Changes in urinary glutathione sulfonamide (GSA) levels between admission and discharge of patients with cystic fibrosis.

There is an urgent need to develop sensitive, non-invasive biomarkers that can track airway inflammatory activity for patients with cystic fibrosis (CF). Urinary glutathione sulfonamide (GSA) levels correlate well with GSA levels in BAL samples and other markers of neutrophilic inflammation, suggesting that this biomarker may be suitable for tracking disease activity in this population. We recruited 102 children (median 11.5 years-old) and 64 adults (median 32.5 years-old) who were admitted to hospital for management of an acute pulmonary exacerbation and/or eradication of infectious agents such as Pseudomonas aeruginosa or Staphylococcus aureus. Our aim was to explore how urinary GSA levels changed across admission timepoints. Urine samples were collected at admission and discharge, and GSA measured by liquid chromatography with mass spectrometry. Paired admission-discharge results were compared using Wilcoxon signed-rank test. Paired admission-discharge samples were available for 53 children and 60 adults. A statistically significant difference was observed between admission-discharge for children and adults. Spearman's correlation analysis identified a correlation between urinary GSA levels and sex and S. aureus infection for children only. Our preliminary findings suggest that urinary GSA is responsive to the resolution of an acute pulmonary exacerbation and therefore warrants further studies in this population.

Azithromycin Augments Bacterial Uptake and Anti-Inflammatory Macrophage Polarization in Cystic Fibrosis.

BACKGROUND: Azithromycin (AZM) is widely being used for treating patients with cystic fibrosis (pwCF) following clinical trials demonstrating improved lung function and fewer incidents of pulmonary exacerba-tions. While the precise mechanisms remain elusive, immunomodulatory actions are thought to be involved. We previously reported impaired phagocytosis and defective anti-inflammatory M2 macrophage polarization in CF. This study systematically analyzed the effect of AZM on the functions of unpolarized and M1/M2 polarized macrophages in CF. METHODS: Monocytes, isolated from the venous blood of patients with CF (pwCF) and healthy controls (HCs), were differentiated into monocyte-derived macrophages (MDMs) and subsequently infected with P. aeruginosa. P. aeruginosa uptake and killing by MDMs in the presence or absence of AZM was studied. M1 and M2 macrophage polarizations were induced and their functions and cytokine release were analyzed. RESULTS: Following AZM treatment, both HC and CF MDMs exhibited a significant increase in P. aeruginosa uptake and killing, however, lysosomal acidification remained unchanged. AZM treatment led to higher activation of ERK1/2 in both HC and CF MDMs. Pharmacological inhibition of ERK1/2 using U0126 significantly reduced P. aeruginosa uptake in HC MDMs. M1 macrophage polarization remained unaffected; however, AZM treatment led to increased IL-6 and IL-10 release in both HC and CF M1 macrophages. AZM also significantly increased the phagocytic index for both pHrodo E. coli and S. aureus in CF M1 macrophages. In CF, AZM treatment promoted anti-inflammatory M2 macrophage polarization, with an increased percentage of CD209+ M2 macrophages, induction of the M2 gene CCL18, along with its secretion in the culture supernatant. However, AZM d'd not restore endocytosis in CF, another essential feature of M2 macrophages. CONCLUSIONS: This study highlights the cellular functions and molecular targets of AZM which may involve an improved uptake of both Gram-positive and Gram-negative bacteria, restored anti-inflammatory macrophage polarization in CF. This may in turn shape the reduced lung inflammation observed in clinical trials. In addition, we confirmed the role of ERK1/2 activation for bacterial uptake.

Standards for the care of people with cystic fibrosis; establishing and maintaining health.

This is the second in a series of four papers updating the European Cystic Fibrosis Society (ECFS) standards for the care of people with CF. This paper focuses on establishing and maintaining health. The guidance is produced using an evidence-based framework and with wide stakeholder engagement, including people from the CF community. Authors provided a narrative description of their topic and statements, which were more directive. These statements were reviewed by a Delphi exercise, achieving good levels of agreement from a wide group for all statements. This guidance reinforces the importance of a multi-disciplinary CF team, but also describes developing models of care including virtual consultations. The framework for health is reinforced, including the need for a physically active lifestyle and the strict avoidance of all recreational inhalations, including e-cigarettes. Progress with cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy is reviewed, including emerging adverse events and advice for dose reduction and interruption. This paper contains guidance that is pertinent to all people with CF regardless of age and eligibility for and access to modulator therapy.

Pregnancy in women with cystic fibrosis and diabetes: An audit of outcomes at two tertiary obstetric hospitals in Australia in the pre-cystic fibrosis transmembrane conductance regulator modulator era.

Background: Pregnancy in women with cystic fibrosis (CF) is becoming more common. Long-term metabolic issues such as diabetes are also becoming more common and have potentially important impacts on pregnancy outcomes. This study aimed to assess the impact of diabetes on pregnancy outcomes for women with CF. Methods: We undertook a retrospective chart audit of pregnancies to women with CF at the two tertiary obstetric hospitals in Southeast Queensland associated with CF and transplant management clinics between 2006 and 2016. Results: A total of 38 pregnancies among 26 women were identified. Four women (five pregnancies) had cystic fibrosis-related diabetes (CFRD) diagnosed prior to pregnancy, and 12 women (15 pregnancies) developed gestational diabetes (GDM) complicating pregnancy. CFRD and GDM were associated with higher rates of delivery complications, prematurity, and the need for neonatal intensive care unit admission. Conclusion: Diabetes is common during pregnancy in women with CF and impacts pregnancy outcomes.

Paraquat ingestion in an adult with cystic fibrosis (CF): Diagnostic and management dilemmas.

N,N'-dimethyl-4,4'bipyridinium dichloride (Paraquat) is a potent herbicide used widely in agriculture. We report the effects of an ingestion of paraquat by a 28 year old male with cystic fibrosis and the diagnostic and management challenges this posed in both the acute and longer term setting. We describe the effects of direct paraquat toxicity on the lung tissue secondary to aspiration and review the long-term sequelae of paraquat, namely osteonecrosis. Our case is the first to describe osteonecrosis of the knee in the context of paraquat toxicity. Survival following ingestion remains poor with a high associated mortality. However, timely treatment with NAC and immunosuppression may impact on survival. In those patients who do survive the acute phase post ingestion, follow-up over years may be required to detect the long-term effects of paraquat on bone health.

Advancing the pipeline of cystic fibrosis clinical trials: a new roadmap with a global trial network perspective.

The growing use of modulator therapies aimed at restoring cystic fibrosis transmembrane conductance regulator (CFTR) protein function in people with cystic fibrosis has fundamentally altered clinical trial strategies needed to advance new therapeutics across an orphan disease population that is now divided by CFTR modulator eligibility. The development of a robust pipeline of nucleic acid-based therapies (NABTs)-initially directed towards the estimated 10% of the cystic fibrosis population who are genetically ineligible for, or intolerant of, CFTR modulators-is dependent on the optimisation of restricted trial participant resources across multiple development programmes, a challenge that will preclude the use of gold standard placebo-controlled trials. Advancement of a full pipeline of symptomatic therapies across the entire cystic fibrosis population will be challenged by smaller effect sizes and uncertainty regarding their clinical importance in a growing modulator-treated population with more mild and stable pulmonary disease. In this Series paper, we aim to lay the foundation for clinical trial strategy and community partnership that must deviate from established and familiar precedent to advance the future pipeline of cystic fibrosis therapeutics.

A systematic review of the clinical impact of small colony variants in patients with cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) is a life-limiting disorder that is characterised by respiratory tract inflammation that is mediated by a range of microbial pathogens. Small colony variants (SCVs) of common respiratory pathogens are being increasingly recognised in CF. The aim of this systematic review is to investigate the prevalence of SCVs, clinical characteristics and health outcomes for patients with CF, and laboratory diagnostic features of SCVs compared to non-small colony variants (NCVs) for a range of Gram-positive and Gram-negative respiratory pathogens. METHODS: A literature search was conducted (PubMed, Web of Science, Embase and Scopus) in April 2020 to identify articles of interest. Data pertaining to demographic characteristics of participants, diagnostic criteria of SCVs, SCV prevalence and impact on lung function were extracted from included studies for analysis. RESULTS: Twenty-five of 673 studies were included in the systematic review. Individuals infected with SCVs of Staphylococcus aureus (S. aureus) were more likely to have had prior use of the broad-spectrum antibiotic trimethoprim sulfamethoxazole (p < 0.001), and the prevalence of SCVs in patients infected with S. aureus was estimated to be 19.3% (95% CI: 13.5% to 25.9%). Additionally, patients infected with SCVs of Gram-negative and Gram-positive pathogens were identified to have a lower forced expiratory volume in one second percentage predicted (-16.8, 95% CI: -23.2 to -10.4) than those infected by NCVs. Gram-positive SCVs were commonly described as small and non-haemolytic, grown on Mannitol salt or blood agar for 24 h at 35°C and confirmed using tube coagulase testing. CONCLUSION: The findings of this systematic review demonstrate that SCVs of S. aureus have a high prevalence in the CF community, and that the occurrence of SCVs in Gram-positive and Gram-negative pathogens is linked to poorer respiratory function. Further investigation is necessary to determine the effect of infection by SCVs on the CF population.

In vitro susceptibility testing of imipenem-relebactam and tedizolid against 102 Mycobacterium abscessus isolates.

OBJECTIVES: Mycobacterium abscessus is an emerging infection in people living with lung diseases, including cystic fibrosis (CF) and bronchiectasis, and it has limited treatment options and low cure rates. The off-label use of novel antibiotics developed for other bacterial pathogens offers potential new therapeutic options. We aimed to describe the in vitro activity of imipenem, imipenem-relebactam and tedizolid against comparator antibiotics in M. abscessus isolates from Australian patients with and without CF. METHODS: We performed susceptibility testing for imipenem-relebactam, tedizolid and comparator antibiotics by Clinical and Laboratory Standards Institute (CLSI) criteria against 102 clinical M. abscessus isolates, including 46 from people with CF. RESULTS: In this study, the minimum inhibitory concentration (MICs) of imipenem-relebactam was one-fold dilution less than of imipenem alone. The MIC50 and MIC90 of imipenem-relebactam were 8 and 16 mg/L, respectively, whereas for imipenem they were 16 and 32 mg/L. Tedizolid had an MIC50 and MIC90 of 2 and 4 mg/L, respectively. Forty non-CF isolates had linezolid susceptibility performed, with MIC50 and MIC90 values of 16 and 32 mg/L, respectively, measured. CONCLUSIONS: This study shows lower MICs for imipenem-relebactam and tedizolid compared to other more commonly used antibiotics and supports their consideration in clinical trials for M. abscessus treatment.

Ceftazidime resistance in Pseudomonas aeruginosa is multigenic and complex.

Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of individual mutations and mutation combinations. Thirty-five mutants with reduced susceptibility to ceftazidime were evolved from two antibiotic-sensitive P. aeruginosa reference strains PAO1 and PA14. Mutations were identified by whole genome sequencing. The evolved mutants tolerated ceftazidime at concentrations between 4 and 1000 times that of the parental bacteria, with most mutants being ceftazidime resistant (minimum inhibitory concentration [MIC] ≥ 32 mg/L). Many mutants were also resistant to meropenem, a carbapenem antibiotic. Twenty-eight genes were mutated in multiple mutants, with dacB and mpl being the most frequently mutated. Mutations in six key genes were engineered into the genome of strain PAO1 individually and in combinations. A dacB mutation by itself increased the ceftazidime MIC by 16-fold although the mutant bacteria remained ceftazidime sensitive (MIC < 32 mg/L). Mutations in ampC, mexR, nalC or nalD increased the MIC by 2- to 4-fold. The MIC of a dacB mutant was increased when combined with a mutation in ampC, rendering the bacteria resistant, whereas other mutation combinations did not increase the MIC above those of single mutants. To determine the clinical relevance of mutations identified through experimental evolution, 173 ceftazidime-resistant and 166 sensitive clinical isolates were analysed for the presence of sequence variants that likely alter function of resistance-associated genes. dacB and ampC sequence variants occur most frequently in both resistant and sensitive clinical isolates. Our findings quantify the individual and combinatorial effects of mutations in different genes on ceftazidime susceptibility and demonstrate that the genetic basis of ceftazidime resistance is complex and multifactorial.

Thoracic Society of Australia and New Zealand (TSANZ) position statement on chronic suppurative lung disease and bronchiectasis in children, adolescents and adults in Australia and New Zealand.

This position statement, updated from the 2015 guidelines for managing Australian and New Zealand children/adolescents and adults with chronic suppurative lung disease (CSLD) and bronchiectasis, resulted from systematic literature searches by a multi-disciplinary team that included consumers. The main statements are: Diagnose CSLD and bronchiectasis early; this requires awareness of bronchiectasis symptoms and its co-existence with other respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease). Confirm bronchiectasis with a chest computed-tomography scan, using age-appropriate protocols and criteria in children. Undertake a baseline panel of investigations. Assess baseline severity, and health impact, and develop individualized management plans that include a multi-disciplinary approach and coordinated care between healthcare providers. Employ intensive treatment to improve symptom control, reduce exacerbation frequency, preserve lung function, optimize quality-of-life and enhance survival. In children, treatment also aims to optimize lung growth and, when possible, reverse bronchiectasis. Individualize airway clearance techniques (ACTs) taught by respiratory physiotherapists, encourage regular exercise, optimize nutrition, avoid air pollutants and administer vaccines following national schedules. Treat exacerbations with 14-day antibiotic courses based upon lower airway culture results, local antibiotic susceptibility patterns, clinical severity and patient tolerance. Patients with severe exacerbations and/or not responding to outpatient therapy are hospitalized for further treatments, including intravenous antibiotics and intensive ACTs. Eradicate Pseudomonas aeruginosa when newly detected in lower airway cultures. Individualize therapy for long-term antibiotics, inhaled corticosteroids, bronchodilators and mucoactive agents. Ensure ongoing care with 6-monthly monitoring for complications and co-morbidities. Undertake optimal care of under-served peoples, and despite its challenges, delivering best-practice treatment remains the overriding aim.

Nontuberculous Mycobacteria in Cystic Fibrosis in the Era of Cystic Fibrosis Transmembrane Regulator Modulators.

Nontuberculous mycobacteria (NTM) are a group of mycobacteria which represent opportunistic pathogens that are of increasing concern in people with cystic fibrosis (pwCF). The acquisition has been traditionally though to be from environmental sources, though recent work has suggested clustered clonal infections do occur and transmission potential demonstrated among pwCF attending CF specialist centers. Guidelines for the screening, diagnosis, and identification of NTM and management of pwCF have been published. The emergence of CF-specific therapies, in particular cystic fibrosis transmembrane regulator (CFTR) modulator drugs, have led to significant improvement in the health and well-being of pwCF and may lead to challenges in sampling the lower respiratory tract including to screen for NTM. This review highlights the epidemiology, modes of acquisition, screening and diagnosis, therapeutic approaches in the context of improved clinical status for pwCF, and the clinical application of CFTR modulator therapies.

Beyond borders: cystic fibrosis survival between Australia, Canada, France and New Zealand.

BACKGROUND: Life expectancy for people with cystic fibrosis (CF) varies considerably both within and between countries. The objective of this study was to compare survival among countries with single-payer healthcare systems while accounting for markers of disease severity. METHODS: This cohort study used data from established national CF registries in Australia, Canada, France and New Zealand from 2015 to 2019. Median age of survival for each of the four countries was estimated using the Kaplan-Meier method. A Cox proportional hazards model was used to compare risk of death between Canada, France and Australia after adjusting for prognostic factors. Due to low number of deaths, New Zealand was not included in final adjusted models. RESULTS: Between 2015 and 2019, a total of 14 842 people (3537 Australia, 4434 Canada, 6411 France and 460 New Zealand) were included. The median age of survival was highest in France 65.9 years (95% CI: 59.8 to 76.0) versus 53.3 years (95% CI: 48.9 to 59.8) for Australia, 55.4 years (95% CI: 51.3 to 59.2) for Canada and 54.8 years (95% CI: 40.7 to not available) for New Zealand. After adjusting for individual-level factors, the risk of death was significantly higher in Canada (HR 1.85, 95% CI: 1.48 to 2.32; p<0.001) and Australia (HR 2.08, 95% CI: 1.64 to 2.64; p<0.001) versus France. INTERPRETATION: We observed significantly higher survival in France compared with countries with single-payer healthcare systems. The median age of survival in France exceeded 60 years of age despite having the highest proportion of underweight patients which may be due to differences in availability of transplant.

Targeted reduction of airborne viral transmission risk in long-term residential aged care.

COVID-19 has demonstrated the devastating consequences of the rapid spread of an airborne virus in residential aged care. We report the use of CO2-based ventilation assessment to empirically identify potential 'super-spreader' zones within an aged care facility, and determine the efficacy of rapidly implemented, inexpensive, risk reduction measures.

Survival of people with cystic fibrosis in Australia.

Survival statistics, estimated using data from national cystic fibrosis (CF) registries, inform the CF community and monitor disease progression. This study aimed to estimate survival among people with CF in Australia and to identify factors associated with survival. This population-based cohort study used prospectively collected data from 23 Australian CF centres participating in the Australian CF Data Registry (ACFDR) from 2005-2020. Period survival analysis was used to calculate median age of survival estimates for each 5-year window from 2005-2009 until 2016-2020. The overall median survival was estimated using the Kaplan-Meier method. Between 2005-2020 the ACFDR followed 4,601 people with CF, noting 516 (11.2%) deaths including 195 following lung transplantation. Out of the total sample, more than half (52.5%) were male and 395 (8.6%) had undergone lung transplantation. Two thirds of people with CF (66.1%) were diagnosed before six weeks of age or by newborn/prenatal screening. The overall median age of survival was estimated as 54.0 years (95% CI: 51.0-57.04). Estimated median survival increased from 48.9 years (95% CI: 44.7-53.5) for people with CF born in 2005-2009, to 56.3 years (95% CI: 51.2-60.4) for those born in 2016-2020. Factors independently associated with reduced survival include receiving a lung transplant, having low FEV1pp and BMI. Median survival estimates are increasing in CF in Australia. This likely reflects multiple factors, including newborn screening, improvement in diagnosis, refinements in CF management and centre-based multidisciplinary care.

Characterizing and correcting immune dysfunction in non-tuberculous mycobacterial disease.

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic, progressive, and growing worldwide health burden associated with mounting morbidity, mortality, and economic costs. Improvements in NTM-PD management are urgently needed, which requires a better understanding of fundamental immunopathology. Here, we examine temporal dynamics of the immune compartment during NTM-PD caused by Mycobacterium avium complex (MAC) and Mycobactereoides abscessus complex (MABS). We show that active MAC infection is characterized by elevated T cell immunoglobulin and mucin-domain containing-3 expression across multiple T cell subsets. In contrast, active MABS infection was characterized by increased expression of cytotoxic T-lymphocyte-associated protein 4. Patients who failed therapy closely mirrored the healthy individual immune phenotype, with circulating immune network appearing to 'ignore' infection in the lung. Interestingly, immune biosignatures were identified that could inform disease stage and infecting species with high accuracy. Additionally, programmed cell death protein 1 blockade rescued antigen-specific IFN-γ secretion in all disease stages except persistent infection, suggesting the potential to redeploy checkpoint blockade inhibitors for NTM-PD. Collectively, our results provide new insight into species-specific 'immune chatter' occurring during NTM-PD and provide new targets, processes and pathways for diagnostics, prognostics, and treatments needed for this emerging and difficult to treat disease.

Rapid fluoroquinolone resistance detection in Pseudomonas aeruginosa using mismatch amplification mutation assay-based real-time PCR.

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 (gyrA248) and GyrA D87N, D87Y and D87H (gyrA259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa.Methods. Following assay validation, the gyrA248 and gyrA259 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.

The Effectiveness of Ultraviolet-C (UV-C) Irradiation on the Viability of Airborne Pseudomonas aeruginosa.

Pseudomonas aeruginosa (Pa) is the predominant bacterial pathogen in people with cystic fibrosis (CF) and can be transmitted by airborne droplet nuclei. Little is known about the ability of ultraviolet band C (UV-C) irradiation to inactivate Pa at doses and conditions relevant to implementation in indoor clinical settings. We assessed the effectiveness of UV-C (265 nm) at up to seven doses on the decay of nebulized Pa aerosols (clonal Pa strain) under a range of experimental conditions. Experiments were done in a 400 L rotating sampling drum. A six-stage Andersen cascade impactor was used to collect aerosols inside the drum and the particle size distribution was characterized by an optical particle counter. UV-C effectiveness was characterized relative to control tests (no UV-C) of the natural decay of Pa. We performed 112 tests in total across all experimental conditions. The addition of UV-C significantly increased the inactivation of Pa compared with natural decay alone at all but one of the UV-C doses assessed. UV-C doses from 246-1968 µW s/cm2 had an estimated effectiveness of approximately 50-90% for airborne Pa. The effectiveness of doses ≥984 µW s/cm2 were not significantly different from each other (p-values: 0.365 to ~1), consistent with a flattening of effectiveness at higher doses. Modelling showed that delivering the highest dose associated with significant improvement in effectiveness (984 µW s/cm2) to the upper air of three clinical rooms would lead to lower room doses from 37-49% of the 8 h occupational limit. Our results suggest that UV-C can expedite the inactivation of nebulized airborne Pa under controlled conditions, at levels that can be delivered safely in occupied settings. These findings need corroboration, but UV-C may have potential applications in locations where people with CF congregate, coupled with other indoor and administrative infection control measures.

Antimicrobial Stewardship in Cystic Fibrosis.

The chronic airway infection and inflammation characteristic of cystic fibrosis (CF) ultimately leads to progressive lung disease, the primary cause of death in persons with CF (pwCF). Despite many recent advances in CF clinical care, efforts to preserve lung function in many pwCF still necessitate frequent antimicrobial use. Incorporating antimicrobial stewardship (AMS) principles into management of pulmonary exacerbations (PEx) would facilitate development of best practices for antimicrobial utilization at CF care centers. However, AMS can be challenging in CF given the unique aspects of chronic, polymicrobial infection in the CF airways, lack of evidence-based guidelines for managing PEx, limited utility for antimicrobial susceptibility testing, and increased frequency of adverse drug events in pwCF. This article describes current evidence-based antimicrobial treatment strategies for pwCF, highlights the potential for AMS to beneficially impact CF care, and provides practical strategies for integrating AMS programs into the management of PEx in pwCF.

Express Yourself: Quantitative Real-Time PCR Assays for Rapid Chromosomal Antimicrobial Resistance Detection in Pseudomonas aeruginosa.

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. Here, we designed and then validated two multiplex quantitative real-time PCR (qPCR) assays to simultaneously detect differential expression of the resistance-nodulation-division efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM, the AmpC ß-lactamase, and the porin OprD, which are commonly associated with chromosomally encoded AMR. Next, qPCRs were tested on 15 sputa from 11 participants with P. aeruginosa respiratory infections to determine AMR profiles in vivo. We confirmed multiplex qPCR testing feasibility directly on sputa, representing a key advancement in in vivo AMR diagnosis. Notably, comparison of sputa with their derived isolates grown in Luria-Bertani broth (±2.5% NaCl) or a 5-antibiotic cocktail showed marked expression differences, illustrating the difficulty in replicating in vivo expression profiles in vitro. Cystic fibrosis sputa showed significantly reduced mexE and mexY expression compared with chronic obstructive pulmonary disease sputa, despite harboring fluoroquinolone- and aminoglycoside-resistant strains, indicating that these loci do not contribute to AMR in vivo. oprD was also significantly downregulated in cystic fibrosis sputa, even in the absence of contemporaneous carbapenem use, suggesting a common adaptive trait in chronic infections that may affect carbapenem efficacy. Sputum ampC expression was highest in participants receiving carbapenems (6.7 to 15×), some of whom were simultaneously receiving cephalosporins, the latter of which would be rendered ineffective by the upregulated ampC. Our qPCR assays provide valuable insights into the P. aeruginosa resistome, and their use on clinical specimens will permit timely treatment alterations that will improve patient outcomes and antimicrobial stewardship measures.