BACKGROUND:Bloodstream infections caused by bacteria with carbapenem resistance are associated with high mortality. Cefiderocol has broad in vitro activity against carbapenem-resistant Gram-negative bacilli. We aimed to assess whether cefiderocol was non-inferior or superior to standard-of-care antibiotics in preventing mortality in patients with bloodstream infection caused by Gram-negative bacilli. METHODS:The GAME CHANGER trial was an open-label, parallel-group, randomised clinical trial in which patients with health-care-associated or hospital-acquired Gram-negative bloodstream infection were recruited from 17 tertiary hospitals with more than 500 beds in Australia, Malaysia, Singapore, Taiwan, Thailand, and Türkiye. Adult patients (aged ≥21 years in Singapore or ≥18 years elsewhere) with positive blood cultures and Gram-negative bacilli seen on Gram stain were eligible. Participants were randomly assigned in a 1:1 ratio to cefiderocol (2 g intravenously every 8 h) or standard of care (chosen by the patient's clinical team) using an online randomisation tool with strata defined by severity of comorbidities and region, with random permuted blocks of unequal size. Patients, treating clinicians, and site investigators were not masked to treatment. The primary outcome was all-cause mortality at 14 days after randomisation. All outcomes were analysed in participants who received at least one dose of assigned antibiotic, grew an aerobic Gram-negative bacillus from the index blood culture, and did not withdraw consent before day 14. The non-inferiority margin was 10%; superiority was to be assessed if non-inferiority was shown, and was evaluated in both the main analysis population and in patients with at least one carbapenem-resistant organism causing bloodstream infection. Missing data were handled by imputing data. The trial was registered on ClinicalTrials.gov (NCT03869437) and is closed to new participants. FINDINGS:513 participants were enrolled between Nov 12, 2019, and Oct 31, 2023 (210 [42%] female and 294 [58%] male). 256 patients were randomly assigned to cefiderocol and 257 were assigned to standard of care. Nine patients (six in the cefiderocol group and three in the standard-of-care group) were excluded from further analysis because they withdrew consent or their blood cultures did not grow an aerobic Gram-negative bacillus. Thus, 504 participants were included in the main analysis population. 20 (8%) of 250 patients in the cefiderocol group had died at 14 days compared with 17 (7%) of 254 patients in the standard-of-care group (absolute risk difference 1%, 95% CI -3 to 6). 127 (25%) of 504 patients had an infection with a carbapenem-resistant organism; at 14 days, nine (14%) of 64 patients in the cefiderocol group had died compared with six (10%) of 63 patients in the standard-of-care group (5%, -7 to 16). There were five treatment-emergent serious adverse events that were either probably or possibly related to the study drug (all in the cefiderocol group): delirium, stupor, rigors, abnormal liver chemistry, and rash, all of which resolved without treatment (except for the rash, which required hydrocortisone and anti-histamines). INTERPRETATION:Among patients with a hospital-acquired or health-care-associated Gram-negative bloodstream infection, cefiderocol resulted in non-inferior 14-day mortality compared with standard of care. Adverse events were similar between groups, although those thought to be related to the study drug only occurred in the cefiderocol group. In both the main analysis population and the carbapenem-resistant subset, cefiderocol was not superior to standard of care. This evidence suggests that cefiderocol is efficacious in patients with health-care-associated Gram-negative bloodstream infection who are at high risk of antibiotic resistance, but more evidence is required to define its efficacy when carbapenem-resistant organisms are the cause. FUNDING:The University of Queensland, Shionogi, The Henderson Foundation, and the National Medical Research Council (Singapore).

Carbapenem-resistant Enterobacterales pose a critical global health threat, exemplified by increasing resistance of uropathogenic E. coli (UPEC) that cause urinary tract infections (UTIs). Here, we investigate the publicly available EnteroBase dataset and identify a signal of increasing UTI caused by phylogroup A E. coli sequence type 167 (ST167). Phylogenetic analysis of ST167 based on whole genome sequence data reveal three major clades (A, B, C), with clade C further resolving into several subclades, notably subclade C2 that possessed high carriage rates of carbapenem and cephalosporin resistance genes. Hierarchical clustering of core genome multi-locus sequence typing reveals ~77% of subclade C2 strains contain <20 allelic differences in 2,513 core genes and harbour two distinct group 1 capsule types, KL124 and KL30, likely originating from Klebsiella. Subclade C2 was predominantly sequenced in North America, and we provide evidence for clonal expansion since 2016. Analysis of UPEC virulence factors reveals complete loss of the type 1 fimbriae genes in strains from clades B and C. Two subclade C2 isolates exhibit significantly reduced capacity to colonise the bladder compared to the reference UPEC strain CFT073 in a murine UTI model. Collectively, our data identifies ST167 as an atypical UPEC clone associated with UTI.

Abstract Background Sleep disruption imposes deleterious physiological and psychological effects. We aimed to describe the relationship between school age respiratory health on adolescent sleep disturbance and mental health symptoms in people living with cystic fibrosis. Methods We conducted a prospective longitudinal study of adolescents aged 10 to 18 years from October 2020 to June 2024. Sleep was evaluated via serial measures of the pediatric daytime sleepiness scale (PDSS) and sleep disturbance scale for children (SDSC), while mental health was assessed with the Patient Health Questionnaire-9 (PHQ-9) for depression and Generalised Anxiety Disorder-7 (GAD-7) for anxiety. Respiratory health was measured alongside, using FEV1 percent predicted and multiple breath washout (MBW). Childhood chest CT scan were previously collected via DEFEND CF (Biomarkers to define the treatment end-points for pulmonary exacerbations in CF) and scored using the Perth-Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF). Linear regression was used to estimate mean difference in sleep and mental health change per school age CT bronchiectasis score. Results 64 adolescents were studied. 56% of participants experienced sleep disturbance symptoms, 45% for mental health symptoms, with no significant association between childhood bronchiectasis score and adolescent PDSS, SDSC scores. Adolescent anxiety and depression scores also did not significantly correlate with childhood bronchiectasis scores. Conclusion While childhood bronchiectasis score did not predict adolescent sleep or mental health, sleep disturbance and symptoms of anxiety and depression were prevalent in the cohort. The need for ongoing monitoring and support in these areas remains crucial for adolescents with cystic fibrosis.

Background. Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, Pseudomonas aeruginosa . Objectives. We aimed to develop two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I ( gyrA 248) and GyrA D87N, D87Y and D87H ( gyrA 259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa . Methods. Following assay validation, the gyrA 248 and gyrA 259 SYBR-MAMAs were tested on 84 Australian clinical P. aeruginosa isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing. Results. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes. Conclusions. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa . An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.

Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterized by increasingly frequent acute pulmonary exacerbations that reduce life quality and length. Human airways are home to a rich polymicrobial environment, which includes members of the obligately anaerobic genus Prevotella . Despite their commonness, surprisingly little is known about the prevalence, role, genomic diversity and antimicrobial resistance (AMR) potential of Prevotella species and strains in healthy and diseased airways. Here, we used comparative genomics to develop a real-time PCR assay to permit rapid Prevotella species identification and quantification from cultures and clinical specimens. Assay specificity was validated across a panel of Prevotella and non- Prevotella species, followed by PCR screening of CF and COPD respiratory-derived cultures. Next, 35 PCR-positive isolates were subjected to whole-genome sequencing. Of eight identified Prevotella species, P. histicola , P. melaninogenica , P. nanceiensis , P. salivae and P. denticola overlapped between participant cohorts. Phylogenomic analysis revealed considerable interhost but limited intrahost diversity, suggesting patient-specific lineages in the lower airways, probably from oral cavity aspirations. Correlation of phenotypic AMR profiles with AMR genes identified excellent correlation between tetQ presence and decreased doxycycline susceptibility, and ermF presence and decreased azithromycin susceptibility and clindamycin resistance. AMR rates were higher in the CF isolates, reflecting greater antibiotic use in this cohort. All tested Prevotella isolates were tobramycin-resistant, providing a potential selection method to improve Prevotella culture retrieval rates. Our addition of 35 airway-derived Prevotella genomes to public databases will enhance ongoing efforts to unravel the role of this diverse and enigmatic genus in both diseased and healthy lungs.

The rise of antimicrobial-resistant (AMR) bacteria is a global health emergency. One critical facet of tackling this epidemic is more rapid AMR diagnosis in serious multidrug-resistant pathogens like Pseudomonas aeruginosa .

ABSTRACTDefective OprD porins contribute to carbapenem resistance and may be important in Pseudomonas aeruginosa adaptation to cystic fibrosis airways. It is unclear whether oprD mutations are fixed in populations of shared strains that are transmitted between patients or whether novel variants arise during infection. We investigated oprD sequences and antimicrobial resistance of two common Australian shared strains, constructed P. aeruginosa mutants with the most common oprD allelic variants and compared characteristics between patients with or without evidence of infection with strains harbouring these variants. Our data show that three independently acquired nonsense mutations arising from a 1-base pair substitution are fixed in strain sub-lineages. These nonsense mutations are likely to contribute to reduced carbapenem susceptibility in the sub-lineages without compromising in vitro fitness. Not only was lung function worse among patients infected with strains harbouring the nonsense mutations than those without, but they also had an increased hazard rate of lung transplantation/death. Our findings further highlight that understanding adaptive changes may help to distinguish patients with greater adverse outcomes despite infection with the same strain.

Pseudomonas aeruginosa chronically infects in the lungs of people with cystic fibrosis and other forms of lung disease. Infections are treated with antibiotics, but over time, the bacteria acquire mutations that reduce their antibiotic susceptibility. The effects of inhibitory amounts of antibiotics in selecting for antibiotic-resistant mutants have been well studied. However, the concentrations of antibiotics that reach infecting bacteria can be sub-inhibitory and but may nonetheless promote emergence of antibiotic-resistant bacteria. Therefore, the aim of this research was to investigate the effects of sub-inhibitory concentrations of antibiotics on the antibiotic susceptibility of P. aeruginosa. Two P. aeruginosa reference strains, PAO1 and PA14, and six isolates from individuals with cystic fibrosis were studied. The bacteria were passaged in the presence of antibiotics (ceftazidime, ciprofloxacin, meropenem or tobramycin) at sub-inhibitory amounts. Fifteen populations of bacteria (up to five per strain) were exposed to each of the four antibiotics. Antibiotic susceptibility was determined following 10 passages on agar supplemented with antibiotic and compared with susceptibility prior to antibiotic exposure. Antibiotic exposure resulted in susceptibility being significantly (>2-fold) reduced for 13 of the 60 populations. Seven samples had reduced susceptibility to ciprofloxacin, three to tobramycin, two to ceftazidime and one to meropenem. Whole-genome sequencing revealed the mutations arising following antibiotic exposure. Mutants with reduced antibiotic susceptibility had mutations in genes known to affect antibiotic resistance, including regulators of efflux pumps (mexR, mexS, mexZ and nalC) and the fusA1 gene that is associated with aminoglycoside resistance. Genes not previously associated with resistance, including gacS, sigX and crfX and two genes with no known function, were also mutated in some isolates with reduced antibiotic susceptibility. Our results show that exposure to sub-inhibitory amounts of antibiotics can select for mutations that reduce the susceptibility of P. aeruginosa to antibiotics and that the profile of mutations is different from that arising during selection with inhibitory antibiotic concentrations. It is likely that exposure to sub-inhibitory amounts of antibiotics during infection contributes to P. aeruginosa becoming antibiotic-resistant.

Patient-derived isolates of the opportunistic pathogen Pseudomonas aeruginosa are frequently resistant to antibiotics due to the presence of sequence variants in resistance-associated genes. However, the frequency of antibiotic resistance and of resistance-associated sequence variants in environmental isolates of P. aeruginosa has not been well studied. Antimicrobial susceptibility testing (ciprofloxacin, ceftazidime, meropenem, tobramycin) of environmental (n=50) and cystic fibrosis (n=42) P. aeruginosa isolates was carried out. Following whole genome sequencing of all isolates, 25 resistance-associated genes were analysed for the presence of likely function-altering sequence variants. Environmental isolates were susceptible to all antibiotics with one exception, whereas patient-derived isolates had significant frequencies of resistance to each antibiotic and a greater number of likely resistance-associated genetic variants. These findings indicate that the natural environment does not act as a reservoir of antibiotic-resistant P. aeruginosa, supporting a model in which antibiotic susceptible environmental bacteria infect patients and develop resistance during infection.

TITLE: Prevalence and impact of oprD mutations in Pseudomonas aeruginosa strains in 1 cystic fibrosis 2 3 Laura J. Sherrard, Bryan A. Wee, Christine Duplancic, Kay A. Ramsay, Keyur A. 4 Dave, Emma Ballard, Claire E. Wainwright, Keith Grimwood, Hanna E. Sidjabat, 5 David M. Whiley, Scott A. Beatson, Timothy J. Kidd†, Scott C. Bell† 6 7 1 QIMR Berghofer Medical Research Institute, Brisbane, QLD 4006, Australia 8 2 Present address: School of Pharmacy, Queen’s University Belfast, Belfast BT9 7BL, United 9 Kingdom 10 3 School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 11 QLD 4072, Australia 12 4 Present address: Usher Institute of Population Health Sciences & Informatics, University of 13 Edinburgh, Edinburgh EH16 4UX, United Kingdom 14 5 Faculty of Medicine, The University of Queensland, Brisbane, QLD 4006, Australia 15 6 Present address: Department of Biochemistry, University of Otago, Dunedin 9016, New 16 Zealand 17 7 Present address: Genome stability Laboratory, Cancer and Ageing Research Program, 18 Institute of Health and Biomedical Innovation, Translational Research Institute, Queensland 19 University of Technology, QLD 4102, Australia 20 8 Department of Respiratory and Sleep Medicine, Queensland Children's Hospital, Brisbane, 21 QLD 4101, Australia 22 9 School of Medicine and Menzies Health Institute Queensland, Griffith University, Gold 23 Coast QLD 4222, Australia 24

Background and objective Aerosol transmission of Pseudomonas aeruginosa has been suggested as a possible mode of respiratory infection spread in patients with cystic fibrosis (CF); however, whether this occurs in other suppurative lung diseases is unknown. Therefore, we aimed to determine if (i) patients with bronchiectasis (unrelated to CF) or chronic obstructive pulmonary disease (COPD) can aerosolize P. aeruginosa during coughing and (ii) if genetically indistinguishable (shared) P. aeruginosa strains are present in these disease cohorts. Methods People with bronchiectasis or COPD and P. aeruginosa respiratory infection were recruited for two studies. Aerosol study: Participants (n = 20) underwent cough testing using validated cough rigs to determine the survival of P. aeruginosa aerosols in the air over distance and duration. Genotyping study: P. aeruginosa sputum isolates (n = 95) were genotyped using the iPLEX20SNP platform, with a subset subjected to the enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) assay to ascertain their genetic relatedness. Results Aerosol study: Overall, 7 of 20 (35%) participants released P. aeruginosa cough aerosols during at least one of the cough aerosol tests. These cough aerosols remained viable for 4 m from the source and for 15 min after coughing. The mean total aerosol count of P. aeruginosa at 2 m was two colony-forming units. Typing study: No shared P. aeruginosa strains were identified. Conclusion Low viable count of P. aeruginosa cough aerosols and a lack of shared P. aeruginosa strains observed suggest that aerosol transmission of P. aeruginosa is an unlikely mode of respiratory infection spread in patients with bronchiectasis and COPD.

A Pseudomonas aeruginosa AUST-02 strain sub-type (M3L7) has been identified in Australia, infects the lungs of some people with cystic fibrosis and is associated with antibiotic resistance. Multiple clonal lineages may emerge during treatment with mutations in chromosomally encoded antibiotic resistance genes commonly observed. Here we describe the within-host diversity and antibiotic resistance of M3L7 during and after antibiotic treatment of an acute pulmonary exacerbation using whole genome sequencing and show both variation and shared mutations in important genes. Eleven isolates from an M3L7 population (n = 134) isolated over 3 months from an individual with cystic fibrosis underwent whole genome sequencing. A phylogeny based on core genome SNPs identified three distinct phylogenetic groups comprising two groups with higher rates of mutation (hypermutators) and one non-hypermutator group. Genomes were screened for acquired antibiotic resistance genes with the result suggesting that M3L7 resistance is principally driven by chromosomal mutations as no acquired mechanisms were detected. Small genetic variations, shared by all 11 isolates, were found in 49 genes associated with antibiotic resistance including frame-shift mutations (mexA, mexT), premature stop codons (oprD, mexB) and mutations in quinolone-resistance determining regions (gyrA, parE). However, whole genome sequencing also revealed mutations in 21 genes that were acquired following divergence of groups, which may also impact the activity of antibiotics and multi-drug efflux pumps. Comparison of mutations with minimum inhibitory concentrations of anti-pseudomonal antibiotics could not easily explain all resistance profiles observed. These data further demonstrate the complexity of chronic and antibiotic resistant P. aeruginosa infection where a multitude of co-existing genotypically diverse sub-lineages might co-exist during and after intravenous antibiotic treatment.

The lungs of individuals with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa that is difficult to eradicate by antibiotic treatment. Two key P. aeruginosa antibiotic resistance mechanisms are the AmpC β-lactamase that degrades β-lactam antibiotics and MexXYOprM, a three-protein efflux pump that expels aminoglycoside antibiotics from the bacterial cells. Levels of antibiotic resistance gene expression are likely to be a key factor in antibiotic resistance but have not been determined during infection. The aims of this research were to investigate the expression of the ampC and mexX genes during infection in patients with CF and in bacteria isolated from the same patients and grown under laboratory conditions. P. aeruginosa isolates from 36 CF patients were grown in laboratory culture and gene expression measured by reverse transcription-quantitative PCR (RT-qPCR). The expression of ampC varied over 20,000-fold and that of mexX over 2,000-fold between isolates. The median expression levels of both genes were increased by the presence of subinhibitory concentrations of antibiotics. To measure P. aeruginosa gene expression during infection, we carried out RT-qPCR using RNA extracted from fresh sputum samples obtained from 31 patients. The expression of ampC varied over 4,000-fold, while mexX expression varied over 100-fold, between patients. Despite these wide variations, median levels of expression of ampC in bacteria in sputum were similar to those in laboratory-grown bacteria. The expression of mexX was higher in sputum than in laboratory-grown bacteria. Overall, our data demonstrate that genes that contribute to antibiotic resistance can be highly expressed in patients, but there is extensive isolate-to-isolate and patient-to-patient variation.

Background Chronic lung infections by Pseudomonas aeruginosa are a significant cause of morbidity and mortality in people with cystic fibrosis (CF). Shared P. aeruginosa strains, that can be transmitted between patients, are of concern and in Australia the AUST-02 shared strain is predominant in individuals attending CF centres in Queensland and Western Australia. M3L7 is a multidrug resistant sub-type of AUST-02 that was recently identified in a Queensland CF centre and was shown to be associated with poorer clinical outcomes. The main aim of this study was to resolve the relationship of the emergent M3L7 sub-type within the AUST-02 group of strains using whole genome sequencing. Results A whole-genome core phylogeny of 63 isolates indicated that M3L7 is a monophyletic sub-lineage within the context of the broader AUST-02 group. Relatively short branch lengths connected all of the M3L7 isolates. A phylogeny based on nucleotide polymorphisms present across the genome showed that the chronological estimation of the most recent common ancestor was around 2001 (± 3 years). SNP differences between sequential M3L7 isolates collected 3-4 years apart from five patients suggested both continuous infection of the same strain and cross-infection of some M3L7 variants between patients. The majority of polymorphisms that were characteristic of M3L7 (i.e. acquired after divergence from all other AUST-02 isolates sequenced) were found to produce non-synonymous mutations in virulence and antibiotic resistance genes. Conclusions M3L7 has recently diverged from a common ancestor indicating descent from a single carrier at a CF treatment centre in Australia. Both adaptation to the lung and transmission of M3L7 between adults attending this centre may have contributed to its rapid dissemination. The study emphasises the importance of clinical management in controlling the emergence of shared strains in CF.

The airborne route is a potential pathway in the person-to-person transmission of bacterial strains among cystic fibrosis (CF) populations. In this cross-sectional study, we investigate the physical properties and survival of common non-Pseudomonas aeruginosa CF pathogens generated during coughing. We conclude that Gram-negative bacteria and Staphylococcus aureus are aerosolised during coughing, can travel up to 4 m and remain viable within droplet nuclei for up to 45 min. These results suggest that airborne person-to-person transmission is plausible for the CF pathogens we measured.