacokinetic simulation was used to evaluate the ability of cefepime dosing guidelines to meet established efficacy targets, whilst maintaining exposure below the determined CIN threshold. RESULTS: In total, 102 cefepime courses were evaluated, with CIN reported in 10. ROC analyses showed that all cefepime pharmacokinetic parameters were strongly predictive of CIN. Cmin of 49 mg/L was identified as the optimal toxicity target, based on its predictive ability (0.88, 95% confidence interval 0.758-0.999, P<0.001) and ease of clinical use. Assessment of cefepime dosing regimens predicted that only 29% of simulated patients achieve therapeutic targets, with patients with impaired renal function more likely to exhibit subtherapeutic concentrations (89%), and patients with normal renal function likely to have potentially toxic exposure (64%). CONCLUSIONS: The findings from this study provide evidence that cefepime exposure is highly predictive of CIN, with Cmin of 49 mg/L being the most appropriate toxicity threshold. Further research is required to optimize cefepime dosing in the context of this therapeutic target.

Assessment of Antimicrobial Activity of Dehydrated Amniotic Membrane in Infectious Keratitis: A Small Retrospective Case Series and In Vitro Study.

PURPOSE: To describe a small case series of infectious keratitis with poor visual outcomes after amniotic membrane (AM) placement and to prospectively evaluate whether AM demonstrates antibacterial activity in vitro against pathogens commonly isolated from infectious corneal ulcers. METHODS: A retrospective case series and in vitro study of antibacterial activity of dehydrated AM using disk diffusion and measurement of inhibitory zones for bacterial assessment and inverted microscopy analysis for Acanthamoeba sp. growth. RESULTS: Three cases of known etiology infectious keratitis are described where the clinical presentation worsened after treatment with AM. In vitro analysis of dehydrated AM, with and without a soft contact lens, demonstrated no inhibition of growth against Pseudomonas aeruginosa or Streptococcus pneumoniae. There was minimal growth inhibition of Staphylococcus aureus, although these zones of inhibition were much smaller than that surrounding the positive control. For Acanthamoeba sp., solubilized, dehydrated AM did not alter cyst density. CONCLUSIONS: In an in vitro analysis, dehydrated AM did not provide evidence for a potentially clinically meaningful antibacterial effect against organisms commonly isolated from corneal ulcers.

Outbreak of Pseudomonas aeruginosa producing VIM carbapenemase in an intensive care unit and its termination by implementation of waterless patient care.

BACKGROUND: Long-term outbreaks of multidrug-resistant Gram-negative bacilli related to hospital-building water systems have been described. However, successful mitigation strategies have rarely been reported. In particular, environmental disinfection or replacement of contaminated equipment usually failed to eradicate environmental sources of Pseudomonas aeruginosa. METHODS: We report the investigation and termination of an outbreak of P. aeruginosa producing VIM carbapenemase (PA-VIM) in the adult intensive care unit (ICU) of a Swiss tertiary care hospital with active case finding, environmental sampling and whole genome sequencing (WGS) of patient and environmental strains. We also describe the implemented control strategies and their effectiveness on eradication of the environmental reservoir. RESULTS: Between April 2018 and September 2020, 21 patients became either infected or colonized with a PA-VIM strain. For 16 of them, an acquisition in the ICU was suspected. Among 131 environmental samples collected in the ICU, 13 grew PA-VIM in sink traps and drains. WGS confirmed the epidemiological link between clinical and environmental strains and the monoclonal pattern of the outbreak. After removing sinks from patient rooms and implementation of waterless patient care, no new acquisition was detected in the ICU within 8 months after the intervention. DISCUSSION: Implementation of waterless patient care with removal of the sinks in patient rooms was successful for termination of a PA-VIM ICU outbreak linked to multiple environmental water sources. WGS provides highly discriminatory accuracy to investigate environment-related outbreaks.

Evaluation of Ceftazidime/Avibactam Administration in Enterobacteriaceae and Pseudomonas aeruginosa Bloodstream Infections by Monte Carlo Simulation.

PURPOSE: To evaluate the administration regimen of ceftazidime/avibactam (CZA) for bloodstream infections caused by Enterobacteriaceae and Pseudomonas aeruginosa. METHODS: The minimal inhibitory concentrations (MICs) of CZA against Enterobacteriaceae and P. aeruginosa isolated from blood cultures at member hospitals in BRICS (Blood Bacterial Resistant Investigation Collaborative System) in 2019 were determined by broth micro-dilution methodology. A 10,000-patient Monte Carlo simulation (MCS) was used to calculate the probability of target attainment (PTA) and cumulative fraction of response (CFR) for different CZA dosage regimens to evaluate their efficacies and optimize the best initial dosage regimen. RESULTS: Altogether, 6487 Enterobacteriaceae and P. aeruginosa strains were isolated from the blood cultures. The overall CZA resistance rate was 2.31%, of which the Enterobacteriaceae and P. aeruginosa rates were 1.57% and 14.29%, respectively. The MCS showed that the greater the MIC value, the worse the therapeutic effect. When the CZA MIC was ≤8 mg/L, the standard dose (2.5g iv q8h) achieved 90% PTA in the subset of patients with creatinine clearance (CrCl) values from 51 to 120 mL/min. Although the high-dose regimen (3.75g iv q8h) achieved 90% PTA in patients with CrCl values from 121 to 190 mL/min, implementing the low-dose regimen (1.25g iv q8h) was also effective for patients in the 51-89 mL/min CrCl range. Generally, the high-dose regimen (3.75g iv q8h) reached 90% CFR against all of the strains. Conversely, in patients with CrCl values of 121-190 mL/min, the standard dose (2.5g iv q8h) failed to reach 90% CFR against some Enterobacteriaceae members and P. aeruginosa. When the dose was reduced to the low-dose regimen (1.25g iv q8h), no patients reached 90% CFR against some Enterobacteriaceae members and P. aeruginosa. CONCLUSION: CZA has good antibacterial activity against Enterobacteriaceae and P. aeruginosa in bloodstream infections. Clinicians could make individualized treatment regimens in accordance with the sensitivity of the strains and the level of renal function in their patients to best predict the drug-related clinical responses.

Population pharmacokinetics of continuous infusion of piperacillin/tazobactam in very elderly hospitalized patients and considerations for target attainment against Enterobacterales and Pseudomonas aeruginosa.

Continuous infusion (CI) piperacillin/tazobactam is frequently used to treat infections in very elderly patients. This study aimed to conduct a population pharmacokinetic analysis of CI piperacillin/tazobactam, and to identify optimal dosages for safe and effective probability of target attainment (PTA) against Enterobacterales and Pseudomonas aeruginosa. Non-linear mixed-effects modelling was performed with Pmetrics. Monte Carlo simulations assessed the steady-state concentration (Css) of increasing piperacillin/tazobactam regimens (from 2.25 to 18 g daily by continuous infusion). Permissible doses were defined as those associated with <10% probability of Css >157.2 mg/L. PTA at the pharmacodynamic targets of free plasma steady-state concentration (fCss)/minimum inhibitory concentration (MIC) ≥1 and ≥4 and cumulative fraction of response (CFR) against EUCAST MIC distribution were also calculated. A total of 141 patients (median age 85 years) provided 217 plasma piperacillin Css. Most patients (55.2%) had hospital-acquired pneumonia and intra-abdominal infections. A one-compartment pharmacokinetic model with parallel linear and Michaelis-Menten elimination best described piperacillin data. Creatinine clearance (CLCR) was the covariate retained by the model. Pharmacokinetic estimates were 6.05 L/h for clearance and 3.39 mg/L for the Michaelis-Menten constant. Permissible doses were up to 4.5, 9, 11.25 and 13.5 g daily by continuous infusion for patients with CLCR of 0-19, 20-39, 40-59 and 60-79 mL/min/1.73 m2, respectively. At the clinical breakpoint of 8 mg/L, the permissible doses only achieved optimal PTA for fCss/MIC ≥1 in patients with CLCR 20-79 mL/min/1.73 m2. Optimal CFRs with the permissible doses were only attained against Escherichia coli and Proteus mirabilis. Permissible dosages and CLCR should be considered for prescribing CI piperacillin/tazobactam in very elderly patients.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.