Lack of correlation between in vitro and within patient measures of P. aeruginosa biofilms in cystic fibrosis.

Current in vitro biofilm modelling of the opportunistic pathogen, Pseudomonas aeruginosa (PA) in people with cystic fibrosis (PwCF) is limited in its ability to mimic the complexities of the cystic fibrosis (CF) lung environment. Recent adaptations of the Microbial Identification after Passive CLARITY Technique (MiPACT) in CF research have allowed for the direct imaging of PA biofilm spatial organization and structure in expectorated sputum. Here, we performed a comparative analysis of in vitro and within patient (ex vivo) measures of PA biofilms using sputa from new onset infected children with CF. MiPACT-fluorescent in situ hybridization (FISH) and fluorescent anti-Psl monoclonal antibody (mAb) staining was performed to directly visualize PA and Psl (exopolysaccharide in PA biofilm matrix) in 11 CF sputum specimens. Corresponding PA isolates, recovered from the same sputum samples, were grown as biofilms in a glass slide chamber model, then visualized by fluorescent live-cell and anti-Psl mAb staining. We observed that PA biovolume, aggregation and Psl antibody binding (normalized per PA biovolume) in CF sputum did not correlate with the in vitro model, although a trend towards significance in the biovolume relationship was observed with the addition of sputum supernatant to the in vitro model.

Predictive modeling of antibiotic eradication therapy success for new-onset Pseudomonas aeruginosa pulmonary infections in children with cystic fibrosis.

Chronic Pseudomonas aeruginosa (Pa) lung infections are the leading cause of mortality among cystic fibrosis (CF) patients; therefore, the eradication of new-onset Pa lung infections is an important therapeutic goal that can have long-term health benefits. The use of early antibiotic eradication therapy (AET) has been shown to clear the majority of new-onset Pa infections, and it is hoped that identifying the underlying basis for AET failure will further improve treatment outcomes. Here we generated machine learning models to predict AET outcomes based on pathogen genomic data. We used a nested cross validation design, population structure control, and recursive feature selection to improve model performance and showed that incorporating population structure control was crucial for improving model interpretation and generalizability. Our best model, controlling for population structure and using only 30 recursively selected features, had an area under the curve of 0.87 for a holdout test dataset. The top-ranked features were generally associated with motility, adhesion, and biofilm formation.

Pseudomonas aeruginosa aggregation and Psl expression in sputum is associated with antibiotic eradication failure in children with cystic fibrosis.

We previously demonstrated that P. aeruginosa isolates that persisted in children with cystic fibrosis (CF) despite inhaled tobramycin treatment had increased anti-Psl antibody binding in vitro compared to those successfully eradicated. We aimed to validate these findings by directly visualizing P. aeruginosa in CF sputum. This was a prospective observational study of children with CF with new-onset P. aeruginosa infection who underwent inhaled tobramycin eradication treatment. Using microbial identification passive clarity technique (MiPACT), P. aeruginosa was visualized in sputum samples obtained before treatment and classified as persistent or eradicated based on outcomes. Pre-treatment isolates were also grown as biofilms in vitro. Of 11 patients enrolled, 4 developed persistent infection and 7 eradicated infection. P. aeruginosa biovolume and the number as well as size of P. aeruginosa aggregates were greater in the sputum of those with persistent compared with eradicated infections (p < 0.01). The amount of Psl antibody binding in sputum was also greater overall (p < 0.05) in samples with increased P. aeruginosa biovolume. When visualized in sputum, P. aeruginosa had a greater biovolume, with more expressed Psl, and formed more numerous, larger aggregates in CF children who failed eradication therapy compared to those who successfully cleared their infection.

Opportunistic Pathogens in Cystic Fibrosis: Epidemiology and Pathogenesis of Lung Infection.

Cystic fibrosis (CF) is one of the most common life-shortening genetic diseases in Caucasians. Due to abnormal accumulation of mucus, respiratory failure caused by chronic infections is the leading cause of mortality in this patient population. The microbiology of these respiratory infections includes a distinct set of opportunistic pathogens, including Pseudomonas aeruginosa, Burkholderia spp., Achromobacter spp., Stenotrophomonas maltophilia, anaerobes, nontuberculous mycobacteria, and fungi. In recent years, culture-independent methods have shown the polymicrobial nature of lung infections, and the dynamics of microbial communities. The unique environment of the CF airway predisposes to infections caused by opportunistic pathogens. In this review, we will highlight how the epidemiology and role in disease of these pathogens in CF differ from that in individuals with other medical conditions. Infectious diseases (ID) physicians should be aware of these differences and the specific characteristics of infections associated with CF.

The role of Psl in the failure to eradicate Pseudomonas aeruginosa biofilms in children with cystic fibrosis.

The exopolysaccharide Psl contributes to biofilm structure and antibiotic tolerance and may play a role in the failure to eradicate Pseudomonas aeruginosa from cystic fibrosis (CF) airways. The study objective was to determine whether there were any differences in Psl in P. aeruginosa isolates that were successfully eradicated compared to those that persisted, despite inhaled tobramycin treatment, in children with CF. Initial P. aeruginosa isolates were collected from children with CF undergoing eradication treatment, grown as biofilms and labeled with 3 anti-Psl monoclonal antibodies (Cam003/Psl0096, WapR001, WapR016) before confocal microscopy visualization. When grown as biofilms, P. aeruginosa isolates from children who failed antibiotic eradication therapy, had significantly increased Psl0096 binding compared to isolates from those who cleared P. aeruginosa. This was confirmed in P. aeruginosa isolates from the SickKids Eradication Cohort as well as the Early Pseudomonas Infection Control (EPIC) trial. Increased anti-Psl antibody binding was associated with bacterial aggregation and tobramycin tolerance. The biofilm matrix represents a potential therapeutic target to improve P. aeruginosa eradication treatment.

Evaluation of digital dispense-assisted broth microdilution antimicrobial susceptibility testing for Pseudomonas aeruginosa isolates.

Antimicrobial susceptibility testing (AST) is essential for detecting resistance in Pseudomonas aeruginosa and other bacterial pathogens. Here we evaluated the performance of broth microdilution (BMD) panels created using a semi-automated liquid handler, the D300e Digital Dispenser (Tecan Group Ltd., CH) that relies on inkjet printing technology. Microtitre panels (96-well) containing nine twofold dilutions of 12 antimicrobials from five classes (ß-lactams, ß-lactam/ß-lactamase inhibitors, aminoglycosides, fluoroquinolones, polymyxins) were prepared in parallel using the D300e Digital Dispenser and standard methods described by CLSI/ISO. To assess performance, panels were challenged with three well characterized quality control organisms and 100 clinical P. aeruginosa isolates. Traditional agreement and error measures were used for evaluation. Essential (EA) and categorical (CA) agreements were 92.7% and 98.0% respectively for P. aeruginosa isolates with evaluable on-scale results. The majority of minor errors that fell outside acceptable EA parameters (≥ ± 1 dilution, 1.9%) were seen with aztreonam (5%) and ceftazidime (4%), however all antimicrobials displayed acceptable performance in this situation. Differences in MIC were often log2 dilution lower for D300e dispensed panels. Major and very major errors were noted for aztreonam (2.6%) and cefepime (1.7%) respectively. The variable performance of D300e panels suggests that further testing is required to confirm their diagnostic utility for P. aeruginosa.

Antimicrobial resistance: Concerns of healthcare providers and people with CF.

BACKGROUND: Chronic lung infections and their treatment pose risks for the development of antimicrobial resistance (AMR) in people with cystic fibrosis (PWCF). In this study, we evaluated the attitudes of healthcare providers' (HCP) and PWCF or their parents' toward AMR within the international CF community. METHODS: HCP and PWCF identified through listservs and CF-related organizations were asked to complete an AMR centered survey, with additional questions on antimicrobial stewardship (AMS) for HCP. Descriptive analyses are reported. RESULTS: The responding 443 HCP and 464 PWCF/Parents were from 30 and 25 countries, respectively. Sixty-two percent of HCP and 56% of PWCF stated they were "very concerned" about AMR, with Pseudomonas spp. and Burkholderia spp. considered the most concerning organisms for both HCP and PWCF/Parents. Non-tuberculous mycobacteria were of greater concern to HCP compared to PWCF/Parents. There was a discrepancy regarding AMR education to PWCF, with 80% of HCP stating having discussed this with PWCF/Parents, but only 50% of PWCF recalling such discussions. CONCLUSION: These results highlight that AMR is relevant to CF HCP and PWCF internationally, indicating that educational tools and research are warranted.

Ecological Succession of Polymicrobial Communities in the Cystic Fibrosis Airways.

Antimicrobial therapies against cystic fibrosis (CF) lung infections are largely aimed at the traditional, well-studied CF pathogens such as Pseudomonas aeruginosa and Burkholderia cepacia complex, despite the fact that the CF lung harbors a complex and dynamic polymicrobial community. A clinical focus on the dominant pathogens ignores potentially important community-level interactions in disease pathology, perhaps explaining why these treatments are often less effective than predicted based on in vitro testing. A better understanding of the ecological dynamics of this ecosystem may enable clinicians to harness these interactions and thereby improve treatment outcomes. Like all ecosystems, the CF lung microbial community develops through a series of stages, each of which may present with distinct microbial communities that generate unique host-microbe and microbe-microbe interactions, metabolic profiles, and clinical phenotypes. While insightful models have been developed to explain some of these stages and interactions, there is no unifying model to describe how these infections develop and persist. Here, we review current perspectives on the ecology of the CF airway and present the CF Ecological Succession (CFES) model that aims to capture the spatial and temporal complexity of CF lung infection, address current challenges in disease management, and inform the development of ecologically driven therapeutic strategies.

Burkholderia cenocepacia ET12 transmission in adults with cystic fibrosis.

This report describes transmission of a Burkholderia cenocepacia ET12 strain (ET12-Bc) at the Toronto Adult Cystic Fibrosis (CF) Centre occurring from 2008 to 2017. Epidemiological and genomic data from 11 patients with CF were evaluated. Isolates were analysed using whole genome sequencing (WGS). Epidemiological investigation and WGS analysis suggested nosocomial transmission, despite enhanced infection control precautions. This was associated with subsequent deaths in 10 patients. ET12-Bc positive patients are no longer cared for on the same unit as ET12-Bc negative patients.

Antimicrobial resistance in cystic fibrosis: A Delphi approach to defining best practices.

BACKGROUND: Antimicrobial susceptibility testing (AST) is a cornerstone of infection management in cystic fibrosis. However, there is little evidence that AST predicts the clinical outcome of CF antimicrobial treatment. It has been suggested there is a need for careful consideration of current AST use by the CF community. METHODS: We engaged a group of experts consisting of pulmonary (adult and pediatric) and infectious disease clinicians, microbiologists, and pharmacists representing a broad international experience. We conducted an iterative systematic survey (Delphi) to determine and quantify consensus regarding key questions facing CF clinicians in the use of respiratory culture results including what tests to order, when to obtain them, and how to act upon the results of the testing. RESULTS: Consensus was reached for many questions but there was not universal agreement to the questions that were addressed. There were some differences with respect to cultures obtained for surveillance compared to when there is clinical worsening. Areas of general consensus include when and how respiratory cultures should be performed, what information should be reported, and when AST should be performed. A key finding is that clinical response to treatment is used to guide treatment decisions rather than AST results. CONCLUSIONS: Recommendations are presented regarding questions related to microbiology testing for patients with CF. We have also offered recommendations for priority research questions.

Microbiology of Cystic Fibrosis Airway Disease.

Although survival of individuals with cystic fibrosis (CF) has been continuously improving for the past 40 years, respiratory failure secondary to recurrent pulmonary infections remains the leading cause of mortality in this patient population. Certain pathogens such as Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and species of the Burkholderia cepacia complex continue to be associated with poorer clinical outcomes including accelerated lung function decline and increased mortality. In addition, other organisms such as anaerobes, viruses, and fungi are increasingly recognized as potential contributors to disease progression. Culture-independent molecular methods are also being used for diagnostic purposes and to examine the interaction of microorganisms in the CF airway. Given the importance of CF airway infections, ongoing initiatives to promote understanding of the epidemiology, clinical course, and treatment options for these infections are needed.

Reconciling Antimicrobial Susceptibility Testing and Clinical Response in Antimicrobial Treatment of Chronic Cystic Fibrosis Lung Infections.

Median cystic fibrosis (CF) survival has increased dramatically over time due to several factors, including greater availability and use of antimicrobial therapies. During the progression of CF lung disease, however, the emergence of multidrug antimicrobial resistance can limit treatment effectiveness, threatening patient longevity. Current planktonic-based antimicrobial susceptibility testing lacks the ability to predict clinical response to antimicrobial treatment of chronic CF lung infections. There are numerous reasons for these limitations including bacterial phenotypic and genotypic diversity, polymicrobial interactions, and impaired antibiotic efficacy within the CF lung environment. The parallels to other chronic diseases such as non-CF bronchiectasis are discussed as well as research priorities for moving forward.

Antimicrobial susceptibility testing (AST) and associated clinical outcomes in individuals with cystic fibrosis: A systematic review.

BACKGROUND: Antimicrobial susceptibility testing (AST) is a cornerstone of infection management. Cystic fibrosis (CF) treatment guidelines recommend AST to select antimicrobial treatments for CF airway infection but its utility in this setting has never been objectively demonstrated. METHODS: We conducted a systematic review of primary published articles designed to address two PICO (patient, intervention, comparator, outcome) questions: 1) "For individuals with CF, is clinical response to antimicrobial treatment of bacterial airways infection predictable from AST results available at treatment initiation?" and 2) "For individuals with CF, is clinical response to antimicrobial treatment of bacterial airways infection affected by the method used to guide antimicrobial selection?" Relationships between AST results and clinical response (changes in pulmonary function, weight, signs and symptoms of respiratory tract infection, and time to next event) were assessed for each article and results were compared across articles when possible. RESULTS: Twenty-five articles describing the results of 20 separate studies, most of which described Pseudomonas aeruginosa treatment, were identified. Thirteen studies described pulmonary exacerbation (PEx) treatment and seven described 'maintenance' of chronic bacterial airways infection. In only three of 16 studies addressing PICO question #1 was there a suggestion that baseline bacterial isolate antimicrobial susceptibility was associated with clinical response to treatment. None of the four studies addressing PICO question #2 suggested that antimicrobial selection methods influenced clinical outcomes. CONCLUSIONS: There is little evidence that AST predicts the clinical outcome of CF antimicrobial treatment, suggesting a need for careful consideration of current AST use by the CF community.

Penicillin-binding protein 3 is a common adaptive target among Pseudomonas aeruginosa isolates from adult cystic fibrosis patients treated with ß-lactams.

OBJECTIVE: Determining the mechanisms that modulate ß-lactam resistance in clinical Pseudomonas aeruginosa (P. aeruginosa) isolates can be challenging, as the molecular profiles identified in mutation-based or expression-based resistance determinant screens may not correlate with in vitro phenotypes. One of the lesser studied resistance mechanisms in P. aeruginosa is the modification of penicillin-binding protein 3 (pbpB/ftsI). This study reported that nonsynonymous polymorphisms within pbpB frequently occur among ß-lactam resistant sputum isolates, and are associated with unique antibiotic susceptibility patterns. METHODS: Longitudinally collected isolates (n = 126) from cystic fibrosis (CF) patients with or without recent ß-lactam therapy or of non-clinical origin were tested for susceptibility to six ß-lactams (aztreonam, ceftazidime, cefsulodin, cefepime, meropenem, and piperacillin). Known ß-lactam resistance mechanisms were characterised by polymerase chain reaction (PCR)-based methods, and polymorphisms in the transpeptidase-encoding domain of pbpB identified by sequencing. RESULTS: Twelve nonsynonymous polymorphisms were detected among 86 isolates (67%) from five CF patients with a history of ß-lactam therapy, compared with one polymorphism in 30 (3.3%) from three patients who had not received ß-lactam treatments. No nonsynonymous polymorphisms were found in ten environmental isolates. Multiple pbpB alleles, often with different combinations of polymorphisms, were detected within the population of strains from each CF patient for up to 2.6 years. Traditional patterns of ampC or mexA de-repression reduced expression of oprD or the presence of extended-spectrum ß-lactamases were not observed in resistant isolates with nonsynonymous polymorphisms in pbpB. CONCLUSION: This study's findings suggest that pbpB is a common adaptive target, and may contribute to the development of ß-lactam resistance in P. aeruginosa.

Burkholderia multivorans septicemia in a pediatric liver transplant patient.

"Cepacia syndrome", caused by Burkholderia cepacia complex and often associated with cystic fibrosis, carries a high mortality rate. It is rare for Burkholderia multivorans, a species within the B. cepacia complex, to cause cepacia syndrome even among patients with cystic fibrosis. This is the first reported fatal case of cepacia syndrome caused by B. multivorans occurring in a pediatric liver transplant recipient who does not have cystic fibrosis. We describe the unique characteristics of this pathogen among the non-cystic fibrosis population and the importance of early recognition and treatment.

A genome-wide association analysis reveals a potential role for recombination in the evolution of antimicrobial resistance in Burkholderia multivorans.

Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomics study of 111 B. multivorans sputum isolates from one CF patient through three stages of infection including an early incident isolate, deep sampling of a one-year period of chronic infection occurring weeks before a lung transplant, and deep sampling of a post-transplant infection. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found the incident isolate was basally related to the rest of the strains and more susceptible to antibiotics from three classes (ß-lactams, aminoglycosides, quinolones). The chronic infection isolates diversified into multiple, distinct genetic lineages and showed reduced antimicrobial susceptibility to the same antibiotics. The post-transplant reinfection isolates derived from the same source as the incident isolate and were genetically distinct from the chronic isolates. They also had a level of susceptibility in between that of the incident and chronic isolates. We identified numerous examples of potential parallel pathoadaptation, in which multiple mutations were found in the same locus or even codon. The set of parallel pathoadaptive loci was enriched for functions associated with virulence and resistance. Our GWAS analysis identified statistical associations between a polymorphism in the ampD locus with resistance to ß-lactams, and polymorphisms in an araC transcriptional regulator and an outer membrane porin with resistance to both aminoglycosides and quinolones. Additionally, these three loci were independently mutated four, three and two times, respectively, providing further support for parallel pathoadaptation. Finally, we identified a minimum of 14 recombination events, and observed that loci carrying putative parallel pathoadaptations and polymorphisms statistically associated with ß-lactam resistance were over-represented in these recombinogenic regions.

Defining antimicrobial resistance in cystic fibrosis.

Antimicrobial resistance (AMR) can present significant challenges in the treatment of cystic fibrosis (CF) lung infections. In CF and other chronic diseases, AMR has a different profile and clinical consequences compared to acute infections and this requires different diagnostic and treatment approaches. This review defines AMR, explains how it occurs, describes the methods used to measure AMR as well as their limitations, and concludes with future directions for research and development in the area of AMR in CF.