key: cord-0932240-e4nmii7z
authors: Sala, M.; Markov, N.; Misharin, A.; Jain, M.
title: 416: Expression of SARS-CoV-2 entry genes is not greater in the nasal mucosa of CF patients
date: 2021-11-30
journal: Journal of Cystic Fibrosis
DOI: 10.1016/s1569-1993(21)01840-3
sha: 2dcf3b928ac957c1ecfeb074abc09a9ef050e6bf
doc_id: 932240
cord_uid: e4nmii7z

nan

Background: Culture-independent analyses of human microbiota have implicated strict and facultative anaerobic bacteria in the onset and progression of cystic fibrosis (CF) lung disease. Oral and supraglotticassociated facultative and obligate anaerobes, Prevotella, Veillonella, Streptococcus spp., have been found to be prevalent within the CF airway and to potentiate the growth and virulence of canonical CF pathogens in vitro, but the mechanistic bases of their contributions to disease pathophysiology are poorly understood because of the inherent limitations of existing laboratory models and the conflicting oxygen demands between anaerobes and host cells. To overcome these limitations, we developed and optimized a dual oxic/anoxic co-culture approach that maintains an oxygen-limited microenvironment at the epithelial interface while host cells are oxygenated basolaterally. Methods: Airway epithelial cells (Calu-3) were assembled in a gaspermeable multiwell plate manifold. The apparatus was moved to an anaerobic chamber while mixed gas (21% O 2 /5% CO 2 /74% N 2 ) was delivered to the base of the plate to oxygenate the basolateral side of the polarized monolayer. Barrier integrity was determined by transepithelial electric resistance, E-cadherin quantification, and ZO-1 immunofluorescence. Proinflammatory cytokines (TNF-α, IL-8, IL-6) were quantified by ELISA. The transcriptomic response of epithelial cells to anoxic culture and anaerobe challenge was determined using RNAseq. Calu-3 cells were treated with an anaerobic bacterial consortium for 24 hours and infected with Pseudomonas aeruginosa PA14 for another 2 hours. Colony-forming units were enumerated to determine PA14 numbers after infection. Results: We first demonstrated that reduced oxygen culture conditions did not compromise the viability, tight junction integrity, or inflammatory response of polarized airway epithelial cells, and RNAseq revealed few changes in their transcriptomic profile. Using individual anaerobes and a mixed community derived from CF airways, we confirmed that the coculture model can sustain host-anaerobe interactions for 24 hours without affecting the viability of host and anaerobic bacterial cells, although in response to anaerobe challenge, apical mucin integrity is significantly altered, as is expression of a panel of inflammatory marker genes. These observations led to our hypothesis that anaerobe-mediated mucin degradation primes the airway epithelium for colonization by canonical pathogens, and treatment of a mucus-overproducing cell line (Calu-3) with a CF anaerobic bacterial consortium before challenge with P. aeruginosa led to a significant increase in pathogen colonization. Conclusion: Altogether, this novel model system offers new insight into the mechanisms of pathogen colonization of the airways and a potential etiological role for aspirated microbiota in CF lung disease. Acknowledgements: Supported by CFF (MOORE20F0) and NHLBI (HL136919). . Furthermore, the HFIM can be used to compare different antibiotic combinations. We evaluated the efficacy of different antibiotic regimens and the relevance of their sequence on killing activity of P. aeruginosa. Methods: Sputum was obtained from a 10-year-old with CF with a 3-year history of smooth and mucoid P. aeruginosa at time of initiation of IV antibiotics. Twenty P. aeruginosa isolates of various morphology were selected for standard minimum inhibitory concentration (MIC) testing, static time kill curve over 24 hours, and HFIM over 7 days. Results: Morphology of 20 isolates showed 6 smooth, 8 mucoid, and 6 highly mucoid P. aeruginosa. Variability in the MIC of the 20 isolates was observed for each antibiotic tested, with antimicrobial susceptibility ranging from susceptible to resistant. Tobramycin MIC ranged from less than 0.5 to 2, ceftazidime less than 0.5 to 4, piperacillin/tazobactam less than 0.5 to 128, and meropenem less than 0.5 to 32 ug/mL. Static time kill showed concentration-dependent killing for each antibiotic, with no differences between smooth and mucoid isolates but an increase in colony-forming units after 8 hours, especially for tobramycin. Comparisons of different HFIM runs showed poor response (killing activitypharmacodynamic response) to meropenem plus tobramycin. When the treatment regimen was switched to piperacillin/tazobactam plus tobramycin, minimal reduction was seen, which resulted in a third switch to ceftazidime plus tobramycin, but switching directly from meropenem plus tobramycin to ceftazidime plus tobramycin resulted in sustained activity. In contrast, when ceftazidime/tobramycin was used as the initial regimen, there was effective and sustained pharmacodynamic activity against P. aeruginosa. Conclusion: The known diversity between P. aeruginosa isolates in the same sputum reduces reliability of standard MIC testing results. In contrast to other studies, we saw no difference in in vitro killing of P. aeruginosa between smooth and mucoid phenotypes. Static time kill curves add additional information about time of occurrence of regrowth and do not necessarily reflect standard MIC results. Finally, the HFIM shows that, when rotating through different antibiotic regimens, the sequence of antibiotic exposure may make a subsequent regimen more or less effective. Background: Mutations in the CFTR gene lead to impaired innate defense, and cystic fibrosis (CF) patients are at risk of respiratory infections, including viral pathogens. The COVID-19 pandemic is caused by SARS-CoV-2, an RNA virus that primarily targets the respiratory system and can damage the health of CF patients. SARS-CoV-2 requires ACE2 for entry into epithelial cells, and its expression is highest in the secretory cells of the nasal epithelium, which is a primary site of SARS-CoV-2 infection. Moreover, a recent study demonstrated existence of a short ACE2 isoform that lacks the receptor binding domain, increases with inflammation, and may decrease susceptibility to infection. It has been proposed that other molecules, including TMPRSS2, IL-6, and cathepsin, are important modulators of SARS-CoV-2 entry and a hyperinflammatory response. We therefore tested whether expression of ACE2 and other genes related to SARS-CoV-2 entry and the inflammatory response are expressed differently in CF patients than in healthy volunteers.

We selected 29 genes identified in the literature to be associated with SARS-CoV-2 entry and assessed whether they were expressed differently in the nasal mucosa (from a curettage without flow sorting) of CF patients than in healthy volunteers. We separated the CF group into 2 cohorts: 1 who were F508del homozygous and 1 who were compound heterozygotes for F508del. The study methods and demographic characteristics of the recruited cohorts have been previously reported [1] . Results: K-means clustering on highly variable genes (ANOVA-like test in DESeq2) identified 3 clusters. Cluster 1 contained samples from healthy volunteer (12 of 12 subjects) and CF F508del (7 of 13 subjects) homozygotes and F508del compound heterozygotes (3 of 10 subjects). Clusters 2 and 3 contained samples from CF F508del homozygote and F508del compound heterozygote patients and were characterized by genes involved in immune response. In silico deconvolution of bulk transcriptomic signatures confirmed that cluster 1 mostly contained epithelial cells and that clusters 2 and 3 were enriched for immune cells. To ensure that sample composition did not influence our analysis, we performed a pairwise comparison using only samples from cluster 1 (epithelial cluster), which contained healthy volunteer, CF F508del homozygote, and F508del compound heterozygote patients. When we assessed the prespecified 29 COVID-19-associated genes, we did not detect any differences between CF patients and healthy volunteers, including ACE2, the short ACE2 isoform, or TMPRSS2. Conclusion: As the pandemic has progressed, there have been a number of reports from around the world highlighting the unexpectedly low incidence of SARS-CoV-2 infection or severe complications in CF patients. Our data suggest that CF patients do not express factors that expose them to higher risk for SARS-CoV2 acquisition or poorer outcomes once infected. Background: Chronic pulmonary infection by bacteria is the primary cause of respiratory failure and death in cystic fibrosis (CF) patients. Pulmonary infections caused by nontuberculous mycobacteria (NTM), especially those of the archetypal rapidly growing mycobacterial pathogen Mycobacterium abscessus, are notoriously difficult to treat, demonstrating persistent infections refractory to antibiotic therapy. We are interested in investigating known physiological mechanisms by which M. abscessus evades immune clearance and increases antibiotic tolerance. Biofilm formation allows chronic pulmonary bacterial pathogens to persist in the face of environmental and host stressors. A critical early step in NTM biofilm formation is cell-cell adhesion, characterized by formation of cellular aggregates in liquid culture. M. abscessus CF isolates classified as rough colony variants contain mutations to disrupt the production of glycopeptidolipids (GPLs), which are key mycomembrane components. These rough colony isolates demonstrate greater wrinkling when plated and are responsible for a majority of chronic M. abscessus infections. Cell-surface remodeling is a known way that M. abscessus adapts to the host lung, but the role of GPL regulation on M. abscessus biofilm formation in the CF environment has not been fully explored. CF sputum is characterized by pockets of hypoxia and is often home to bacteria growing as aggregates. We are exploring the connection between M. abscessus biofilm formation and the tightly controlled hypoxia-induced dormancy response system to regulate aggregation to increase survival in low-oxygen environments. The 2-component DosSR dormancy response system senses low-oxygen environments using the DosS sensor and, through the DosR response regulator, promotes transcriptional changes to increase survival under hypoxia. Methods: We hypothesize that hypoxic conditions activate dormancy survival (Dos), which in turn downregulates GPL production and modification through an unknown Dos regulon component. In this model, GPL downregulation exposes underlying membrane adhesins and increases membrane hydrophobicity, resulting in greater cell-cell adhesion and biofilm formation. To elucidate the regulatory ability of these systems on biofilm formation and dispersal, we have created a library of mutants with disruption of key GPL biosynthesis and Dos regulon components. We use a novel in vitro aggregation assay that quantifies changes in aggregation throughout culture maturity, allowing for exploration of the early stages of biofilm formation. Results: We have determined that a GPL-deficient mutant of the model NTM Mycobacterium smegmatis constitutively aggregates, and a DosR knockout mutant showed a defect in aggregation. In addition, by varying culture volumes, we observed that decreasing culture oxygenation increases culture aggregation. Conclusion: By identifying key regulatory components of M. abscessus biofilm formation and how they influence early biofilm formation, we hope to identify targets for novel therapeutics to prevent or disperse biofilms. Background: Linezolid is an oxazolidinone antibiotic given orally or parenterally for severe Staphylococcus aureus infections. Resistance to linezolid is considered rare in S. aureus but could emerge with mutations to the 23S rRNA or by acquisition of the 23S methyltransferase Cfr. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). The goals of this study were to determine the incidence of linezolid resistance in CF and determine molecular mechanisms of linezolid resistance in this population. Methods: With IRB approval, we collected retrospective microbiology reports for patients attending the University of Iowa CF Center between 2008 and 2018. We identified subjects with S. aureus resistant to linezolid (minimum inhibitory concentration >4) and obtained susceptible and resistant isolates from these patients, which we stored in the pathology laboratory. After retesting susceptibility to linezolid by broth microdilution assay, we sequenced whole genomes on the Illumina platform and determined multilocus sequence type (ST) in Bactopia. We assembled sequences de novo with SPAdes and used HISAT2 to align assemblies to a collection of reference genes. We identified nucleotide changes between linezolid resistant and susceptible isolates using Arraystar. Results: We identified 4 patients with CF who had linezolid-resistant S. aureus between 2008 and 2018. During this interval, 111 patients were treated with linezolid. We sequenced 9 linezolid-resistant and 21 susceptible isolates from these 4 subjects. These subjects had 8 to 20 different orders for linezolid. None of the resistant strains encoded Cfr. Each individual's linezolid-resistant strain was distinct and was genetically similar to their linezolid-susceptible S. aureus, suggesting that linezolid resistance evolved independently in the 4 patients. Three resistant strains developed on a ST5 background and one on ST105. One subject had 4 linezolid-resistant cultures, all of which occurred with deletion of the DNA repair genes mutS and mutL. These isolates exhibited hypermutation and had mutations in multiple ribosomal subunits. Another subject had the G2603 T 23S rRNA variant previously associated with linezolid resistance. In 2 subjects, the genetic basis for linezolid resistance was unclear. Linezolid resistance did not persist in 3 subjects, suggesting that this adaptation may decrease the overall fitness of S. aureus.

Pseudomonas aeruginosa evolutionary adaptation and diversification in cystic fibrosis chronic lung infections

The role of Pseudomonas aeruginosa lipopolysaccharide in bacterial pathogenesis and physiology

Genetic basis and physiological effects of lipid A hydroxylation in Pseudomonas aeruginosa PAO1

Acknowledgements: Supported by NIHAI147314 and CFF grants Ernst18G0 and Ernst18I0.