A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

The changing epidemiology of pulmonary infection in children and adolescents with cystic fibrosis: an 18-year experience.

The impact of evolving treatment regimens, airway clearance strategies, and antibiotic combinations on the incidence and prevalence of respiratory infection in cystic fibrosis (CF) in children and adolescents remains unclear. The incidence, prevalence, and prescription trends from 2002 to 2019 with 18,339 airway samples were analysed. Staphylococcus aureus [- 3.86% (95% CI - 5.28-2.43)] showed the largest annual decline in incidence, followed by Haemophilus influenzae [- 3.46% (95% CI - 4.95-1.96)] and Pseudomonas aeruginosa [- 2.80%95% CI (- 4.26-1.34)]. Non-tuberculous mycobacteria and Burkholderia cepacia showed a non-significant increase in incidence. A similar pattern of change in prevalence was observed. No change in trend was observed in infants < 2 years of age. The mean age of the first isolation of S. aureus (p < 0.001), P. aeruginosa (p < 0.001), H. influenza (p < 0.001), Serratia marcescens (p = 0.006) and Aspergillus fumigatus (p = 0.02) have increased. Nebulised amikacin (+ 3.09 ± 2.24 prescription/year, p = 0.003) and colistin (+ 1.95 ± 0.3 prescriptions/year, p = 0.032) were increasingly prescribed, while tobramycin (- 8.46 ± 4.7 prescriptions/year, p < 0.001) showed a decrease in prescription. Dornase alfa and hypertonic saline nebulisation prescription increased by 16.74 ± 4.1 prescriptions/year and 24 ± 4.6 prescriptions/year (p < 0.001). There is a shift in CF among respiratory pathogens and prescriptions which reflects the evolution of cystic fibrosis treatment strategies over time.

Airway commensal bacteria in cystic fibrosis inhibit the growth of P. aeruginosa via a released metabolite.

In cystic fibrosis (CF), Pseudomonas aeruginosa infection plays a critical role in disease progression. Although multiple studies suggest that airway commensals might be able to interfere with pathogenic bacteria, the role of the distinct commensals in the polymicrobial lung infections is largely unknown. In this study, we aimed to identify airway commensal bacteria that may inhibit the growth of P. aeruginosa. Through a screening study with more than 80 CF commensal strains across 21 species, more than 30 commensal strains from various species have been identified to be able to inhibit the growth of P. aeruginosa. The underlying mechanisms were investigated via genomic, metabolic and functional analysis, revealing that the inhibitory commensals may affect the growth of P. aeruginosa by releasing a large amount of acetic acid. The data provide information about the distinct roles of airway commensals and provide insights into novel strategies for controlling airway infections.

Challenges and mitigation strategies associated with Burkholderia cepacia complex contamination in pharmaceutical manufacturing.

Burkholderia cepacia complex (BCC) is a Gram-negative, non-spore-forming bacterium with more than 20 opportunistic pathogenic species, most commonly found in soil and water. Due to their rapid mutation rates, these organisms are adaptable and possess high genomic plasticity. BCC can cause life-threatening infections in immunocompromised individuals, such as those with cystic fibrosis, chronic granulomatous disease, and neonates. BCC contamination is a significant concern in pharmaceutical manufacturing, frequently causing non-sterile product recalls. BCC has been found in purified water, cosmetics, household items, and even ultrasound gel used in veterinary practices. Pharmaceuticals, personal care products, and cleaning solutions have been implicated in numerous outbreaks worldwide, highlighting the risks associated with intrinsic manufacturing site contamination. Regulatory compliance, product safety, and human health protection depend on testing for BCC in pharmaceutical manufacturing. Identification challenges exist, with BCC often misidentified as other bacteria like non-lactose fermenting Escherichia coli or Pseudomonas spp., particularly in developing countries where reporting BCC in pharmaceuticals remains limited. This review comprehensively aims to address the organisms causing BCC contamination, genetic diversity, identification challenges, regulatory requirements, and mitigation strategies. Recommendations are proposed to aid pharmaceutical chemists in managing BCC-associated risks and implementing prevention strategies within manufacturing processes.

Ecological patterns and processes of temporal turnover within lung infection microbiota.

BACKGROUND: Chronic infection and consequent airway inflammation are the leading causes of morbidity and early mortality for people living with cystic fibrosis (CF). However, lower airway infections across a range of chronic respiratory diseases, including in CF, do not follow classical 'one microbe, one disease' concepts of infection pathogenesis. Instead, they are comprised of diverse and temporally dynamic lung infection microbiota. Consequently, temporal dynamics need to be considered when attempting to associate lung microbiota with changes in disease status. Set within an island biogeography framework, we aimed to determine the ecological patterns and processes of temporal turnover within the lung microbiota of 30 paediatric and adult CF patients prospectively sampled over a 3-year period. Moreover, we aimed to ascertain the contributions of constituent chronic and intermittent colonizers on turnover within the wider microbiota. RESULTS: The lung microbiota within individual patients was partitioned into constituent chronic and intermittent colonizing groups using the Leeds criteria and visualised with persistence-abundance relationships. This revealed bacteria chronically infecting a patient were both persistent and common through time, whereas intermittently infecting taxa were infrequent and rare; respectively representing the resident and transient portions of the wider microbiota. It also indicated that the extent of chronic colonization was far greater than could be appreciated with microbiological culture alone. Using species-time relationships to measure temporal turnover and Vellend's rationalized ecological processes demonstrated turnover in the resident chronic infecting groups was conserved and underpinned principally by the deterministic process of homogenizing dispersal. Conversely, intermittent colonizing groups, representing newly arrived immigrants and transient species, drove turnover in the wider microbiota and were predominately underpinned by the stochastic process of drift. For adult patients, homogenizing dispersal and drift were found to be significantly associated with lung function. Where a greater frequency of homogenizing dispersal was observed with worsening lung function and conversely drift increased with better lung function. CONCLUSIONS: Our work provides a novel ecological framework for understanding the temporal dynamics of polymicrobial infection in CF that has translational potential to guide and improve therapeutic targeting of lung microbiota in CF and across a range of chronic airway diseases. Video Abstract.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

MEK1/2 inhibition decreases pro-inflammatory responses in macrophages from people with cystic fibrosis and mitigates severity of illness in experimental murine methicillin-resistant Staphylococcus aureus infection.

Chronic pulmonary bacterial infections and associated inflammation remain a cause of morbidity and mortality in people with cystic fibrosis (PwCF) despite new modulator therapies. Therapies targeting host factors that dampen detrimental inflammation without suppressing immune responses critical for controlling infections remain limited, while the development of lung infections caused by antimicrobial resistant bacteria is an increasing global problem, and a significant challenge in CF. Pharmacological compounds targeting the mammalian MAPK proteins MEK1 and MEK2, referred to as MEK1/2 inhibitor compounds, have potential combined anti-microbial and anti-inflammatory effects. Here we examined the immunomodulatory properties of MEK1/2 inhibitor compounds PD0325901, trametinib, and CI-1040 on CF innate immune cells. Human CF macrophage and neutrophil phagocytic functions were assessed by quantifying phagocytosis of serum opsonized pHrodo red E. coli, Staphylococcus aureus, and zymosan bioparticles. MEK1/2 inhibitor compounds reduced CF macrophage pro-inflammatory cytokine production without impairing CF macrophage or neutrophil phagocytic abilities. Wild-type C57BL6/J and Cftr tm1kth (F508del homozygous) mice were used to evaluate the in vivo therapeutic potential of PD0325901 compared to vehicle treatment in an intranasal methicillin-resistant Staphylococcus aureus (MRSA) infection with the community-acquired MRSA strain USA300. In both wild-type and CF mice, PD0325901 reduced inflammation associated body mass loss. Wild-type mice treated with PD0325901 had significant reduction in neutrophil-mediated inflammation compared to vehicle treatment groups, with preserved clearance of bacteria in lung, liver, or spleen 1 day after infection in either wild-type or CF mouse models. In summary, this study provides the first data evaluating the therapeutic potential of MEK1/2 inhibitor to modulate CF immune cells and demonstrates that MEK1/2 inhibitors diminish pro-inflammatory responses without impairing host defense mechanisms required for acute pathogen clearance.

Unusual Cause of Hemoptysis in a Woman With Cystic Fibrosis.

CASE PRESENTATION: A 20-year-old patient with cystic fibrosis (CF) complicated by pansinusitis, pancreatic insufficiency, and diabetes presented to the local ED after an episode of large-volume hemoptysis at home. At baseline, she had advanced lung disease (FEV1, 0.97 L; 31% predicted) and upper lobe-predominant fibrocavitary changes. She was intermittently followed at a regional lung transplant center. She was previously evaluated for transplant but was not listed at the time of this presentation because of nontuberculous mycobacteria infection. She had never used tobacco, without reports of recreational inhaled drug use. Her mother had CF, and one of her brothers died in 2018 at age 24 of respiratory failure resulting from the disease.

CFTR is required for zinc-mediated antibacterial defense in human macrophages.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion transporter required for epithelial homeostasis in the lung and other organs, with CFTR mutations leading to the autosomal recessive genetic disease CF. Apart from excessive mucus accumulation and dysregulated inflammation in the airways, people with CF (pwCF) exhibit defective innate immune responses and are susceptible to bacterial respiratory pathogens such as Pseudomonas aeruginosa. Here, we investigated the role of CFTR in macrophage antimicrobial responses, including the zinc toxicity response that is used by these innate immune cells against intracellular bacteria. Using both pharmacological approaches, as well as cells derived from pwCF, we show that CFTR is required for uptake and clearance of pathogenic Escherichia coli by CSF-1-derived primary human macrophages. CFTR was also required for E. coli-induced zinc accumulation and zinc vesicle formation in these cells, and E. coli residing in macrophages exhibited reduced zinc stress in the absence of CFTR function. Accordingly, CFTR was essential for reducing the intramacrophage survival of a zinc-sensitive E. coli mutant compared to wild-type E. coli. Ectopic expression of the zinc transporter SLC30A1 or treatment with exogenous zinc was sufficient to restore antimicrobial responses against E. coli in human macrophages. Zinc supplementation also restored bacterial killing in GM-CSF-derived primary human macrophages responding to P. aeruginosa, used as an in vitro macrophage model relevant to CF. Thus, restoration of the zinc toxicity response could be pursued as a therapeutic strategy to restore innate immune function and effective host defense in pwCF.

Cystic Fibrosis Mice Are Highly Susceptible to Repeated Acute Pseudomonas aeruginosa Pneumonia after Intranasal Inoculation.

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that controls chloride current. A number of different CFTR transgenic mouse lines have been developed and subjected to both acute and chronic infection models. However, prior studies showed no substantial differences in bacterial clearance between CF and non-CF mice after single inoculations. Here, using F508del transgenic CF mice, we examined the role of repeated acute Pseudomonas aeruginosa (PA) infection, with the second inoculation 7 days after the first. We found that CF mice were more susceptible to PA infection than non-CF mice following the second inoculation, with non-CF mice showing better neutrophil recruitment and effector functions. We further investigated the characteristics of lung immune cells using single-cell RNA sequencing, finding that non-CF lung neutrophils had more prominent upregulation of adhesion molecules including intercellular adhesion molecule-1 (ICAM-1) compared to CF lung neutrophils. Although people with CF are often colonized with bacteria and have high numbers of neutrophils in the airways during chronic infection, these data suggest that CF neutrophils have deficient effector functions in the setting of repeated acute infection.

In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores.

The multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cells. We demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection. IMPORTANCE: Multidrug-resistant Pseudomonas aeruginosa is a versatile bacterium that frequently causes lung infections. This pathogen is life-threatening to mechanically-ventilated patients in intensive care units and is a debilitating burden for individuals with cystic fibrosis. However, the role of P. aeruginosa virulence factors and their regulation during infection are not fully understood. Previous murine lung infection studies have demonstrated that the production of siderophores (e.g., pyoverdine and pyochelin) is necessary for full P. aeruginosa virulence. In this report, we provide further mechanistic insight into this phenomenon. We characterize distinct and novel ways these siderophores contribute to virulence using an in vitro human lung epithelial cell culture model.

Prevalence and variability of siderophore production in the Achromobacter genus.

Achromobacter spp. are opportunistic pathogens of environmental origin increasingly isolated in patients with underlying conditions like cystic fibrosis (CF). Despite recent advances, their virulence factors remain incompletely studied, and siderophore production has not yet been investigated in this genus. The aim of this study was to evaluate the production of siderophores in a large collection of Achromobacter spp. and evaluate the variability according to the origin of the strain and species. A total of 163 strains were studied, including 128 clinical strains (CF and non-CF patients) and 35 strains of environmental origin. Siderophores were quantified by the liquid chrome azurol-sulphonate assay. Species were identified by nrdA gene-based phylogeny. Strains were assigned to 20 species, with Achromobacter xylosoxidans being the most represented (51.5% of strains). Siderophore production was observed in 72.4% of the strains, with amounts ranging from 10.1% to 90% siderophore units. A significantly higher prevalence of siderophore-producing strains and greater production of siderophores were observed for clinical strains compared with strains of environmental origin. Highly variable observations were made according to species: A. xylosoxidans presented unique characteristics (one of the highest prevalence of producing strains and highest amounts produced, particularly by CF strains). Siderophores are important factors for bacterial growth commonly produced by members of the Achromobacter genus. The significance of the observations made during this study must be further investigated. Indeed, the differences observed according to species and the origin of strains suggest that siderophores may represent important determinants of the pathophysiology of Achromobacter spp. infections and also contribute to the particular epidemiological success of A. xylosoxidans in human infections. IMPORTANCE: Achromobacter spp. are recognized as emerging opportunistic pathogens in humans with various underlying diseases, including cystic fibrosis (CF). Although their pathophysiological traits are increasingly studied, their virulence factors remain incompletely described. Particularly, siderophores that represent important factors of bacterial growth have not yet been studied in this genus. A population-based study was performed to explore the ability of members of the Achromobacter genus to produce siderophores, both overall and in relevant subgroups (Achromobacter species; strain origin, either clinical-from CF or non-CF patients-or environmental). This study provides original data showing that siderophore production is a common trait of Achromobacter strains, particularly observed among clinical strains. The major species, Achromobacter xylosoxidans, encompassed both one of the highest prevalence of siderophore-producing strains and strains producing the largest amounts of siderophores, particularly observed for CF strains. These observations may represent additional advantages accounting for the epidemiological success of this species.

Genomic editing in Burkholderia multivorans by CRISPR/Cas9.

Burkholderia cepacia complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in Burkholderia, these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in Burkholderia multivorans. Several modifications were implemented, including selection marker replacement, the optimization of araB promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the sacB gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (rpfR, bceF, cepR, and bcsB) from two strains of B. multivorans, as well as its usefulness in the targeted insertion of the gfp gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other Burkholderia cepacia complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCEBurkholderia encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, Burkholderia multivorans, a member of the Burkholderia cepacia complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the B. multivorans species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in B. multivorans, but it can likely be extended to other Burkholderia species.

The role of hypermutation and collateral sensitivity in antimicrobial resistance diversity of Pseudomonas aeruginosa populations in cystic fibrosis lung infection.

Pseudomonas aeruginosa is an opportunistic pathogen which causes chronic, drug-resistant lung infections in cystic fibrosis (CF) patients. In this study, we explore the role of genomic diversification and evolutionary trade-offs in antimicrobial resistance (AMR) diversity within P. aeruginosa populations sourced from CF lung infections. We analyzed 300 clinical isolates from four CF patients (75 per patient) and found that genomic diversity is not a consistent indicator of phenotypic AMR diversity. Remarkably, some genetically less diverse populations showed AMR diversity comparable to those with significantly more genetic variation. We also observed that hypermutator strains frequently exhibited increased sensitivity to antimicrobials, contradicting expectations from their treatment histories. Investigating potential evolutionary trade-offs, we found no substantial evidence of collateral sensitivity among aminoglycoside, beta-lactam, or fluoroquinolone antibiotics, nor did we observe trade-offs between AMR and growth in conditions mimicking CF sputum. Our findings suggest that (i) genomic diversity is not a prerequisite for phenotypic AMR diversity, (ii) hypermutator populations may develop increased antimicrobial sensitivity under selection pressure, (iii) collateral sensitivity is not a prominent feature in CF strains, and (iv) resistance to a single antibiotic does not necessarily lead to significant fitness costs. These insights challenge prevailing assumptions about AMR evolution in chronic infections, emphasizing the complexity of bacterial adaptation during infection.IMPORTANCEUpon infection in the cystic fibrosis (CF) lung, Pseudomonas aeruginosa rapidly acquires genetic mutations, especially in genes involved in antimicrobial resistance (AMR), often resulting in diverse, treatment-resistant populations. However, the role of bacterial population diversity within the context of chronic infection is still poorly understood. In this study, we found that hypermutator strains of P. aeruginosa in the CF lung undergoing treatment with tobramycin evolved increased sensitivity to tobramycin relative to non-hypermutators within the same population. This finding suggests that antimicrobial treatment may only exert weak selection pressure on P. aeruginosa populations in the CF lung. We further found no evidence for collateral sensitivity in these clinical populations, suggesting that collateral sensitivity may not be a robust, naturally occurring phenomenon for this microbe.

An in vitro medium for modeling gut dysbiosis associated with cystic fibrosis.

The gut physiology of pediatric and adult persons with cystic fibrosis (pwCF) is altered relative to healthy persons. The CF gut is characterized, in part, as having excess mucus, increased fat content, acidic pH, increased inflammation, increased antibiotic perturbation, and the potential for increased oxygen availability. These physiological differences shift nutritional availability and the local environment for intestinal microbes, thus likely driving significant changes in microbial metabolism, colonization, and competition with other microbes. The impact of any specific change in this physiological landscape is difficult to parse using human or animal studies. Thus, we have developed a novel culture medium representative of the CF gut environment, inclusive of all the aforementioned features. This medium, called CF-MiPro, maintains CF gut microbiome communities, while significantly shifting nonCF gut microbiome communities toward a CF-like microbial profile, characterized by low Bacteroidetes and high Proteobacteria abundance. This medium is able to maintain this culture composition for up to 5 days of passage. Additionally, microbial communities passaged in CF-MiPro produce significantly less immunomodulatory short-chain fatty acids (SCFA), including propionate and butyrate, than communities passaged in MiPro, a culture medium representative of healthy gut physiology, confirming not only a shift in microbial composition but also altered community function. Our results support the potential for this in vitro culture medium as a new tool for the study of CF gut dysbiosis. IMPORTANCE Cystic fibrosis is an autosomal recessive disease that disrupts ion transport at mucosal surfaces, leading to mucus accumulation and altered physiology of both the lungs and the intestines, among other organs, with the resulting altered environment contributing to an imbalance of microbial communities. Culture media representative of the CF airway have been developed and validated; however, no such medium exists for modeling the CF intestine. Here, we develop and validate a first-generation culture medium inclusive of features that are altered in the CF colon. Our findings suggest this novel medium, called CF-MiPro, as a maintenance medium for CF gut microbiome samples and a flexible tool for studying key drivers of CF-associated gut dysbiosis.

Positioning the preventive potential of microbiome treatments for cystic fibrosis in the context of current therapies.

Antibiotics and cystic fibrosis transmembrane conductance regulator (CFTR) modulators play a pivotal role in cystic fibrosis (CF) treatment, but both have limitations. Antibiotics are linked to antibiotic resistance and disruption of the airway microbiome, while CFTR modulators are not widely accessible, and structural lung damage and pathogen overgrowth still occur. Complementary strategies that can beneficially modulate the airway microbiome in a preventive way are highly needed. This could be mediated via oral probiotics, which have shown some improvement of lung function and reduction of airway infections and exacerbations, as a cost-effective approach. However, recent data suggest that specific and locally administered probiotics in the respiratory tract might be a more targeted approach to prevent pathogen outgrowth in the lower airways. This review aims to summarize the current knowledge on the CF airway microbiome and possibilities of microbiome treatments to prevent bacterial and/or viral infections and position them in the context of current CF therapies.

Promising antibacterial efficacy of arenicin peptides against the emerging opportunistic pathogen Mycobacterium abscessus.

BACKGROUND: Mycobacterium abscessus, a fast-growing non-tuberculous mycobacterium, is an emerging opportunistic pathogen responsible for chronic bronchopulmonary infections in people with respiratory diseases such as cystic fibrosis (CF). Due to its intrinsic polyresistance to a wide range of antibiotics, most treatments for M. abscessus pulmonary infections are poorly effective. In this context, antimicrobial peptides (AMPs) active against bacterial strains and less prompt to cause resistance, represent a good alternative to conventional antibiotics. Herein, we evaluated the effect of three arenicin isoforms, possessing two or four Cysteines involved in one (Ar-1, Ar-2) or two disulfide bonds (Ar-3), on the in vitro growth of M. abscessus. METHODS: The respective disulfide-free AMPs, were built by replacing the Cysteines with alpha-amino-n-butyric acid (Abu) residue. We evaluated the efficiency of the eight arenicin derivatives through their antimicrobial activity against M. abscessus strains, their cytotoxicity towards human cell lines, and their hemolytic activity on human erythrocytes. The mechanism of action of the Ar-1 peptide was further investigated through membrane permeabilization assay, electron microscopy, lipid insertion assay via surface pressure measurement, and the induction of resistance assay. RESULTS: Our results demonstrated that Ar-1 was the safest peptide with no toxicity towards human cells and no hemolytic activity, and the most active against M. abscessus growth. Ar-1 acts by insertion into mycobacterial lipids, resulting in a rapid membranolytic effect that kills M. abscessus without induction of resistance. CONCLUSION: Overall, the present study emphasized Ar-1 as a potential new alternative to conventional antibiotics in the treatment of CF-associated bacterial infection related to M. abscessus.

Time-dependent pharmacodynamics of amikacin on Mycobacterium abscessus growth and resistance emergence.

Mycobacterium abscessus pulmonary disease is increasing in prevalence globally, particularly for individuals with cystic fibrosis. These infections are challenging to treat due to a high rate of resistance. Amikacin is critical to treatment, but the development of toxicity, amikacin resistance, and treatment failure are significant challenges. Amikacin has been characterized previously as peak-dependent and extended-interval dosing is commonly used. In our hollow fiber infection model of M. abscessus, amikacin exhibited time-dependent rather than the expected peak-dependent pharmacodynamics. Humanized amikacin exposures with more frequent, short-interval dosing (continuous infusion or every 12 hours) yielded improved microbiological response compared to extended-interval dosing (every 24 hours or 1-3 times per week). Short-interval dosing inhibited growth with a mean (SD) maximum Δlog10 colony forming units of -4.06 (0.52), significantly more than extended-interval dosing (P = 0.0013) every 24 hours, -2.40 (0.58), or 1-3 times per week, -2.39 (0.38). Growth recovery, an indicator of resistance emergence, occurred at 6.56 (0.70) days with short-interval dosing but was significantly earlier with extended-interval dosing (P = 0.0032) every 24 hours, 3.88 (0.85) days, and 1-3 times per week, 3.27 (1.72) days. Microbiological response correlated best with the pharmacodynamic index of %T > minimum inhibitory concentration (MIC), with an EC80 for growth inhibition of ~40%T > MIC. We used a previously published population model of amikacin to determine the probability of achieving 40%T > MIC and show that current dosing strategies are far below this target, which may partially explain why treatment failure remains so high for these infections. These data support a cautious approach to infrequent amikacin dosing for the treatment of M. abscessus.IMPORTANCEPulmonary disease caused by Mycobacterium abscessus complex (MABSC) is increasing worldwide, particularly in patients with cystic fibrosis. MABSC is challenging to treat due to high levels of antibiotic resistance. Treatment requires 2-4 antibiotics over more than 12 months and has a significant risk of toxicity but still fails to eradicate infection in over 50% of patients with cystic fibrosis. Antibiotic dosing strategies have been largely informed by common bacteria such as Pseudomonas aeruginosa. The "pharmacodynamic" effects of amikacin, a backbone of MABSC treatment, were thought to be related to maximum "peak" drug concentration, leading to daily or three times weekly dosing. However, we found that amikacin MABSC kill and growth recovery, an indicator of antibiotic resistance, are dependent on how long amikacin concentrations are above the minimum inhibitory concentration, not how high the peak concentration is. Therefore, we recommend a re-evaluation of amikacin dosing to determine if increased frequency can improve efficacy.