SARS-CoV-2 antibodies and their neutralizing capacity against live virus in human milk after COVID-19 infection and vaccination: prospective cohort studies.

BACKGROUND: There is limited understanding of the impact of coronavirus disease 2019 (COVID-19) infection and vaccination type and interval on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) human milk antibodies and their neutralizing capacity. OBJECTIVES: These cohort studies aimed to determine the presence of antibodies and live virus neutralizing capacity in milk from females infected with COVID-19, unexposed milk bank donors, and vaccinated females and examine impacts of vaccine interval and type. METHODS: Milk was collected from participants infected with COVID-19 during pregnancy or lactation (Cohort-1) and milk bank donors (Cohort-2) from March 2020-July 2021 at 3 sequential 4-wk intervals and COVID-19 vaccinated participants with varying dose intervals (Cohort-3) (January-October 2021). Cohort-1 and Cohort-3 were recruited from Sinai Health (patients) and through social media. Cohort-2 included Ontario Milk Bank donors. Milk was examined for SARS-CoV-2 antibodies and live virus neutralization. RESULTS: Of females with COVID-19, 53% (Cohort-1, n = 55) had anti-SARS-CoV-2 IgA antibodies in ≥1 milk sample. IgA+ samples (40%) were more likely neutralizing than IgA- samples (odds ratio [OR]: 2.18; 95% confidence interval [CI]: 1.03, 4.60; P = 0.04); however, 25% of IgA- samples were neutralizing. Both IgA positivity and neutralization decreased ∼6 mo after symptom onset (0-100 compared with 201+ d: IgA OR: 14.30; 95% CI: 1.08, 189.89; P = 0.04; neutralizing OR: 4.30; 95% CI: 1.55, 11.89; P = 0.005). Among milk bank donors (Cohort-2, n = 373), 4.3% had IgA antibodies; 23% of IgA+ samples were neutralizing. Vaccination (Cohort-3, n = 60) with mRNA-1273 and shorter vaccine intervals (3 to <6 wk) resulted in higher IgA and IgG than BNT162b2 (P < 0.04) and longer intervals (6 to <16 wk) (P≤0.02), respectively. Neutralizing capacity increased postvaccination (P = 0.04) but was not associated with antibody positivity. CONCLUSIONS: SARS-CoV-2 infection and vaccination (type and interval) impacted milk antibodies; however, antibody presence did not consistently predict live virus neutralization. Although human milk is unequivocally the best way to nourish infants, guidance on protection to infants following maternal infection/vaccination may require more nuanced messaging. This study was registered at clinicaltrials.gov as NCT04453969 and NCT04453982.

Carbonate chemistry and carbon sequestration driven by inorganic carbon outwelling from mangroves and saltmarshes.

Mangroves and saltmarshes are biogeochemical hotspots storing carbon in sediments and in the ocean following lateral carbon export (outwelling). Coastal seawater pH is modified by both uptake of anthropogenic carbon dioxide and natural biogeochemical processes, e.g., wetland inputs. Here, we investigate how mangroves and saltmarshes influence coastal carbonate chemistry and quantify the contribution of alkalinity and dissolved inorganic carbon (DIC) outwelling to blue carbon budgets. Observations from 45 mangroves and 16 saltmarshes worldwide revealed that >70% of intertidal wetlands export more DIC than alkalinity, potentially decreasing the pH of coastal waters. Porewater-derived DIC outwelling (81 ± 47 mmol m-2 d-1 in mangroves and 57 ± 104 mmol m-2 d-1 in saltmarshes) was the major term in blue carbon budgets. However, substantial amounts of fixed carbon remain unaccounted for. Concurrently, alkalinity outwelling was similar or higher than sediment carbon burial and is therefore a significant but often overlooked carbon sequestration mechanism.

Network connectivity and local transcriptomic vulnerability underpin cortical atrophy progression in Parkinson's disease.

Parkinson's disease pathology is hypothesized to spread through the brain via axonal connections between regions and is further modulated by local vulnerabilities within those regions. The resulting changes to brain morphology have previously been demonstrated in both prodromal and de novo Parkinson's disease patients. However, it remains unclear whether the pattern of atrophy progression in Parkinson's disease over time is similarly explained by network-based spreading and local vulnerability. We address this gap by mapping the trajectory of cortical atrophy rates in a large, multi-centre cohort of Parkinson's disease patients and relate this atrophy progression pattern to network architecture and gene expression profiles. Across 4-year follow-up visits, increased atrophy rates were observed in posterior, temporal, and superior frontal cortices. We demonstrated that this progression pattern was shaped by network connectivity. Regional atrophy rates were strongly related to atrophy rates across structurally and functionally connected regions. We also found that atrophy progression was associated with specific gene expression profiles. The genes whose spatial distribution in the brain was most related to atrophy rate were those enriched for mitochondrial and metabolic function. Taken together, our findings demonstrate that both global and local brain features influence vulnerability to neurodegeneration in Parkinson's disease.

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.

A Prolonged Outbreak of Human Adenovirus A31 (HAdV-A31) Infection on a Pediatric Hematopoietic Stem Cell Transplantation Ward with Whole Genome Sequencing Evidence of International Linkages.

A surge in hematopoietic stem cell transplantation (HSCT) human adenovirus A31 (HAdV-A31) infections was initially observed in late 2014/2015 at SickKids (SK) Hospital, Toronto, Canada. In response, enhanced laboratory monitoring for all adenovirus infections was conducted. Positive samples underwent genotyping, viral culture, and, in selected cases, whole-genome sequencing (WGS). HAdV-A31 specimens/DNA obtained from four international pediatric HSCT centers also underwent WGS. During the SK outbreak period (27 October 2014 to 31 October 2018), 17/20 HAdV-A31 isolates formed a distinct clade with 0 to 8 mutations between the closest neighbors. Surveillance before and after the outbreak detected six additional HAdV-A31 HSCT cases; three of the four sequenced cases clustered within the outbreak clade. Two SK outbreak isolates were identical to sequences from two patients in an outbreak in England. Three SK non-outbreak sequences also had high sequence similarity to strains from three international centers. Environmental PCR testing of the HSCT ward showed significant adenovirus contamination. Despite intense infection control efforts, we observed re-occurrence of infection with the outbreak strain. Severe but nonfatal infection was observed more commonly with HAdV-A31 compared to other genotypes, except HAdV-C1. Our findings strongly implicate nosocomial spread of HAdV-A31 over 10 years on a HSCT unit and demonstrate the value of WGS in defining and mapping the outbreak. Close linkages among strains in different countries suggest international dissemination, though the mechanism is undetermined. This large, extended outbreak emphasizes the pre-eminent role of HAdV-A31 in causing intractable pediatric HSCT outbreaks of severe illness worldwide.

Elucidating sources of atmospheric NOX pollution in a heavily urbanized environment using multiple stable isotopes.

Atmospheric deposition of nitrogen (N) from rain and aerosols can be a significant non-point source - particularly in urbanized coastal areas and contribute to coastal eutrophication and hypoxia. Here, we present geochemical and isotopic data from surface waters coupled with an 18-month time series of geochemical and isotopic data measured on wet and dry deposition over Hong Kong from June 2018. Dual stable isotopes of nitrate (δ15N-NO3- and δ18O-NO3-) of rain and total suspended particulates (TSP) were analyzed to trace the sources and understand seasonal pattern of atmospheric nitrate. The δ15N of TSP, δ15N-NO3 in rain and TSP ranged from +0.94 to +17.6‰, -4.1 to +3.0‰ and -1.3 to +9.0‰ respectively. δ15N varied seasonally with higher values in winter and lower values in summer. This variation can be explained by a change in the sources of atmospheric NOx driven by the East Asian Monsoon. It was found that most NOx comes from coal burning in winter and a mix of vehicle emissions, fossil fuel combustion and lightning in summer. Moreover, the estimated dry and wet deposition of nitrate and ammonium in Hong Kong is around 18 kg N ha-1 annually, which is of the same order of magnitude as N released by sewage effluents and groundwater. This implies that atmospheric N deposition over the N-limited waters of the eastern side of Hong Kong could contribute significantly to the N budget. Therefore, atmospheric N deposition may alter the local N marine cycling, thus monitoring its impact is crucial for water quality in Southern China.

Assessment of panfungal PCR performance with formalin-fixed paraffin-embedded tissue specimens†.

We reviewed the performance of a panfungal ITS-2 PCR and Sanger sequencing assay performed on 88 FFPE specimens at The Hospital for Sick Children (Toronto, Canada) in 2019. A potential fungal pathogen was identified by ITS PCR in 62.7 and 2.9% of positive and negative direct slide examination of tissue specimens, respectively. ITS amplicons were detected in 87/88 specimens, with 53/88 (60.2%) considered as 'positive-contaminants' and 34/88 (38.6%) as 'positive-potential pathogen' upon sequencing. Potential pathogens included Blastomyces dermatitidis (17.1%), Cryptococcus neoformans (17.1%), Histoplasma capsulatum (14.3%) and Mucormycetes (11.4%). Laboratories should only perform ITS PCR on FFPE tissues if fungal elements have been confirmed on histopathology slides. LAY SUMMARY: In this study, we examined how well a DNA-based test could detect DNA from fungi in archived human biopsy tissues. The best performance was achieved if fungi were seen in the tissue under a microscope before being tested. Our results indicate that we should only use this test if these conditions are met.

Relationship between impulsivity, uncontrolled eating and body mass index: a hierarchical model.

BACKGROUND: Impulsivity increases the risk for obesity and weight gain. However, the precise role of impulsivity in the aetiology of overeating behavior and obesity is currently unknown. Here we examined the relationships between personality-related measures of impulsivity, Uncontrolled Eating, body mass index (BMI), and longitudinal weight changes. In addition, we analyzed the associations between general impulsivity domains and cortical thickness to elucidate brain vulnerability factors related to weight gain. METHODS: Students (N = 2318) in their first year of university-a risky period for weight gain-completed questionnaire measures of impulsivity and eating behavior at the beginning of the school year. We also collected their weight at the end of the term (N = 1177). Impulsivity was divided into three factors: stress reactivity, reward sensitivity and lack of self-control. Using structural equation models, we tested a hierarchical relationship, in which impulsivity traits were associated with Uncontrolled Eating, which in turn predicted BMI and weight change. Seventy-one participants underwent T1-weighted MRI to investigate the correlation between impulsivity and cortical thickness. RESULTS: Impulsivity traits showed positive correlations with Uncontrolled Eating. Higher scores in Uncontrolled Eating were in turn associated with higher BMI. None of the impulsivity-related measurements nor Uncontrolled Eating were correlated with longitudinal weight gain. Higher stress sensitivity was associated with increased cortical thickness in the superior temporal gyrus. Lack of self-control was positively associated with increased thickness in the superior medial frontal gyrus. Finally, higher reward sensitivity was associated with lower thickness in the inferior frontal gyrus. CONCLUSION: The present study provides a comprehensive characterization of the relationships between different facets of impulsivity and obesity. We show that differences in impulsivity domains might be associated with BMI via Uncontrolled Eating. Our results might inform future clinical strategies aimed at fostering self-control abilities to prevent and/or treat unhealthy weight gain.

Failed Eradication Therapy of New-Onset Pseudomonas aeruginosa Infections in Children With Cystic Fibrosis Is Associated With Bacterial Resistance to Neutrophil Functions.

BACKGROUND: Antibiotics, such as inhaled tobramycin, are used to eradicate new-onset Pseudomonas aeruginosa (PA) infections in patients with cystic fibrosis (CF) but frequently fail due to reasons poorly understood. We hypothesized that PA isolates' resistance to neutrophil antibacterial functions was associated with failed eradication in patients harboring those strains. METHODS: We analyzed all PA isolates from a cohort of 39 CF children with new-onset PA infections undergoing tobramycin eradication therapy, where 30 patients had eradicated and 9 patients had persistent infection. We characterized several bacterial phenotypes and measured the isolates' susceptibility to neutrophil antibacterial functions using in vitro assays of phagocytosis and intracellular bacterial killing. RESULTS: PA isolates from persistent infections were more resistant to neutrophil functions, with lower phagocytosis and intracellular bacterial killing compared to those from eradicated infections. In multivariable analyses, in vitro neutrophil responses were positively associated with twitching motility, and negatively with mucoidy. In vitro neutrophil phagocytosis was a predictor of persistent infection following tobramycin even after adjustment for clinical risk factors. CONCLUSIONS: PA isolates from new-onset CF infection show strain-specific susceptibility to neutrophil antibacterial functions, and infection with PA isolates resistant to neutrophil phagocytosis is an independent risk factor for failed tobramycin eradication.

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

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.

Evidence and Urgency Related EEG Signals during Dynamic Decision-Making in Humans.

A successful class of models link decision-making to brain signals by assuming that evidence accumulates to a decision threshold. These evidence accumulation models have identified neuronal activity that appears to reflect sensory evidence and decision variables that drive behavior. More recently, an additional evidence-independent and time-variant signal, called urgency, has been hypothesized to accelerate decisions in the face of insufficient evidence. However, most decision-making paradigms tested with fMRI or EEG in humans have not been designed to disentangle evidence accumulation from urgency. Here we use a face-morphing decision-making task in combination with EEG and a hierarchical Bayesian model to identify neural signals related to sensory and decision variables, and to test the urgency-gating model. Forty females and 34 males took part (mean age, 23.4 years). We find that an evoked potential time locked to the decision, the centroparietal positivity, reflects the decision variable from the computational model. We further show that the unfolding of this signal throughout the decision process best reflects the product of sensory evidence and an evidence-independent urgency signal. Urgency varied across subjects, suggesting that it may represent an individual trait. Our results show that it is possible to use EEG to distinguish neural signals related to sensory evidence accumulation, decision variables, and urgency. These mechanisms expose principles of cognitive function in general and may have applications to the study of pathologic decision-making such as in impulse control and addictive disorders.SIGNIFICANCE STATEMENT Perceptual decisions are often described by a class of models that assumes that sensory evidence accumulates gradually over time until a decision threshold is reached. In the present study, we demonstrate that an additional urgency signal impacts how decisions are formed. This endogenous signal encourages one to respond as time elapses. We found that neural decision signals measured by EEG reflect the product of sensory evidence and an evidence-independent urgency signal. A nuanced understanding of human decisions, and the neural mechanisms that support it, can improve decision-making in many situations and potentially ameliorate dysfunction when it has gone awry.

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.

Does effort-cost decision-making relate to real-world motivation in people living with HIV?

INTRODUCTION: Low motivation is frequent in older people with HIV, yet poorly understood. Effort-cost decision-making (ECDM) tasks inspired by behavioral economics have shown promise as indicators of motivation or apathy. These tasks assess the willingness to exert effort to earn a monetary reward, providing an estimate of the subjective "cost" of effort for each participant. Here we sought evidence for a relationship between ECDM task performance and self-reported motivation in a cross-sectional study involving 80 middle-aged and older people with well-controlled HIV infection, a chronic health condition with a high burden of mental and cognitive health challenges. METHODS: Participants attending a regular follow-up visit for a Canadian longitudinal study of brain health in HIV completed a computerized ECDM task and a self-report measure of motivation. Other brain health measures were available, collected for the parent study (cognition, depression, anxiety, and vitality, as well as self-reported time spent on real-world leisure activities). RESULTS: Contrary to our hypothesis, we found no relationship between ECDM performance and self-reported motivation. However, those willing to accept higher effort in the ECDM task also reported more time engaged in real-world activities. This association had a small-to-moderate effect size. CONCLUSIONS: The behavioral economics construct of subjective cost of effort, measured with a laboratory ECDM task, does not relate to motivation in people living with chronic HIV. However, the task shows some relationship with real-world goal-directed behavior, suggesting this construct has potential clinical relevance. More work is needed to understand how the subjective cost of effort plays out in clinical symptoms and everyday activities.

Pseudomonas aeruginosa Strain-sharing in Early Infection Among Children With Cystic Fibrosis.

BACKGROUND: We previously identified Pseudomonas aeruginosa isolates with characteristics typical of chronic infection in some early infections in children with cystic fibrosis (CF), suggesting that these isolates may have been acquired from other patients. Our objective was to define the extent of P. aeruginosa strain-sharing in early CF infections and its impact on antibiotic eradication treatment failure rates. METHODS: We performed whole genome sequencing on isolates from early pediatric CF pulmonary infections and from the following comparator groups in the same hospital: chronic CF infection, sink drains, sterile site infections, and asymptomatic carriage. Univariate logistic regression was used to assess factors associated with treatment failure. RESULTS: In this retrospective, observational study, 1029 isolates were sequenced. The CF clones strain B and clone C were present. In 70 CF patients with early infections, 14 shared strains infected 29 (41%) patients over 5 years; 16% (n = 14) of infections had mixed strains. In the 70 children, approximately one-third of shared-strain infections were likely due to patient-to-patient transmission. Mixed-strain infections were associated with strain-sharing (odds ratio, 8.50; 95% confidence interval, 2.2-33.4; P = .002). Strain-sharing was not associated with antibiotic eradication treatment failure; however, nosocomial strain transmission was associated with establishment of chronic infection in a CF sibling pair. CONCLUSIONS: Although early P. aeruginosa CF infection is thought to reflect acquisition of diverse strains from community reservoirs, we identified frequent early CF strain-sharing that was associated with the presence of mixed strains and instances of possible patient-to-patient transmission.

Orbitofrontal-Striatal Structural Alterations Linked to Negative Symptoms at Different Stages of the Schizophrenia Spectrum.

Negative symptoms such as anhedonia and apathy are among the most debilitating manifestations of schizophrenia (SZ). Imaging studies have linked these symptoms to morphometric abnormalities in 2 brain regions implicated in reward and motivation: the orbitofrontal cortex (OFC) and striatum. Higher negative symptoms are generally associated with reduced OFC thickness, while higher apathy specifically maps to reduced striatal volume. However, it remains unclear whether these tissue losses are a consequence of chronic illness and its treatment or an underlying phenotypic trait. Here, we use multicentre magnetic resonance imaging data to investigate orbitofrontal-striatal abnormalities across the SZ spectrum from healthy populations with high schizotypy to unmedicated and medicated first-episode psychosis (FEP), and patients with chronic SZ. Putamen, caudate, accumbens volume, and OFC thickness were estimated from T1-weighted images acquired in all 3 diagnostic groups and controls from 4 sites (n = 337). Results were first established in 1 discovery dataset and replicated in 3 independent samples. There was a negative correlation between apathy and putamen/accumbens volume only in healthy individuals with schizotypy; however, medicated patients exhibited larger putamen volume, which appears to be a consequence of antipsychotic medications. The negative association between reduced OFC thickness and total negative symptoms also appeared to vary along the SZ spectrum, being significant only in FEP patients. In schizotypy, there was increased OFC thickness relative to controls. Our findings suggest that negative symptoms are associated with a temporal continuum of orbitofrontal-striatal abnormalities that may predate the occurrence of SZ. Thicker OFC in schizotypy may represent either compensatory or pathological mechanisms prior to the disease onset.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software.

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

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.