In vitro activity of cefiderocol in Pseudomonas aeruginosa isolates from people with cystic fibrosis recovered during three multicentre studies in Spain.

OBJECTIVES: Despite the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators, Pseudomonas aeruginosa is still a major pathogen in people with cystic fibrosis (pwCF). We determine the activity of cefiderocol and comparators in a collection of 154 P. aeruginosa isolates recovered from pwCF during three multicentre studies performed in 17 Spanish hospitals in 2013, 2017 and 2021. METHODS: ISO broth microdilution was performed and MICs were interpreted with CLSI and EUCAST criteria. Mutation frequency and WGS were also performed. RESULTS: Overall, 21.4% were MDR, 20.8% XDR and 1.3% pandrug-resistant (PDR). Up to 17% of the isolates showed a hypermutator phenotype. Cefiderocol demonstrated excellent activity; only 13 isolates (8.4%) were cefiderocol resistant by EUCAST (none using CLSI). A high proportion of the isolates resistant to ceftolozane/tazobactam (71.4%), meropenem/vaborbactam (70.0%), imipenem/relebactam (68.0%) and ceftazidime/avibactam (55.6%) were susceptible to cefiderocol. Nine out of 13 cefiderocol-resistant isolates were hypermutators (P < 0.001). Eighty-three STs were detected, with ST98 being the most frequent. Only one isolate belonging to the ST175 high-risk clone carried blaVIM-2. Exclusive mutations affecting genes involved in membrane permeability, AmpC overexpression (L320P-AmpC) and efflux pump up-regulation were found in cefiderocol-resistant isolates (MIC = 4-8 mg/L). Cefiderocol resistance could also be associated with mutations in genes related to iron uptake (tonB-dependent receptors and pyochelin/pyoverdine biosynthesis). CONCLUSIONS: Our results position cefiderocol as a therapeutic option in pwCF infected with P. aeruginosa resistant to most recent ß-lactam/ß-lactamase inhibitor combinations.

Synergistic effects of inhaled aztreonam plus tobramycin on hypermutable cystic fibrosis Pseudomonas aeruginosa isolates in a dynamic biofilm model evaluated by mechanism-based modelling and whole genome sequencing.

OBJECTIVE: Hypermutable Pseudomonas aeruginosa strains are highly prevalent in chronic lung infections of patients with cystic fibrosis (CF). Acute exacerbations of these infections have limited treatment options. This study aimed to investigate inhaled aztreonam and tobramycin against clinical hypermutable P. aeruginosa strains using the CDC dynamic in vitro biofilm reactor (CBR), mechanism-based mathematical modelling (MBM) and genomic studies. METHODS: Two CF multidrug-resistant strains were investigated in a 168 h CBR (n = 2 biological replicates). Regimens were inhaled aztreonam (75 mg 8-hourly) and tobramycin (300 mg 12-hourly) in monotherapies and combination. The simulated pharmacokinetic profiles of aztreonam and tobramycin (t1/2 = 3 h) were based on published lung fluid concentrations in patients with CF. Total viable and resistant counts were determined for planktonic and biofilm bacteria. MBM of total and resistant bacterial counts and whole genome sequencing were completed. RESULTS: Both isolates showed reproducible bacterial regrowth and resistance amplification for the monotherapies by 168 h. The combination performed synergistically, with minimal resistant subpopulations compared to the respective monotherapies at 168 h. Mechanistic synergy appropriately described the antibacterial effects of the combination regimen in the MBM. Genomic analysis of colonies recovered from monotherapy regimens indicated noncanonical resistance mechanisms were likely responsible for treatment failure. CONCLUSION: The combination of aztreonam and tobramycin was required to suppress the regrowth and resistance of planktonic and biofilm bacteria in all biological replicates of both hypermutable multidrug-resistant P. aeruginosa CF isolates. The developed MBM could be utilised for future investigations of this promising inhaled combination.

OriPlex: Origami-enabled multiplexed detection of respiratory pathogens.

Origami biosensors leverage paper foldability to develop total analysis systems integrated in a single piece of paper. This capability can also be utilized to incorporate additional features that would be difficult to achieve with rigid substrates. In this article, we report a new design for 3D origami biosensors called OriPlex, which leverages the foldability of filter paper for the multiplexed detection of bacterial pathogens. OriPlex immunosensors detect pathogens by folding nanoparticle reservoirs containing different types of nanoprobes. This releases antibody-coated nanoparticles in a central channel where targets are captured through physical interactions. The OriPlex concept was demonstrated by detecting the respiratory pathogens Pseudomonas aeruginosa (PA) and Klebsiella pneumoniae (KP) with a limit of detection of 3.4·103 cfu mL-1 and 1.4·102 cfu mL-1, respectively, and with a turn-around time of 25 min. Remarkably, the OriPlex biosensors allowed the multiplexed detection of both pathogens spiked into real bronchial aspirate (BAS) samples at a concentration of 105 cfu mL-1 (clinical infection threshold), thus demonstrating their suitability for diagnosing lower tract respiratory infections. The results shown here pave the way for implementing OriPlex biosensors as a screening test for detecting superbugs requiring personalized antibiotics in suspected cases of nosocomial pneumonia.

In vitro activity of cefiderocol against European Pseudomonas aeruginosa and Acinetobacter spp., including isolates resistant to meropenem and recent ß-lactam/ß-lactamase inhibitor combinations.

Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter spp. represent major threats and have few approved therapeutic options. Non-|fermenting Gram-negative isolates were collected from hospitalized inpatients from 49 sites in 6 European countries between 01 January 2020 and 31 December 2020 and underwent susceptibility testing against cefiderocol and ß-lactam/ß-lactamase inhibitor combinations. Meropenem-resistant (MIC >8 mg/L), cefiderocol-susceptible isolates were analyzed by PCR, and cefiderocol-resistant isolates were analyzed by whole-genome sequencing to identify resistance mechanisms. Overall, 1,451 (950 P. aeruginosa; 501 Acinetobacter spp.) isolates were collected, commonly from the respiratory tract (42.0% and 39.3%, respectively). Cefiderocol susceptibility was higher than |ß|-|l|a|c|t|a|m|/|ß|-|l|a|c|t|a|mase| inhibitor combinations against P. aeruginosa (98.9% vs 83.3%-91.4%), and P. |aeruginosa resistant to meropenem (n = 139; 97.8% vs 12.2%-59.7%), ß-lactam/ß-lactamase inhibitor combinations (93.6%-98.1% vs 10.7%-71.8%), and both meropenem and ceftazidime-avibactam (96.7% vs 5.0%-||45.0%) or |ceftolozane-tazobactam (98.4% vs 8.1%-54.8%), respectively. Cefiderocol and sulbactam-durlobactam susceptibilities were high against Acinetobacter spp. (92.4% and 97.0%) and meropenem-resistant Acineto|bacter |spp. (n = 227; 85.0% and 93.8%) but lower against sulbactam-durlobactam- (n |= 15; 13.3%) and cefiderocol- (n = 38; 65.8%) resistant isolates, respectively. Among meropenem-resistant P. aeruginosa and Acinetobacter spp., the most common ß-||lactamase genes were metallo-ß-lactamases [30/139; blaVIM-2 (15/139)] and oxacillinases [215/227; blaOXA-23 (194/227)], respectively. Acquired ß-lactamase genes were identified in 1/10 and 32/38 of cefiderocol-resistant P. aeruginosa and Acinetobacter spp., and pirA-like or piuA mutations in 10/10 and 37/38, respectively. Conclusion: cefiderocol susceptibility was high against P. aeruginosa and Acinetobacter spp., including meropenem-resistant isolates and those resistant to recent ß-lactam/ß-lactamase inhibitor combinations common in first-line treatment of European non-fermenters. IMPORTANCE: This was the first study in which the in vitro activity of cefiderocol and non-licensed ß-lactam/ß-lactamase inhibitor combinations were directly compared against Pseudomonas aeruginosa and Acinetobacter spp., including meropenem- and ß-lactam/ß-lactamase inhibitor combination-resistant isolates. A notably large number of European isolates were collected. Meropenem resistance was defined according to the MIC breakpoint for high-dose meropenem, ensuring that data reflect antibiotic activity against isolates that would remain meropenem resistant in the clinic. Cefiderocol susceptibility was high against non-fermenters, and there was no apparent cross resistance between cefiderocol and ß-lactam/ß-lactamase inhibitor combinations, with the exception of sulbactam-durlobactam. These results provide insights into therapeutic options for infections due to resistant P. aeruginosa and Acinetobacter spp. and indicate how early susceptibility testing of cefiderocol in parallel with ß-lactam/ß-lactamase inhibitor combinations will allow clinicians to choose the effective treatment(s) from all available options. This is particularly important as current treatment options against non-fermenters are limited.

In vitro activity of human defensins HNP-1 and hBD-3 against multidrug-resistant ESKAPE Gram-negatives of clinical origin and selected peptidoglycan recycling-defective mutants.

The use of immune compounds as antimicrobial adjuvants is a classic idea recovering timeliness in the current antibiotic resistance scenario. However, the activity of certain antimicrobial peptides against ESKAPE Gram-negatives has not been sufficiently investigated. The objective of this study was to determine the activities of human defensins HNP-1 and hBD-3 alone or combined with permeabilizing/peptidoglycan-targeting agents against clinical ESKAPE Gram-negatives [Acinetobacter baumannii (AB), Enterobacter cloacae (EC), Klebsiella pneumoniae (KP), and acute/chronic Pseudomonas aeruginosa (PA)]. Lethal concentrations (LCs) of HNP-1 and hBD-3 were determined in four collections of multidrug resistant EC, AB, KP, and PA clinical strains (10-36 isolates depending on the collection). These defensins act through membrane permeabilization plus peptidoglycan building blockade, enabling that alterations in peptidoglycan recycling may increase their activity, which is why different recycling-defective mutants were also included. Combinations with physiological lysozyme and subinhibitory colistin for bactericidal activities determination, and with meropenem for minimum inhibitory concentrations (MICs), were also assessed. HNP-1 showed undetectable activity (LC > 32 mg/L for all strains). hBD-3 showed appreciable activities: LC ranges 2-16, 8-8, 8->32, and 8->32 mg/L for AB, EC, KP, and PA, being PA strains from cystic fibrosis significantly more resistant than acute origin ones. None of the peptidoglycan recycling-defective mutants showed greater susceptibility to HNP-1/hBD-3. Combination with colistin or lysozyme did not change their bactericidal power, and virtually neither did meropenem + hBD-3 compared to meropenem MICs. This is the first study comparatively analyzing the HNP-1/hBD-3 activities against the ESKAPE Gram-negatives, and demonstrates interesting bactericidal capacities of hBD-3 mostly against AB and EC. IMPORTANCE: In the current scenario of critical need for new antimicrobials against multidrug-resistant bacteria, all options must be considered, including classic ideas such as the use of purified immune compounds. However, information regarding the activity of certain human defensins against ESKAPE Gram-negatives was incomplete. This is the first study comparatively assessing the in vitro activity of two membrane-permeabilizing/peptidoglycan construction-blocking defensins (HNP-1 and hBD-3) against relevant clinical collections of ESKAPE Gram-negatives, alone or in combination with permeabilizers, additional peptidoglycan-targeting attacks, or the blockade of its recycling. Our data suggest that hBD-3 has a notable bactericidal activity against multidrug-resistant Acinetobacter baumannii and Enterobacter cloacae strains that should be considered as potential adjuvant option. Our results suggest for the first time an increased resistance of Pseudomonas aeruginosa strains from chronic infection compared to acute origin ones, and provide new clues about the predominant mode of action of hBD-3 against Gram-negatives (permeabilization rather than peptidoglycan-targeting).

Transferable AmpCs in Klebsiella pneumoniae: interplay with peptidoglycan recycling, mechanisms of hyperproduction, and virulence implications.

Chromosomal and transferable AmpC ß-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to ß-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most ß-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable ß-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC ß-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for ß-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.

Activity of cefiderocol and innovative ß-lactam/ß-lactamase inhibitor combinations against isogenic strains of Escherichia coli expressing single and double ß-lactamases under high and low permeability conditions.

OBJECTIVES: To analyse the impact of the most clinically relevant ß-lactamases and their interplay with low outer membrane permeability on the activity of cefiderocol, ceftazidime/avibactam, aztreonam/avibactam, cefepime/enmetazobactam, cefepime/taniborbactam, cefepime/zidebactam, imipenem/relebactam, meropenem/vaborbactam, meropenem/xeruborbactam and meropenem/nacubactam against recombinant Escherichia coli strains. METHODS: We constructed 82 E. coli laboratory transformants expressing the main ß-lactamases circulating in Enterobacterales (70 expressing single ß-lactamase and 12 producing double carbapenemase) under high (E. coli TG1) and low (E. coli HB4) permeability conditions. Antimicrobial susceptibility testing was determined by reference broth microdilution. RESULTS: Aztreonam/avibactam, cefepime/zidebactam, cefiderocol, meropenem/xeruborbactam and meropenem/nacubactam were active against all E. coli TG1 transformants. Imipenem/relebactam, meropenem/vaborbactam, cefepime/taniborbactam and cefepime/enmetazobactam were also highly active, but unstable against most of MBL-producing transformants. Combination of ß-lactamases with porin deficiency (E. coli HB4) did not significantly affect the activity of aztreonam/avibactam, cefepime/zidebactam, cefiderocol or meropenem/nacubactam, but limited the effectiveness of the rest of carbapenem- and cefepime-based combinations. Double-carbapenemase production resulted in the loss of activity of most of the compounds tested, an effect particularly evident for those E. coli HB4 transformants in which MBLs were present. CONCLUSIONS: Our findings highlight the promising activity that cefiderocol and new ß-lactam/ß-lactamase inhibitors have against recombinant E. coli strains expressing widespread ß-lactamases, including when these are combined with low permeability or other enzymes. Aztreonam/avibactam, cefiderocol, cefepime/zidebactam and meropenem/nacubactam will help to mitigate to some extent the urgency of new compounds able to resist MBL action, although NDM enzymes represent a growing challenge against which drug development efforts are still needed.

Filling knowledge gaps related to AmpC-dependent ß-lactam resistance in Enterobacter cloacae.

Enterobacter cloacae starred different pioneer studies that enabled the development of a widely accepted model for the peptidoglycan metabolism-linked regulation of intrinsic class C cephalosporinases, highly conserved in different Gram-negatives. However, some mechanistic and fitness/virulence-related aspects of E. cloacae choromosomal AmpC-dependent resistance are not completely understood. The present study including knockout mutants, ß-lactamase cloning, gene expression analysis, characterization of resistance phenotypes, and the Galleria mellonella infection model fills these gaps demonstrating that: (i) AmpC enzyme does not show any collateral activity impacting fitness/virulence; (ii) AmpC hyperproduction mediated by ampD inactivation does not entail any biological cost; (iii) alteration of peptidoglycan recycling alone or combined with AmpC hyperproduction causes no attenuation of E. cloacae virulence in contrast to other species; (iv) derepression of E. cloacae AmpC does not follow a stepwise dynamics linked to the sequential inactivation of AmpD amidase homologues as happens in Pseudomonas aeruginosa; (v) the enigmatic additional putative AmpC-type ß-lactamase generally present in E. cloacae does not contribute to the classical cephalosporinase hyperproduction-based resistance, having a negligible impact on phenotypes even when hyperproduced from multicopy vector. This study reveals interesting particularities in the chromosomal AmpC-related behavior of E. cloacae that complete the knowledge on this top resistance mechanism.

A randomized controlled mHealth trial that evaluates social comparison-oriented gamification to improve physical activity, sleep quantity, and quality of life in young adults.

INTRODUCTION: The integration of gamification in mHealth interventions presents a novel approach to enhance user engagement and health outcomes. This study aims to evaluate whether comparison-oriented gamification can effectively improve various aspects of health and well-being, including physical activity, sedentary behavior, sleep, and overall quality of life among young adults. METHODS: Potential 107 young adults (from 19 to 28 years old) participated in an 8-week trial. Participants were assigned to either a gamified mHealth intervention (LevantApp) with daily leaderboards and progress bars (n = 53, 26 % dropped-out), or a control condition without gamification (n = 52, 29 % dropped-out). Physical activity (number of steps, moderate and moderate-to-vigorous physical activity -MVPA-) and sleep quantity were measured objectively via accelerometry and subjectively using the International Physical Activity Questionnaire(IPAQ), Pittsburgh Sleep Quality Index(PSQI), Sedentary Behavior Questionnaire(SBQ), and Short Form Health Survey(SF-36). RESULTS: This mHealth intervention with social comparison-oriented gamification significantly improved moderate physical activity to a greater extent than the control group. Additionally, the intervention group showed improvements in the number of steps, moderate physical activity, sedentary time, emotional wellbeing, and social functioning. However, no significant group by time interaction was observed. No significant differences were observed in sleep quality or quantity. CONCLUSION: s: The LevantApp gamified mHealth intervention was effective in improving moderate physical activity, physical functioning, and role-emotional in young adults. No significant effects were found on step counts, MVPA or sleep, suggesting that while gamification can enhance specific aspects of physical activity and quality of life, its impact may vary across different outcomes.

Nonlethal Molecular Nanomachines Potentiate Antibiotic Activity Against Gram-Negative Bacteria by Increasing Cell Permeability and Attenuating Efflux.

Antibiotic resistance is a pressing public health threat. Despite rising resistance, antibiotic development, especially for Gram-negative bacteria, has stagnated. As the traditional antibiotic research and development pipeline struggles to address this growing concern, alternative solutions become imperative. Synthetic molecular nanomachines (MNMs) are molecular structures that rotate unidirectionally in a controlled manner in response to a stimulus, such as light, resulting in a mechanical action that can propel molecules to drill into cell membranes, causing rapid cell death. Due to their broad destructive capabilities, clinical translation of MNMs remains challenging. Hence, here, we explore the ability of nonlethal visible-light-activated MNMs to potentiate conventional antibiotics against Gram-negative bacteria. Nonlethal MNMs enhanced the antibacterial activity of various classes of conventional antibiotics against Gram-negative bacteria, including those typically effective only against Gram-positive strains, reducing the antibiotic concentration required for bactericidal action. Our study also revealed that MNMs bind to the negatively charged phospholipids of the bacterial inner membrane, leading to permeabilization of the cell envelope and impairment of efflux pump activity following light activation of MNMs. The combined effects of MNMs on membrane permeability and efflux pumps resulted in increased antibiotic accumulation inside the cell, reversing antibiotic resistance and attenuating its development. These results identify nonlethal MNMs as pleiotropic antibiotic enhancers or adjuvants. The combination of MNMs with traditional antibiotics is a promising strategy against multidrug-resistant Gram-negative infections. This approach can reduce the amount of antibiotics needed and slow down antibiotic resistance development, thereby preserving the effectiveness of our current antibiotics.

Emergence of cefiderocol resistance during ceftazidime/avibactam treatment caused by a large genomic deletion, including ampD and piuCD genes, in Pseudomonas aeruginosa.

We report the emergence of cefiderocol resistance during the treatment of a ST312 Pseudomonas aeruginosa respiratory infection with ceftazidime/avibactam. whole genome sequencing (WGS) revealed that resistance was caused by a large genomic deletion, including PiuDC (iron transport system) and AmpD (ampC negative regulator), driven by the integration of phage DNA. Thus, our findings alert that this type of deletion could be an efficient (two mechanisms in one step) specific cefiderocol resistance mechanism that might occur nonspecifically upon treatment with ß-lactams that select for AmpC overexpression.

Prevalence of sports supplements consumption and its association with food choices among female elite football players.

OBJECTIVES: The present study aimed to 1) investigate the consumption of Sports Supplements (SSs) among female elite football players, 2) evaluate the influence of age on SS consumption, and 3) determine the relationship between the consumption of SSs and dietary choices among elite football players. METHODS: A total of 126 female football players of Primera Iberdrola and Reto Iberdrola who participated in this descriptive, observational, and cross-sectional study completed a self-administered questionnaire on SSs and the Athletes' Food Choices Questionnaire. RESULTS: Overall, 84.1% of participants consumed supplements, mainly for improved sports performance (68.3%) and health (34.1%). The main sources of purchase were the Internet (34.9%) and specialized shops (23.8%), and players were commonly advised by a dietitian-nutritionist to use SSs (56.3%). The SSs most often consumed included whey protein (30.2%), sports drinks (28.6%), creatine monohydrate (28.6%), sports bars (27.8%), and caffeine (27.8%). Older players consumed more supplements at the time of data compilation. Players predominantly acquired these supplements by using the Internet and reported benefits from their use (all P ≤ 0.036). Additionally, players who consumed SSs conveyed more concern about their food choices. CONCLUSIONS: A high prevalence of female football players consumed SSs, particularly SSs supported by robust scientific evidence. Older players had higher supplement consumption rates. The use of SSs was related to food choices through nutritional characteristics of foods, knowledge about health and nutrition, weight control with the help of food, and the performance benefits players could acquire.

Deciphering mechanisms affecting cefepime-taniborbactam in vitro activity in carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas spp. isolates recovered during a surveillance study in Spain.

PURPOSE: To characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates. METHODS: CPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing. RESULTS: A reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers. CONCLUSIONS: The reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

Pseudomonasaeruginosa antimicrobial susceptibility profiles, resistance mechanisms and international clonal lineages: update from ESGARS-ESCMID/ISARPAE Group.

SCOPE: Pseudomonas aeruginosa, a ubiquitous opportunistic pathogen considered one of the paradigms of antimicrobial resistance, is among the main causes of hospital-acquired and chronic infections associated with significant morbidity and mortality. This growing threat results from the extraordinary capacity of P. aeruginosa to develop antimicrobial resistance through chromosomal mutations, the increasing prevalence of transferable resistance determinants (such as the carbapenemases and the extended-spectrum ß-lactamases), and the global expansion of epidemic lineages. The general objective of this initiative is to provide a comprehensive update of P. aeruginosa resistance mechanisms, especially for the extensively drug-resistant (XDR)/difficult-to-treat resistance (DTR) international high-risk epidemic lineages, and how the recently approved ß-lactams and ß-lactam/ß-lactamase inhibitor combinations may affect resistance mechanisms and the definition of susceptibility profiles. METHODS: To address this challenge, the European Study Group for Antimicrobial Resistance Surveillance (ESGARS) from the European Society of Clinical Microbiology and Infectious Diseases launched the 'Improving Surveillance of Antibiotic-Resistant Pseudomonas aeruginosa in Europe (ISARPAE)' initiative in 2022, supported by the Joint programming initiative on antimicrobial resistance network call and included a panel of over 40 researchers from 18 European Countries. Thus, a ESGARS-ISARPAE position paper was designed and the final version agreed after four rounds of revision and discussion by all panel members. QUESTIONS ADDRESSED IN THE POSITION PAPER: To provide an update on (a) the emerging resistance mechanisms to classical and novel anti-pseudomonal agents, with a particular focus on ß-lactams, (b) the susceptibility profiles associated with the most relevant ß-lactam resistance mechanisms, (c) the impact of the novel agents and resistance mechanisms on the definitions of resistance profiles, and (d) the globally expanding XDR/DTR high-risk lineages and their association with transferable resistance mechanisms. IMPLICATION: The evidence presented herein can be used for coordinated epidemiological surveillance and decision making at the European and global level.

To update or not to update the ESCMID guidelines for the diagnosis and treatment of biofilm infections - That is the question! The opinion of the ESGB board.

Background: The work on the ESGB guidelines for diagnosis and treatment of biofilm infections began in 2012 and the result was published in 2014. The guidelines have been and still are frequently cited in the literature proving its usefulness for people working with biofilm infections. At the ESGB Biofilm conference in Mallorca 2022 (Eurobiofilms2022) the board of the ESGB decided to evaluate the 2014-guidelines and relevant publications since 2014 based on a lecture given at the Eurobiofilms2022. Guideline methods: The Delphi method for working on production of guidelines and the current ESCMID rules for guidelines are presented. The criteria for evaluation of relevant literature are very strict and especially for treatment, most clinicians and regulatory authorities require convincing results from Level I (randomized controlled trials) publications to justify changes of treatments. The relevant new biofilm literature and the relevant biofilm presentations from the Eurobiofilms meetings and ECCMID conferences was used for evaluating the contemporary relevance of the ESGB 2014 guidelines. Diagnosis of biofilm infections: Several infectious diseases have been recognized as biofilm infections since 2014, but the diagnostic methods and therapeutic strategies are still the same as recommended in the 2014 ESGB guidelines which are summarized in this opinion paper. Treatment of biofilm infections: Some promising new in vitro and in vivo (animal experiments) observations and reports for therapy of biofilm infections are mentioned, but they still await clinical trials. Conclusion: The interim opinion at the present time (2022) is therefore, that the guidelines do not need revision now, but there is a need for survey articles discussing new methods of diagnosis and treatment of biofilm infections in order - hopefully - to give inspiration to conduct clinical trials which may lead to progress in diagnosis and treatment of patients with biofilm infections.

Corrigendum: CARB-ES-19 multicenter study of carbapenemase-producing Klebsiella pneumoniae and Escherichia coli from all Spanish provinces reveals interregional spread of high-risk clones such as ST307/OXA-48 and ST512/KPC-3.

[This corrects the article DOI: 10.3389/fmicb.2022.918362.].

Enhancing early functional independence following cardiac surgery: a quality improvement programme.

Early mobility and activity programmes following cardiac surgery are vital for improved patient outcomes, as they accelerate the recovery of functional capacity and walking distance. We observed that only 5.3% of our patients achieved a Functional Independence Measurement (FIM) score of 80% or more by the third postoperative day (POD). Additionally, the average 6-minute walk distance achieved by the fourth POD was only 188 m. Therefore, a quality improvement (QI) project was implemented with the aim of attaining a FIM score of 80% by the third POD for more than 80% of patient underwent/undergoing cardiac surgery without complications.A model-for-improvement framework was used to drive continuous improvement. This project was implemented in February 2021. Baseline data were prospectively collected between November 2020 and January 2021 (preintervention). Outcomes were analysed using standard control chart rules to detect changes over time. Unpaired Student t-tests assessed significant differences in mean levels between two groups, (preintervention vs postintervention).χ2 tests were conducted between the two groups according to gender and patient satisfaction scores.The percentage of patients who achieved a FIM score of 80% or more by the third POD gradually increased to 91.4% 5 months following programme implementation and was sustained thereafter. The mean patient FIM score significantly improved to 81.20±3.77 (p<0.001) by the third POD. Similarly, the mean 6-minute walk distance increased to 267.90±36.10 m (p<0.001) by the fourth POD. The percentage of patients who displayed the level of confidence needed to carry out activities of daily living (ADL) and exercises independently at home increased to 89.4% (p<0.001) by the fifth POD. No adverse events associated with the mobility and activity programme were reported.This QI project demonstrated a substantial improvement in patient functional independence, walking distance and the level of confidence needed to independently carry out ADL and exercises following cardiac surgery.

A paper biosensor for overcoming matrix effects interfering with the detection of sputum pyocyanin with competitive immunoassays.

Detecting sputum pyocyanin (PYO) with a competitive immunoassay is a promising approach for diagnosing Pseudomonas aeruginosa respiratory infections. However, it is not possible to perform a negative control to evaluate matrix-effects in competitive immunoassays, and the highly complex sputum matrix often interferes with target detection. Here, we show that these issues are alleviated by performing competitive immunoassays with a paper biosensor. The biosensing platform consists of a paper reservoir, which contains antibody-coated gold nanoparticles, and a substrate containing a competing recognition element, which is a piece of paper modified with an albumin-antigen conjugate. Detection of PYO with a limit of detection of 4.7·10-3 µM and a dynamic range between 4.7·10-1 µM and 47.6 µM is accomplished by adding the sample to the substrate with the competing element and pressing the reservoir against it for 5 min. When tested with patient samples, the biosensor was able to qualitatively differentiate spiked from non-spiked samples, whereas ELISA did not show a clear cut-off between them. Furthermore, the relative standard deviation was lower when determining sputum with the paper-based biosensor. These features, along with a mild liquefaction step that circumvents the use of harsh chemicals or instruments, make our biosensor a good candidate for diagnosing Pseudomonas infections at the bedside through the detection of sputum PYO.

The balance between antibiotic resistance and fitness/virulence in Pseudomonas aeruginosa: an update on basic knowledge and fundamental research.

The interplay between antibiotic resistance and bacterial fitness/virulence has attracted the interest of researchers for decades because of its therapeutic implications, since it is classically assumed that resistance usually entails certain biological costs. Reviews on this topic revise the published data from a general point of view, including studies based on clinical strains or in vitro-evolved mutants in which the resistance phenotype is seen as a final outcome, i.e., a combination of mechanisms. However, a review analyzing the resistance/fitness balance from the basic research perspective, compiling studies in which the different resistance pathways and respective biological costs are individually approached, was missing. Here we cover this gap, specifically focusing on Pseudomonas aeruginosa, a pathogen that stands out because of its extraordinary capacity for resistance development and for which a considerable number of recent and particular data on the interplay with fitness/virulence have been released. The revised information, split into horizontally-acquired vs. mutation-driven resistance, suggests a great complexity and even controversy in the resistance-fitness/virulence balance in the acute infection context, with results ranging from high costs linked to certain pathways to others that are seemingly cost-free or even cases of resistance mechanisms contributing to increased pathogenic capacities. The elusive mechanistic basis for some enigmatic data, knowledge gaps, and possibilities for therapeutic exploitation are discussed. The information gathered suggests that resistance-fitness/virulence interplay may be a source of potential antipseudomonal targets and thus, this review poses the elementary first step for the future development of these strategies harnessing certain resistance-associated biological burdens.

Mutation Analysis in Regulator DNA-Binding Regions for Antimicrobial Efflux Pumps in 17,000 Pseudomonas aeruginosa Genomes.

Mutations leading to upregulation of efflux pumps can produce multiple drug resistance in the pathogen Pseudomonas aeruginosa. Changes in their DNA binding regions, i.e., palindromic operators, can compromise pump depression and subsequently enhance resistance against several antibacterials and biocides. Here, we have identified (pseudo)palindromic repeats close to promoters of genes encoding 13 core drug-efflux pumps of P. aeruginosa. This framework was applied to detect mutations in these repeats in 17,292 genomes. Eighty-nine percent of isolates carried at least one mutation. Eight binary genetic properties potentially related to expression were calculated for mutations. These included palindromicity reduction, mutation type, positioning within the repeat and DNA-bending shift. High-risk ST298, ST308 and ST357 clones commonly carried four conserved mutations while ST175 and the cystic fibrosis-linked ST649 clones showed none. Remarkably, a T-to-C transition in the fourth position of the upstream repeat for mexEF-oprN was nearly exclusive of the high-risk ST111 clone. Other mutations were associated with high-risk sublineages using sample geotemporal metadata. Moreover, 1.5% of isolates carried five or more mutations suggesting they undergo an alternative program for regulation of their effluxome. Overall, P. aeruginosa shows a wide range of operator mutations with a potential effect on efflux pump expression and antibiotic resistance.