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Since the 1989 discovery that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), there has been substantial progress toward understanding the molecular basis for CF lung disease, leading to the discovery and development of new therapeutic approaches. However, the earliest impact of the loss of CFTR function on airway physiology and structure and its relationship to initial infection and inflammation are poorly understood. Universal newborn screening for CF in the United States represents an unprecedented opportunity for investigating CF clinical manifestations very early in life. Recently developed animal models with pulmonary phenotypic manifestations also provide a window into the early consequences of this genetic disorder. For these reasons, the National Heart, Lung, and Blood Institute (NHLBI) convened a working group of extramural experts, entitled "Future Research Directions in Early CF Lung Disease" on September 21-22, 2010, to identify future research directions of great promise in CF. The priority areas identified included (1) exploring pathogenic mechanisms of early CF lung disease; (2) leveraging newborn screening to elucidate the natural history of early lung disease; (3) developing a spectrum of biomarkers of early lung disease that reflects CF pathophysiology, clinical outcome, and response to treatment; (4) exploring the role of genetics/genomics (e.g., modifier genes, gene-environmental interactions, and epigenetics) in early CF pathogenesis; (5) defining early microbiological events in CF lung disease; and (6) elucidating the initial airway inflammatory, remodeling, and repair mechanisms in CF lung disease. [ABSTRACT FROM AUTHOR]

Background: Intermittent viral exacerbations in patients with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa (PA) infection are associated with increased bacterial load. A few clinical studies suggest that rhinoviruses (RV) are associated with the majority of viral-related exacerbations in CF and require prolonged intravenous antibiotic treatment. These observations imply that acute RV infection may increase lower respiratory symptoms by increasing planktonic bacterial load. However, the underlying mechanisms are not known.Methods: Primary CF airway epithelial cells differentiated into mucociliary phenotype were infected with mucoid PA (MPA) followed by RV and examined for bacterial density, biofilm mass, levels of chemokines and hydrogen peroxide (H2O2). The need for dual oxidase 2, a component of NADPH oxidase, in RV-induced generation of H2O2 in CF cells was assessed using gene-specific siRNA.Results: Superinfection with RV increased chemokine responses in CF mucociliary-differentiated airway epithelial cells with pre-existing MPA infection in the form of biofilm. This was associated with the presence of planktonic bacteria at both the apical and basolateral epithelial cell surfaces. Further, RV-induced generation of H2O2 via dual oxidase 2 in CF cells was sufficient for dispersal of planktonic bacteria from the biofilm. Inhibition of NADPH oxidase reduced bacterial transmigration across mucociliary-differentiated CF cells and the interleukin-8 response in MPA- and RV-infected cells.Conclusion: This study shows that acute infection with RV liberates planktonic bacteria from biofilm. Planktonic bacteria, which are more proinflammatory than their biofilm counterparts, stimulate increased chemokine responses in CF airway epithelial cells which, in turn, may contribute to the pathogenesis of CF exacerbations. [ABSTRACT FROM AUTHOR]

RATIONALE: Chronic infection with Burkholderia cepacia complex bacteria in cystic fibrosis is associated with accelerated decline in pulmonary function and increased mortality. Clinical implications of the recently characterized genomovar VI, B. dolosa, are unknown. OBJECTIVES: Characterization of impact of B. dolosa on pulmonary function and mortality in cystic fibrosis. METHODS: We compared patients chronically infected with B. dolosa (n = 31) with unmatched patients with B. multivorans (n = 24) and with age- and sex-matched control subjects without Burkholderia species (n = 58). We analyzed rates of pulmonary function decline (% predicted FEV(1)) using a random effects model assuming segmented linear trends. All available FEV(1) measurements from 5 yr (median, 4.8) before until 2.5 yr (median, 1.5) after the first positive culture for Burkholderia (reference date) were analyzed. Survival was compared using the Kaplan-Meier method and proportional hazards model. MEASUREMENTS AND MAIN RESULTS: Baseline FEV(1) and rate of decline were similar in the cohorts. Decline in FEV(1) after the reference date accelerated in patients with B. dolosa (-2.3 percentage points/yr pre vs. -7.1 post, p = 0.002), but was unchanged in the B. multivorans and control patients (-2.3 vs. -0.8 post, p = 0.38, and -2.1 pre vs. -0.5 post, p = 0.20, respectively). The probability of dying within 18 mo of the reference date was 13, 7, and 3% for B. dolosa, B. multivorans, and control patients, respectively (B. dolosa vs. control hazard ratio, 10.8; 95% confidence interval, 1.3-92.8; p = 0.03). CONCLUSIONS: B. dolosa chronic infection in cystic fibrosis is associated with accelerated loss of lung function and decreased survival. [ABSTRACT FROM AUTHOR]

In our curated panel of Burkholderia cepacia complex isolates, Burkholderia multivorans strain AU28442 was unusually highly β-lactam resistant. To explore the molecular mechanisms leading to this phenotype, we performed whole genome sequencing (WGS) and microbiological and biochemical assays. WGS analysis revealed that strain AU28442 produced two β-lactamases, AmpC22 and a novel PenA-like β-lactamase denominated PenA39. Additionally, the strain presented frame-shift mutations in the genes encoding penicillin binding proteins 3 (PBP3) and 4 (PBP4). The antibiotic susceptibilities of the parent AU28442 strain carrying bla PenA39 vs the isogenic E. coli strain producing bla PenA39 were discrepant with ceftazidime MICs of >512 and 1 μg/mL, respectively. Accordingly, PenA39 was found to poorly hydrolyze β-lactams with k cat values of ≤8.8 s -1 . An overlay of the crystal structure of PenA39 with PenA1 revealed a shift in the SDN loop in the variant, which may affect the catalytic efficiency of PenA39 toward substrates and inhibitors. Moreover, microscopic examination of AU28442 revealed shortened rod-shaped cells compared to B. multivorans ATCC 17616, which carries a full complement of intact PBPs. Further complementation assays confirmed that the loss of PBP3 and PBP4 was the main factor contributing to the high-level β-lactam resistance observed in B. multivorans AU28442. This information allowed us to revert susceptibility by pairing a potent β-lactamase inhibitor with a β-lactam with promiscuous PBP binding. This detailed characterization of B. multivorans provides an illustration of the myriad ways in which bacteria under antibiotic selection can develop resistance and demonstrates a mechanism to overcome it.

Background: Infection control guidelines for cystic fibrosis (CF) stress cleaning of environmental surfaces and patientcare equipment in CF clinics. This multicenter study measured cleanliness of frequently touched surfaces in CF clinics using an ATP bioluminescence assay to assess the effectiveness of cleaning/disinfection and the impact of feedback., Methods: Eight surfaces were tested across 19 clinics (10 pediatric, 9 adult) over 5 rounds of testing. Rounds 1 and 2 served as uncleaned baseline, and Round 3 occurring after routine cleaning. Rounds 4 and 5 were performed after feedback provided to staff and measured after cleaning. Pass rates defined as <250 relative light units were the primary outcome., Results: Of the 750 tests performed, 72% of surfaces passed at baseline, and 79%, 83%, and 85% of surfaces passed in Rounds 3, 4, and 5, respectively. The overall pass-rate was significantly higher in adult compared to pediatric clinics (86% vs 71%; P < 0.001). In pediatric clinics, blood pressure equipment and computer keyboards in the pulmonary function lab consistently passed, but the exam room patient/visitor chairs consistently failed in all rounds. In adult clinics blood pressure equipment, keyboards in exam rooms and exam tables passed in all rounds and no surface consistently failed., Conclusion: We demonstrate the feasibility of an ATP bioluminescence assay to measure cleanliness of patient care equipment and surfaces in CF clinics. Pass rates improved after cleaning and feedback for certain surfaces. We found that surfaces are more challenging to keep clean in clinics taking care of younger patients.

Ice machines can harbor water-related organisms, and the use of ice or tap water for clinical care activities has been associated with infections in health care settings. During 2021-2022, a total of 23 cases of infection by Burkholderia multivorans (sequence type ST659) were reported at two southern California hospitals and linked to contaminated ice and water from ice machines. In addition to these 23 cases, this report also includes 23 previously unreported cases of B. multivorans ST659 infections that occurred during 2020-2024: 13 at a northern California hospital, eight at a hospital in Colorado, and two additional cases at one of the southern California hospitals. The same brand of ice machine and brands of filters, descaling, and sanitizing products were used by all four hospitals; B. multivorans was isolated from samples collected from ice machines in two of the hospitals. Whole genome sequencing indicated that all clinical and ice machine isolates were highly genetically similar (0-14 single nucleotide variant differences across 81% of the selected reference genome). Recommendations from public health officials to halt the outbreak included avoiding ice and tap water during clinical care activities. An investigation is ongoing to determine possible sources of ice machine contamination. During outbreaks of water-related organisms in health care facilities, health care personnel should consider avoiding the use of tap water, including ice and water from ice machines, for patient care., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Matthew J. Arduino reports honoraria and travel grants from the Society for Healthcare Epidemiology of America and the Association for Professionals in Infection Control and Epidemiology. Lindsay J. Caverly and John J. LiPuma report receiving grants from the Cystic Fibrosis Foundation during the investigation. No other potential conflicts of interest were disclosed.

Nontuberculous mycobacteria (NTM) in drinking water are a significant public health concern. However, an incomplete understanding of the factors that influence the occurrence of NTM in drinking water limits our ability to characterize risk and prevent infection. This study sought to evaluate the influence of season and water treatment, distribution, and stagnation on NTM in drinking water. Samples were collected source-to-tap in a full-scale, chloraminated drinking water system approximately monthly from December 2019 to November 2020. NTM were characterized using culture-dependent (plate culture with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry [MALDI-TOF MS] isolate analysis) and culture-independent methods (quantitative PCR and genome-resolved metagenomics). Sampling locations included source waters, three locations within the treatment plant, and five buildings receiving water from the distribution system. Building plumbing samples consisted of first draw, 5-min flush, and full flush cold-water samples. As the study took place during the COVID-19 pandemic, the influence of reduced water usage in three of the five buildings was also investigated. The highest NTM densities source-to-tap were found in the summer first draw building water samples (10 7 gene copies/L), which also had the lowest monochloramine concentrations. Flushing was found to be effective for reducing NTM and restoring disinfectant residuals, though flush times necessary to improve water quality varied by building. Clinically relevant NTM species, including Mycobacterium avium , were recovered via plate culture, with increased occurrence observed in buildings with higher water age. Four of five NTM metagenome-assembled genomes were identified to the species level and matched identified isolates.IMPORTANCENTM infections are increasing in prevalence, difficult to treat, and associated with high morbidity and mortality rates. Our lack of understanding of the factors that influence NTM occurrence in drinking water limits our ability to prevent infections, accurately characterize risk, and focus remediation efforts. In this study, we comprehensively evaluated NTM in a full-scale drinking water system, showing that various steps in treatment and distribution influence NTM presence. Stagnant building water contained the highest NTM densities source-to-tap and was associated with low disinfectant residuals. We illustrated the differences in NTM detection and characterization obtained from culture-based and culture-independent methods, highlighting the complementarity between these approaches. We demonstrated that focusing NTM mitigation efforts in building plumbing systems, which have the highest NTM densities source-to-tap, has potential for immediate positive effects. We also identified steps during treatment that increase NTM levels, which provides beneficial information for utilities seeking to reduce NTM in finished water., Competing Interests: The authors declare no conflict of interest.

SUMMARYThis guidance presents recommendations for clinical microbiology laboratories for processing respiratory samples from people with cystic fibrosis (pwCF). Appropriate processing of respiratory samples is crucial to detect bacterial and fungal pathogens, guide treatment, monitor the epidemiology of cystic fibrosis (CF) pathogens, and assess therapeutic interventions. Thanks to CF transmembrane conductance regulator modulator therapy, the health of pwCF has improved, but as a result, fewer pwCF spontaneously expectorate sputum. Thus, the collection of sputum samples has decreased, while the collection of other types of respiratory samples such as oropharyngeal and bronchoalveolar lavage samples has increased. To optimize the detection of microorganisms, including Pseudomonas aeruginosa , Staphylococcus aureus , Haemophilus influenzae , and Burkholderia cepacia complex; other less common non-lactose fermenting Gram-negative bacilli, e.g., Stenotrophomonas maltophilia , Inquilinus , Achromobacter , Ralstonia , and Pandoraea species; and yeasts and filamentous fungi, non-selective and selective culture media are recommended for all types of respiratory samples, including samples obtained from pwCF after lung transplantation. There are no consensus recommendations for laboratory practices to detect, characterize, and report small colony variants (SCVs) of S. aureus , although studies are ongoing to address the potential clinical impact of SCVs. Accurate identification of less common Gram-negative bacilli, e.g., S. maltophilia , Inquilinus , Achromobacter , Ralstonia , and Pandoraea species, as well as yeasts and filamentous fungi, is recommended to understand their epidemiology and clinical importance in pwCF. However, conventional biochemical tests and automated platforms may not accurately identify CF pathogens. MALDI-TOF MS provides excellent genus-level identification, but databases may lack representation of CF pathogens to the species-level. Thus, DNA sequence analysis should be routinely available to laboratories for selected clinical circumstances. Antimicrobial susceptibility testing (AST) is not recommended for every routine surveillance culture obtained from pwCF, although selective AST may be helpful, e.g., for unusual pathogens or exacerbations unresponsive to initial therapy. While this guidance reflects current care paradigms for pwCF, recommendations will continue to evolve as CF research expands the evidence base for laboratory practices., Competing Interests: Lisa Saiman receives grant funding (provided to institution) from the U.S. CF Foundation and serves as a consultant to the CF Foundation. Valerie Waters receives grant funding from the U.S. CF Foundation and CF Canada. John J. LiPuma receives grant funding from the U.S. CF Foundation. Lucas R. Hoffman receives grant funding from the U.S. CF Foundation. Kevin Alby has no conflicts of interest to report. Sean X. Zhang receives grant funding from the U.S. CF Foundation. Yvonne C. Yau receives grant funding from the U.S. CF Foundation and CF Canada. Damian G. Downey receives grants from the CF Trust and U.S. CF Foundation. He receives support from the European CF Society and U.S. CF Foundation for travel to meetings. He is the Director of the European CF Society Clinical Trials Network. Isabelle Sermet-Gaudelus receives funding from the European Cystic Fibrosis Society and the UK Cystic Fibrosis Trust. Jean-Philippe Bouchara receives funding from Vaincre la Mucoviscidose, La Pierre Le Bigaut, Association Grégory Lemarchal, and Anjou Muco. Timothy J Kidd has no conflicts of interest to report. Scott C. Bell receives grant funding from the U.S. CF Foundation, the Australian National Health and Medical Research Council, and the Medical Research Future's Fund. A. Whitney Brown is an employee of the U.S. CF Foundation.

In pharmaceutical manufacturing, ensuring product safety involves the detection and identification of microorganisms with human pathogenic potential, including Burkholderia cepacia complex (BCC), Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma spp., some of which may be missed or not identified by traditional culture-dependent methods. In this study, we employed a metagenomic approach to detect these taxa, avoiding the limitations of conventional cultivation methods. We assessed the groundwater microbiome's taxonomic and functional features from samples collected at two locations in the spring and summer. All datasets comprised 436-557 genera with Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Cyanobacteria accounting for > 95% of microbial DNA sequences. The aforementioned species constituted less than 18.3% of relative abundance. Escherichia and Salmonella were mainly detected in Hot Springs, relative to Jefferson, while Clostridium and Pseudomonas were mainly found in Jefferson relative to Hot Springs. Multidrug resistance efflux pumps and BlaR1 family regulatory sensor-transducer disambiguation dominated in Hot Springs and in Jefferson. These initial results provide insight into the detection of specified microorganisms and could constitute a framework for the establishment of comprehensive metagenomic analysis for the microbiological evaluation of pharmaceutical-grade water and other non-sterile pharmaceutical products, ensuring public safety., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

While online monitoring of physicochemical parameters has widely been incorporated into drinking water treatment systems, online microbial monitoring has lagged behind, resulting in the use of surrogate parameters (disinfectant residual, applied dose, concentration × time, CT) to assess disinfection system performance. Online flow cytometry (online FCM) allows for automated quantification of total and intact microbial cells. This study sought to investigate the feasibility of online FCM for full-scale drinking water ozone disinfection system performance monitoring. A water treatment plant with high lime solids turbidity in the ozone contactor influent was selected to evaluate the online FCM in challenging conditions. Total and intact cell counts were monitored for 40 days and compared to surrogate parameters (ozone residual, ozone dose, and CT) and grab sample assay results for cellular adenosine triphosphate (cATP), heterotrophic plate counts (HPC), impedance flow cytometry, and 16S rRNA gene sequencing. Online FCM provided insight into the dynamics of the full-scale ozone system, including offering early warning of increased contactor effluent cell concentrations, which was not observed using surrogate measures. Positive correlations were observed between online FCM intact cell counts and cATP levels (Kendall's tau=0.40), HPC (Kendall's tau=0.20), and impedance flow cytometry results (Kendall's tau=0.30). Though a strong correlation between log intact cell removal and CT was not observed, 16S rRNA gene sequencing results showed that passage through the ozone contactor significantly changed the microbial community (p < 0.05). Potential causes of the low overall cell inactivation in the contactor and the significant changes in the microbial community after ozonation include regrowth in the later chambers of the contactor and varied ozone resistance of drinking water microorganisms. This study demonstrates the suitability of direct, online microbial analysis for monitoring full-scale disinfection systems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lutgarde Raskin reports supplies were provided by Sysmex America Inc. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Polymicrobial infection of the airways is a hallmark of obstructive lung diseases such as cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Pulmonary exacerbations (PEx) in these conditions are associated with accelerated lung function decline and higher mortality rates. Understanding PEx ecology is challenged by high inter-patient variability in airway microbial community profiles. We analyze bacterial communities in 880 CF sputum samples collected during an observational prospective cohort study and develop microbiome descriptors to model community reorganization prior to and during 18 PEx. We identify two microbial dysbiosis regimes with opposing ecology and dynamics. Pathogen-governed PEx show hierarchical community reorganization and reduced diversity, whereas anaerobic bloom PEx display stochasticity and increased diversity. A simulation of antimicrobial treatment predicts better efficacy for hierarchically organized communities. This link between PEx, microbiome organization, and treatment success advances the development of personalized clinical management in CF and, potentially, other obstructive lung diseases., (© 2024. The Author(s).)

A collection of 161 Ralstonia isolates, including 90 isolates from persons with cystic fibrosis, 27 isolates from other human clinical samples, 8 isolates from the hospital environment, 7 isolates from industrial samples, and 19 environmental isolates, was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and yielded confident species level identification scores for only 62 (39%) of the isolates, including four that proved misidentified subsequently. Whole-genome sequence analysis of 32 representative isolates for which no confident MALDI-TOF MS species level identification was obtained revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively, and provided the basis for updating an in-house MALDI-TOF MS database. A reanalysis of all mass spectra with the updated MALDI-TOF MS database increased the percentage of isolates with confident species level identification up to 77%. The antimicrobial susceptibility of 30 isolates mainly representing novel human clinical and environmental Ralstonia species was tested toward 17 antimicrobial agents and demonstrated that the novel Ralstonia species were generally multi-resistant, yet susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline. An analysis of genomic antimicrobial resistance genes in 32 novel and publicly available genome sequences revealed broadly distributed beta-lactam resistance determinants.IMPORTANCEThe present study demonstrated that a commercial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification database can be tailored to improve the identification of Ralstonia species. It also revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively. An analysis of minimum inhibitory concentration values demonstrated that the novel Ralstonia species were generally multi-resistant but susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline., Competing Interests: The authors declare no conflict of interest.

Rationale: Rates of viral respiratory infection (VRI) are similar in people with cystic fibrosis (CF) and the general population; however, the associations between VRI and CF pulmonary exacerbations (PEx) require further elucidation. Objectives: To determine VRI prevalence during CF PEx and evaluate associations between VRI, clinical presentation, and treatment response. Methods: The STOP2 (Standardized Treatment of Pulmonary Exacerbations II) study was a multicenter randomized trial to evaluate different durations of intravenous antibiotic therapy for PEx. In this ancillary study, participant sputum samples from up to three study visits were tested for respiratory viruses using multiplex polymerase chain reactions. Baselines and treatment-associated changes in mean lung function (percent predicted forced expiratory volume in 1 s) , respiratory symptoms (Chronic Respiratory Infection Symptom Score), weight, and C-reactive protein were compared as a function of virus detection. Odds of PEx retreatment within 30 days and future PEx hazard were modeled by logistic and Cox proportional hazards regression, respectively. Results: A total of 1,254 sputum samples from 621 study participants were analyzed. One or more respiratory viruses were detected in sputum samples from 245 participants (39.5%). Virus-positive participants were more likely to be receiving CF transmembrane conductance regulator modulator therapy (45% vs. 34%) and/or chronic azithromycin therapy (54% vs. 44%) and more likely to have received treatment for nontuberculous Mycobacterium infection in the preceding 2 years (7% vs. 3%). At study visit 1, virus-positive participants were more symptomatic (mean Chronic Respiratory Infection Symptom Score, 53.8 vs. 51.1), had evidence of greater systemic inflammation (log 10 C-reactive protein concentration, 1.32 log 10 mg/L vs. 1.23 log 10 mg/L), and had a greater drop in percent predicted forced expiratory volume in 1 second from the prior 6-month baseline (5.8 vs. 3.6). Virus positivity was associated with reduced risk of future PEx (hazard ratio, 0.82; 95% confidence interval, 0.69-0.99; P  = 0.034) and longer median time to next PEx (255 d vs. 172 d; P  = 0.021) compared with virus negativity. Conclusions: More than one-third of STOP2 participants treated for a PEx had a positive test result for a respiratory virus with more symptomatic initial presentation compared with virus-negative participants, but favorable long-term outcomes. More refined phenotyping of PEx, taking VRIs into account, may aid in optimizing personalized management of PEx.Clinical trial registered with www.clinicaltrials.gov (NCT02781610).

Background: Care guidelines for cystic fibrosis (CF) have been developed to enhance consistent care and to improve health outcomes. We determined if adherence to CF care guidelines predicted P. aeruginosa incidence rates (Pa-IR) at U.S. CF centers in 2018., Methods: This cross-sectional CF Foundation Patient Registry study included 82 adult and 132 pediatric centers. Adherence to 12 guidelines was defined categorically (guideline met) or as a continuous measure (proportion of patients being treated/evaluated per guideline). Association of adherence to individual guidelines with Pa-IR, accounted for center and patient characteristics relevant to Pa-IR and were modeled using random forests and weighted-least-squares (WLS) analyses., Results: The mean Pa-IR was 0.2 cases/patient-years at risk (SE 0.0074) for all centers combined. Guideline adherence was lowest for ≥4 bacterial cultures/year (54% of centers) and annual oral glucose tolerance test (OGTT) (48% of centers), and highest for annual non-tuberculous mycobacteria (NTM) sputum culture (98%). The mean number of guidelines met was 6.7 and higher for pediatric (7.3) than adult (5.6) centers, (p<0.001). The number of guidelines met correlated negatively with Pa-IR (β=-0.007, p = 0.043). Macrolide prescription and annual OGTT per guideline were associated with lower and higher Pa-IR, respectively. Centers with lower center-wide lung function, higher proportion of pwCF with low body-mass index, and location in the Southwest had higher Pa-IR., Conclusion: Overall adherence to guidelines was high except for performing ≥4 bacterial cultures/year and OGTT. Higher Pa-IR was associated with center characteristics and lower guideline adherence. The lower Pa-IR with greater adherence to guidelines suggests that focusing on quality care can positively impact Pa-IR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: Home spirometry is increasingly used to monitor lung function in people with cystic fibrosis (pwCF). Although decreases in lung function in the setting of increased respiratory symptoms are consistent with a pulmonary exacerbation (PEx), the interpretation of home spirometry during asymptomatic periods of baseline health is unclear. The aims of this study were to determine the variation in home spirometry in pwCF during asymptomatic periods of baseline health and to identify associations between this variation and PEx., Methods: Near-daily home spirometry measurements were obtained from a cohort of pwCF enrolled in a long-term study of the airway microbiome. Associations between the degree of variation in home spirometry and the time to next PEx were evaluated., Results: Thirteen subjects (mean age of 29 years and mean percent predicted forced expiratory volume in one second [ppFEV 1 ] of 60) provided a median of 204 spirometry readings taken during 40 periods of baseline health. The mean week-to-week within-subject level of variation in ppFEV 1 was 15.2 ± 6.2%. The degree of variation in ppFEV 1 during baseline health was not associated with time to PEx., Conclusions: Variation in ppFEV 1 measured with near-daily home spirometry in pwCF during periods of baseline health exceeded the variation in ppFEV 1 expected in clinic spirometry (based on ATS guidelines). The degree of variation in ppFEV 1 during baseline health was not associated with time to PEx. These data are relevant for guiding interpretation of home spirometry., Competing Interests: Declaration of Competing Interest There are no conflicts of interests for any authors in regard to this study., (Copyright © 2023 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: Opportunistic bacterial infection is a hallmark of cystic fibrosis (CF) lung disease and early mortality. Poorly characterized prevalence changes have accompanied two decades of health improvements, with CFTR modulators likely to further affect infection epidemiology., Methods: Bacterial prevalence change trends across birth cohorts were assessed with linear regression using 2001-2019 US CF Foundation Patient Registry data. Informative missingness was assessed, as was age-to-age infection status., Results: Bacterial prevalence constantly changed from 2001 to 2019, with changes differing across birth cohorts. Informative censoring affected prevalence change for some organisms. Age-to-age infection status changes were greater than net changes in bacterial prevalence and varied by age., Conclusions: CF infection epidemiology changed over two decades and will continue to do so. Understanding how modulators affect infection epidemiology will require creative designs for longitudinal prevalence change studies emphasizing prevalence changes independent of effects on lung biology., Competing Interests: Declaration of Competing Interest The authors have no competing interests relative to this content to report. MWK received salary support to his institution from the Cystic Fibrosis Foundation and the National Institutes of Health (UL1TR002548). JJL received salary support from the Cystic Fibrosis Foundation (CAVERL22AB0)., (Copyright © 2023 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

The novel clinical-stage β-lactam-β-lactamase inhibitor combination, cefepime-taniborbactam, demonstrates promising activity toward many Gram-negative bacteria producing class A, B, C, and/or D β-lactamases. We tested this combination against a panel of 150 Burkholderia cepacia complex (Bcc) and Burkholderia gladioli strains. The addition of taniborbactam to cefepime shifted cefepime minimum inhibitory concentrations toward the provisionally susceptible range in 59% of the isolates tested. Therefore, cefepime-taniborbactam possessed similar activity as first-line agents, ceftazidime and trimethoprim-sulfamethoxazole, supporting further development., Competing Interests: D.A.S. and G.M. are employees of Venotorx Pharmaceuticals, Inc. Venatorx Pharmaceuticals, Inc., provided funding as a research grant to K.M.P-W. to conduct this study.

Background: The progression of lung disease in people with cystic fibrosis (pwCF) has been associated with a decrease in the diversity of airway bacterial communities. How often low diversity communities occur in advanced CF lung disease and how they may be associated with clinical outcomes is not clear, however., Methods: We sequenced a region of the bacterial 16S ribosomal RNA gene to characterize bacterial communities in sputum from 190 pwCF with advanced lung disease (FEV 1 ≤40% predicted), with particular attention to the prevalence and relative abundance of dominant genera. We evaluated relationships between community diversity and clinical outcomes., Results: Although most of the 190 pwCF with advanced lung disease had airway bacterial communities characterized by low diversity with a dominant genus, a considerable minority (40%) did not. The absence of a dominant genus, presence of methicillin-susceptible Staphylococcus aureus, and greater bacterial richness positively correlated with lung function. Higher relative abundance of the dominant genus and greater antimicrobial use negatively correlated with lung function. PwCF with a low diversity community and dominant genus had reduced lung transplant-free survival compared to those without (median survival of 1.6 vs 2.9 years)., Conclusions: A considerable proportion of pwCF with advanced lung disease do not have airway bacterial communities characterized by low diversity and a dominant genus and these individuals had better survival. An understanding of the antecedents of low diversity airway communities- and the impact these may have on lung disease trajectory - may provide avenues for improved management strategies., Competing Interests: Declaration of Competing Interest There are no conflicts of interests for any authors in regard to this study., (Copyright © 2023 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Background: There is increased interest in bacteriophage (phage) therapy to treat infections caused by antibiotic-resistant bacteria. A lung transplant recipient with cystic fibrosis and Burkholderia multivorans infection was treated with inhaled phage therapy for 7 days before she died., Methods: Phages were given via nebulization through the mechanical ventilation circuit. Remnant respiratory specimens and serum were collected. We quantified phage and bacterial deoxyribonucleic acid (DNA) using quantitative polymerase chain reaction, and tested phage neutralization in the presence of patient serum. We performed whole genome sequencing and antibiotic and phage susceptibility testing on 15 B. multivorans isolates. Finally, we extracted lipopolysaccharide (LPS) from two isolates and visualized their LPS using gel electrophoresis., Results: Phage therapy was temporally followed by a temporary improvement in leukocytosis and hemodynamics, followed by worsening leukocytosis on day 5, deterioration on day 7, and death on day 8. We detected phage DNA in respiratory samples after 6 days of nebulized phage therapy. Bacterial DNA in respiratory samples decreased over time, and no serum neutralization was detected. Isolates collected between 2001 and 2020 were closely related but differed in their antibiotic and phage susceptibility profiles. Early isolates were not susceptible to the phage used for therapy, while later isolates, including two isolates collected during phage therapy, were susceptible. Susceptibility to the phage used for therapy was correlated with differences in O-antigen profiles of an early versus a late isolate., Conclusions: This case of clinical failure of nebulized phage therapy highlights the limitations, unknowns, and challenges of phage therapy for resistant infections., (© 2023 The Authors. Transplant Infectious Disease published by Wiley Periodicals LLC.)

Ralstonia insidiosa and Chryseobacterium gleum are bacterial species commonly found in potable water systems, and these two species contribute to the robustness of biofilm formation in a model six-species community from the International Space Station (ISS) potable water system. Here, we set about characterizing the interaction between these two ISS-derived strains and examining the extent to which this interaction extends to other strains and species in these two genera. The enhanced biofilm formation between the ISS strains of R. insidiosa and C. gleum is robust to starting inoculum and temperature and occurs in some but not all tested growth media, and evidence does not support a soluble mediator or coaggregation mechanism. These findings shed light on the ISS R. insidiosa and C. gleum interaction, though such enhancement is not common between these species based on our examination of other R. insidiosa and C. gleum strains, as well as other species of Ralstonia and Chryseobacterium . Thus, while the findings presented here increase our understanding of the ISS potable water model system, not all our findings are broadly extrapolatable to strains found outside of the ISS. IMPORTANCE Biofilms present in drinking water systems and terminal fixtures are important for human health, pipe corrosion, and water taste. Here, we examine the enhanced biofilm of cocultures for two very common bacteria from potable water systems: Ralstonia insidiosa and Chryseobacterium gleum. While strains originally isolated on the International Space Station show enhanced dual-species biofilm formation, terrestrial strains do not show the same interaction properties. This study contributes to our understanding of these two species in both dual-culture and monoculture biofilm formation.

Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic human pathogens that are inherently multidrug resistant, limiting treatment options for infections. Here, a novel diazabicyclooctane, ETX0462, was evaluated for activity against Bcc and B . gladioli . Ninety-eight percent of the isolates examined in this study were susceptible. ETX0462 was found to demonstrate in vitro activity superior to that of currently available treatment options (e.g., trimethoprim-sulfamethoxazole and ceftazidime).

In July 2021, the Virginia Department of Health notified CDC of a cluster of eight invasive infections with Burkholderia stabilis, a bacterium in the Burkholderia cepacia complex (BCC), among hospitalized patients at hospital A. Most patients had undergone ultrasound-guided procedures during their admission. Culture of MediChoice M500812 nonsterile ultrasound gel used in hospital A revealed contamination of unopened product with B. stabilis that matched the whole genome sequencing (WGS) of B. stabilis strains found among patients. CDC and hospital A, in collaboration with partner health care facilities, state and local health departments, and the Food and Drug Administration (FDA), identified 119 B. stabilis infections in 10 U.S. states, leading to the national recall of all ultrasound gel products produced by Eco-Med Pharmaceutical (Eco-Med), the manufacturer of MediChoice M500812. Additional investigation of health care facility practices revealed frequent use of nonsterile ultrasound gel to assist with visualization in preparation for or during invasive, percutaneous procedures (e.g., intravenous catheter insertion). This practice could have allowed introduction of contaminated ultrasound gel into sterile body sites when gel and associated viable bacteria were not completely removed from skin, leading to invasive infections. This outbreak highlights the importance of appropriate use of ultrasound gel within health care settings to help prevent patient infections, including the use of only sterile, single-use ultrasound gel for ultrasonography when subsequent percutaneous procedures might be performed., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Nontuberculous mycobacteria (NTM) are frequently present in municipal drinking water and building plumbing, and some are believed to cause respiratory tract infections through inhalation of NTM-containing aerosols generated during showering. However, the present understanding of NTM transfer from water to air is insufficient to develop NTM risk mitigation strategies. This study aimed to characterize the contribution of shower water to the abundance of viable NTM in indoor air. Shower water and indoor air samples were collected, and 16S rRNA and rpoB genes were sequenced. The sequencing results showed that running the shower impacted the bacterial community structure and NTM species composition in indoor air by transferring certain bacteria from water to air. A mass balance model combined with NTM quantification results revealed that on average 1/132 and 1/254 of NTM cells in water were transferred to air during 1 hour of showering using a rain and massage showerhead, respectively. A large fraction of the bacteria transferred from water to air were membrane-damaged, i.e. they had compromised membranes based on analysis by live/dead staining and flow cytometry. However, the damaged NTM in air were recoverable as shown by growth in a culture medium mimicking the respiratory secretions of people with cystic fibrosis, implying a potential infection risk by NTM introduced to indoor air during shower running. Among the recovered NTM, Mycobacterium mucogenicum was the dominant species as determined by rpoB gene sequencing. Overall, this study lays the groundwork for future pathogen risk management and public health protection in the built environment., (© The Author(s) 2022. Published by Oxford University Press on behalf of National Academy of Sciences.)

We describe the follow-up investigation of an outbreak of endophthalmitis due to Burkholderia contaminans following cataract surgery in a single clinic. Whole-genome sequence analysis of bacteria recovered from affected patients and the clinic identified the clinic's ventilation system as the source of infection.

Antimicrobial susceptibility testing (AST) has been used to guide therapy of airway infection in persons with cystic fibrosis (CF) for decades. However, evidence that AST adds benefit to treatment outcomes in CF is lacking. In fact, the routine use of AST has potential to exacerbate inappropriate antibiotic use. Several features of airway infection in CF contribute to the limitations of AST in predicting treatment outcomes, providing rationale for abandoning this practice altogether. Other features of CF infection suggest, however, that select use of AST can provide worthwhile guidance to antibiotic selection., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Chronic polymicrobial airway infections are a hallmark of cystic fibrosis (CF) lung disease. Antibiotic therapy is a primary treatment of CF pulmonary exacerbations (PEx); however, the impact of episodic antibiotic treatment on airway bacterial communities has not been well described. We analyzed sputum samples from adults with CF obtained immediately before and during antibiotic treatment of PEx. Sequencing of the V4 region of the bacterial 16S ribosomal RNA gene was used to assess changes in bacterial community structure during antibiotic treatment. The peak impact of antibiotic treatment was observed by day four or five of treatment. These findings advance our understanding of bacterial community dynamics during antibiotic treatment of PEx and complement recent and ongoing studies evaluating the optimal duration of antibiotic therapy for PEx., (Copyright © 2022. Published by Elsevier B.V.)

Tebipenem-pivoxil hydrobromide, an orally bioavailable carbapenem, is currently in clinical development for the treatment of extended-spectrum β-lactamase- and AmpC-producing Enterobacterales. Previously, tebipenem was found to possess antimicrobial activity against the biothreat pathogens, Burkholderia pseudomallei and Burkholderia mallei . Thus, herein, tebipenem was evaluated against a panel of 150 curated strains of Burkholderia cepacia complex (Bcc) and Burkholderia gladioli , pathogens that infect people who are immunocompromised or have cystic fibrosis. Using the provisional susceptibility breakpoint of 0.12 mg/L for tebipenem, 100% of the Bcc and B . gladioli tested as being provisionally resistant to tebipenem. Bcc and B . gladioli possess two inducible chromosomal β-lactamases, PenA and AmpC. Using purified PenA1 and AmpC1, model β-lactamases expressed in Burkholderia multivorans ATCC 17616, PenA1 was found to slowly hydrolyze tebipenem, while AmpC1 was inhibited by tebipenem with a k 2 / K value of 1.9 ± 0.1 × 10 3 M -1 s -1 . In addition, tebipenem was found to be a weak inducer of bla PenA1 expression. The combination of the slow hydrolysis by PenA1 and weak induction of bla PenA1 likely compromises the potency of tebipenem against Bcc and B . gladioli .

Stenotrophomonas maltophilia is a non-fermenting, Gram-negative bacillus that has emerged as an opportunistic nosocomial pathogen. Its intrinsic multidrug resistance makes treating infections caused by S. maltophilia a great clinical challenge. Clinical management is further complicated by its molecular heterogeneity that is reflected in the uneven distribution of antibiotic resistance and virulence determinants among different strains, the shortcomings of available antimicrobial susceptibility tests and the lack of standardized breakpoints for the handful of antibiotics with in vitro activity against this microorganism. Herein, we provide an update on the most recent literature concerning these issues, emphasizing the impact they have on clinical management of S. maltophilia infections., (Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy 2022.)

Bacterial infection and inflammation of the airways are the leading causes of morbidity and mortality in persons with cystic fibrosis (CF). The ecology of the bacterial communities inhabiting CF airways is poorly understood, especially with respect to how community structure, dynamics, and microbial metabolic activity relate to clinical outcomes. In this study, the bacterial communities in 818 sputum samples from 109 persons with CF were analyzed by sequencing bacterial 16S rRNA gene amplicons. We identified eight alternative community types (pulmotypes) by using a Dirichlet multinomial mixture model and studied their temporal dynamics in the cohort. Across patients, the pulmotypes displayed chronological patterns in the transition among each other. Furthermore, significant correlations between pulmotypes and patient clinical status were detected by using multinomial mixed effects models, principal components regression, and statistical testing. Constructing pulmotype-specific metabolic activity profiles, we found that pulmotype microbiota drive distinct community functions including mucus degradation or increased acid production. These results indicate that pulmotypes are the result of ordered, underlying drivers such as predominant metabolism, ecological competition, and niche construction and can form the basis for quantitative, predictive models supporting clinical treatment decisions., (© 2021. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Background: Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P. aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time., Methods: Twenty-two sequential P. aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time., Results: The majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits., Conclusions: The overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P. aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF., Competing Interests: The authors have declared that no competing interests exist.

The last resort for treating multidrug-resistant (MDR) Pseudomonas aeruginosa and other MDR Gram-negative bacteria is a class of antibiotics called the polymyxins; however, polymyxin-resistant isolates have emerged. In response, antimicrobial peptides (AMPs) and their synthetic mimetics have been investigated as alternative therapeutic options. Oligothioetheramides (oligoTEAs) are a class of synthetic, sequence-defined oligomers composed of N -allylacrylamide monomers and an abiotic dithiol backbone that is resistant to serum degradation. Characteristic of other AMP mimetics, the precise balance between charge and hydrophobicity has afforded cationic oligoTEAs potent antimicrobial activity, particularly for the compound BDT-4G, which consists of a 1,4-butanedithiol backbone and guanidine pendant groups, the latter of which provides a cationic charge at physiological pH. However, the activity and mechanism of cationic oligoTEAs against MDR Gram-negative isolates have yet to be fully investigated. Herein, we demonstrated the potent antimicrobial activity of BDT-4G against clinical isolates of P. aeruginosa with a range of susceptibility profiles, assessed the kinetics of bactericidal activity, and further elucidated its mechanism of action. Activity was also evaluated against a panel of polymyxin-resistant isolates, including intrinsically-resistant species. We demonstrate that BDT-4G can evade some of the mechanisms conferring resistance to polymyxin B and thus may have therapeutic potential.

The IR Biotyper and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using ClinProTools software (MALDI-TOF MS-ClinProTools) are two novel typing methods that rely on the analysis of carbohydrate and peptide residues in intact bacterial cells. These two methods have shown promising results in the rapid and accurate typing of bacteria. In this study, we evaluated these novel typing methods in comparison with genotypic typing for cluster analysis of Burkholderia cenocepacia epidemic strain ET12, isolated from adult cystic fibrosis patients. Sixty-six isolates of B. cenocepacia were used in this study, 35 of which were identified as the ET12 strain and 31 as non-ET12 strains by repetitive-element PCR (rep-PCR). Twelve isolates were used for the creation of typing models using IR Biotyper and MALDI-TOF MS-ClinProTools, and 54 isolates were used for external validation of the typing models. The IR Biotyper linear discriminant analysis (LDA) model had a diagnostic sensitivity of 84.6% for typing the epidemic strain, ET12. At a cutoff of 70%, MALDI-TOF MS-ClinProTools had 87.5% diagnostic sensitivity in detecting the ET12 strain ( P =  1.00). Both methods had a diagnostic specificity of ≥80% for detecting the ET12 strain. In conclusion, IR Biotyper and MALDI-TOF MS-ClinProTools offer rapid typing using proteomics and analysis of small cellular molecules with a low running cost. Our pilot study showed suboptimal accuracy of both methods for typing outbreak strains of B. cenocepacia. Extending the spectral region analyzed by the IR Biotyper can improve the accuracy and has the potential of improving the generalizability of this technique for typing other organisms. IMPORTANCE Respiratory infections due to Burkholderia cenocepacia, particularly the ET12 epidemic strain, are considered sentinel events for persons with cystic fibrosis, as they are often associated with person-to-person transmission and accelerated decline in lung function and early mortality. Current typing methods are generally only available at reference centers, with long turn-around-times, which can affect the identification of outbreaks and critical patient triage. This pilot study aims to add to the growing literature illustrating the potential utility of Fourier transform infrared spectroscopy (FTIR), a novel rapid method, for the successful typing of clinically significant bacteria. In this study, we evaluated its utility to discriminate between the ET12 clone and non-ET12 isolates of B. cenocepacia and compared it to proteomics cluster analysis using MALDI-TOF MS and ClinProTools software. Both methods had encouraging but suboptimal accuracy (≥85% sensitivity and ≥83% specificity), which will likely be improved by extending the spectral region analyzed by the IR Biotyper with updated software.

The Burkholderia cepacia complex (BCC) is known for causing serious lung infections in people with cystic fibrosis (CF). These infections can require lung transplantation, eligibility for which may be guided by antimicrobial susceptibility testing (AST). While the Clinical and Laboratory Standards Institute recommends AST for BCC, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) does not, due to poor method performance and correlation with clinical outcomes. Furthermore, limited data exist on the performance of automated AST methods for BCC. To address these issues, reproducibility and accuracy were evaluated for disk diffusion (DD), broth microdilution (BMD), and MicroScan WalkAway using 50 B. cenocepacia and 50 B. multivorans isolates collected from people with CF. The following drugs were evaluated in triplicate: chloramphenicol (CAM), ceftazidime (CAZ), meropenem (MEM), trimethoprim-sulfamethoxazole (TMP-SMX), minocycline (MIN), levofloxacin (LVX), ciprofloxacin (CIP), and piperacillin-tazobactam (PIP-TAZ). BMD reproducibility was ≥ 95% for MEM and MIN only, and MicroScan WalkAway reproducibility was similar to BMD. DD reproducibility was < 90% for all drugs tested when a 3 mm cut-off was applied. When comparing the accuracy of DD to BMD, only MEM met all acceptance criteria. TMP-SMX and LVX had high minor errors, CAZ had unacceptable very major errors (VME), and MIN, PIP-TAZ, and CIP had both unacceptable minor errors and VMEs. For MicroScan WalkAway, no drugs met acceptance criteria. Analyses also showed that errors were not attributed to one species. In general, our data agree with EUCAST recommendations.

A recent workshop titled "Developing Models to Study Polymicrobial Infections," sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

The Burkholderia cepacia complex (Bcc) and Burkholderia gladioli are opportunistic pathogens that most commonly infect persons with cystic fibrosis or compromised immune systems. Members of the Burkholderia genus are intrinsically multidrug resistant (MDR), possessing both a PenA carbapenemase and an AmpC β-lactamase, rendering treatment of infections due to these species problematic. Here, we tested the β-lactam-β-lactamase inhibitor combination imipenem-relebactam against a panel of MDR Bcc and B. gladioli strains. The addition of relebactam to imipenem dramatically lowered the MICs for Bcc and B. gladioli: only 16% of isolates tested susceptible to imipenem, while 71.3% were susceptible to the imipenem-relebactam combination. While ceftazidime-avibactam remained the most potent combination drug against this panel of Bcc and B. gladioli strains, imipenem-relebactam was active against 71.4% of the ceftazidime-avibactam-resistant isolates. Relebactam demonstrated potent inactivation of Burkholderia multivorans PenA1, with an apparent K i ( K i app ) value of 3.2 μM. Timed mass spectrometry revealed that PenA1 formed a very stable adduct with relebactam, without any detectable desulfation for as long as 24 h. Based on our results, imipenem-relebactam may represent an alternative salvage therapy for Bcc and B. gladioli infections, especially in cases where the isolates are resistant to ceftazidime-avibactam.

Lessons learnt from the pandemic show that telehealth for cystic fibrosis allows multidisciplinary visits but better means for monitoring of lung function and microbiology are needed https://bit.ly/2V7g09x., Competing Interests: Conflict of interest: M.S. Muhlebach reports grants from the Cystic Fibrosis Foundation during the conduct of the study and service in a consulting group for Nabriva Pharmaceuticals outside the submitted work. Conflict of interest: F. Cunningham has nothing to disclose. Conflict of interest: J.J. LiPuma has nothing to disclose. Conflict of interest: J.J. Zhou reports grants from the US Cystic Fibrosis Foundation during the conduct of the study. Conflict of interest: L. Saiman reports grants from the Cystic Fibrosis Foundation during the conduct of the study., (Copyright ©The authors 2021.)

Simple and rapid detection of Burkholderia cepacia complex (BCC) bacteria, a common cause of pharmaceutical product recalls, is essential for consumer safety. In this study, we developed and evaluated a ribB -based colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of BCC in (i) nuclease-free water after 361 days, (ii) 10 μg/mL chlorhexidine gluconate (CHX) solutions, and (iii) 50 μg/mL benzalkonium chloride (BZK) solutions after 184 days. The RibB 5 primer specifically detected 20 strains of BCC but not 36 non-BCC strains. The limit of detection of the LAMP assay was 1 pg/μL for Burkholderia cenocepacia strain J2315. Comparison of LAMP with a qPCR assay using 1440 test sets showed higher sensitivity: 60.6% in nuclease-free water and 42.4% in CHX solution with LAMP vs. 51.3% and 31.1%, respectively, with qPCR. These results demonstrate the potential of the ribB -based LAMP assay for the rapid and sensitive detection of BCC in pharmaceutical manufacturing.

Rationale: Pulmonary infections with nontuberculous mycobacteria (NTM) are increasingly prevalent in people with cystic fibrosis (CF). Clinical outcomes following NTM acquisition are highly variable, ranging from transient self-resolving infection to NTM pulmonary disease associated with significant morbidity. Relationships between airway microbiota and variability of NTM outcomes in CF are unclear., Objective: To identify features of CF airway microbiota associated with outcomes of NTM infection., Methods: 188 sputum samples, obtained from 24 subjects with CF, each with three or more samples collected from 3.5 years prior to, and up to 6 months following incident NTM infection, were selected from a sample repository. Sputum DNA underwent bacterial 16S rRNA gene sequencing. Airway microbiota were compared based on the primary outcome, a diagnosis of NTM pulmonary disease, using Wilcoxon rank-sum testing, autoregressive integrated moving average modelling and network analyses., Measurements and Main Results: Subjects with and without NTM pulmonary disease were similar in clinical characteristics, including age and lung function at the time of incident NTM infection. Time-series analyses of sputum samples prior to incident NTM infection identified positive correlations between Pseudomonas , Streptococcus , Veillonella , Prevotella and Rothia with diagnosis of NTM pulmonary disease and with persistent NTM infection. Network analyses identified differences in clustering of taxa between subjects with and without NTM pulmonary disease, and between subjects with persistent versus transient NTM infection., Conclusions: CF airway microbiota prior to incident NTM infection are associated with subsequent outcomes, including diagnosis of NTM pulmonary disease, and persistence of NTM infection. Associations between airway microbiota and NTM outcomes represent targets for validation as predictive markers and for future therapies., Competing Interests: Conflict of interest: L.J. Caverly has nothing to disclose. Conflict of interest: M. Zimbric has nothing to disclose. Conflict of interest: M. Azar has nothing to disclose. Conflict of interest: K. Opron has nothing to disclose. Conflict of interest: J.J. LiPuma has nothing to disclose., (Copyright ©The authors 2021.)

Introduction. In recent years, the Herbaspirillum genus has emerged as a pathogen in healthcare-related infections and has became stablished as an opportunistic pathogen. Hypothesis/Gap Statement. Little is known about the pathogenesis induced by Herbaspirillum genus. Aim. To evaluate the cytotoxic effects of genus Herbaspirillum , its ability to adhere to lung human cells and the ability of environmental and clinical strains of Herbaspirillum to induce pneumonia in mice. Methodology. Environmental and clinical isolates of Herbaspirillum were examined for their cytotoxic effects on the Calu-3 cell lineage. Cytotoxic activity of secretome was tested using MTT/neutral red assays and cell morphology analysis. Herbaspirillum adhesion on Calu-3 cells was assessed using bright-field microscopy and cell-associated bacteria were counted. A mouse model of acute lung infection was done using a clinical and an environmental strain. Adult male mice were used, and the pneumonia was inducted by intra-tracheal inoculation of 10 8 or 10 9 bacteria. Mice weight variations were evaluated at the end of the experiment. Bronchoalveolar lavage was collected and evaluated for total and differential cytology. A histological examination of lungs was performed giving a histological score. Results. The secretomes of all the strains induced morphological alterations in cells, but only H. seropedicae SmR1 were cytotoxic in MTT and neutral red assays. Clinical strains of H. frisingense AU14459 and H. hutttiense subsp. huttiense AU11883 exhibited low adherence to lung cells, while SmR1 was non-adhesive. Following intratracheal inoculation, mice treated with 10 9 c.f.u. of the SmR1 and AU11883 strains lost 18 and 6% of their weight over 7 days, respectively, and presented moderate clinical signs. Infected mice showed inflammatory cell infiltration in the perivascular and peribroncheal/peribronchiolar spaces. Bronchoalveolar fluid of mice inoculated with SmR1 10 9 c.f.u. presented an increase in total leucocyte cells and in neutrophils population. Conclusion. These in vivo and in vitro results provide insights into how some Herbaspirillum strains cause infection in humans, providing a basis for the characterization of pathogenesis studies on this emerging infectious agent.

Background: Molecular diagnostics have led to the identification of a broad range of bacterial species in cystic fibrosis (CF) including Inquilinus. The clinical significance of Inquilinus in CF has not been thoroughly characterized., Methods: Retrospective, case-control study of persons with CF from two CF centers with at least one respiratory culture positive for Inquilinus spp. compared with age-matched CF controls with chronic Pseudomonas aeruginosa. Percent predicted forced expiratory volume in one second (ppFEV1) and body mass index percentile (BMI) were modeled from time of first positive culture up to 5 years later. Rates of pulmonary exacerbations were compared. Inquilinus isolates were genotyped to evaluate strain diversity., Results: Seventeen patients with Inquilinus infection were identified with a mean age of 13 years at first positive culture. Most cases had multiple cultures positive for Inquilinus. ppFEV1 was not different between cases versus controls (80.2% vs 81.6%, p = 0.97 at baseline, 67.5% vs. 73.3%, p = 0.82 at 5 years). Patients were undernourished and BMI percentiles did not differ between groups (30.7% vs 43.4%, p = 0.32 at baseline, 37.9% vs. 37.6%, p = 0.98 at 5 years). There was no difference in the pulmonary exacerbation rate (3.0/year vs 2.5/year, p = 0.34). Genotyping showed diverse genetic strains between patients., Conclusions: Inquilinus can present in childhood and is often associated with chronic infection in CF. Lung function and nutrition status at time of detection, lung function decline, and pulmonary exacerbation rates in Inquilinus cases were similar to those with chronic P. aeruginosa, a well-established CF pathogen., Competing Interests: Declarations of Competing Interest None., (Copyright © 2020. Published by Elsevier B.V.)

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, Burkholderia mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli , which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen β-lactamase, which confers resistance to β-lactams. The β-lactam-β-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. In this study, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured MICs of SUL-DUR against BCC and B. gladioli ( n  = 150), B. mallei ( n  = 30), and B. pseudomallei ( n  = 28), studied the kinetics of inhibition of the PenA1 β-lactamase from B. multivorans and the PenI β-lactamase from B. pseudomallei by durlobactam, tested for bla PenA1 induction by SUL-DUR, and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 μg/ml. Durlobactam potently inhibited PenA1 and PenI with second-order rate constant for inactivation ( k 2 /K ) values of 3.9 × 10 6 M -1 s -1 and 2.6 × 10 3 M -1 s -1 and apparent K i ( K i app ) of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest that SUL-DUR may be useful as a treatment for Burkholderia infections., (Copyright © 2021 American Society for Microbiology.)