Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

RATIONALE: Pseudomonas aeruginosa (P.a.) is the major bacterial pathogen colonizing the airways of adult cystic fibrosis (CF) patients and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evades the immune system and antibiotic therapy. While the ability of P.a. to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. OBJECTIVES: To detect and characterize intracellular P.a. in CF airway epithelium from human lung explant tissues. METHODS: We sampled the lung explant tissues from CF patients undergoing lung transplantation and non-CF lung donor control. We analyzed lung tissue sections for the presence of intracellular P.a. by quantitative culture and microscopy, in parallel to histopathology and airway morphometry. MEASUREMENTS AND MAIN RESULTS: P.a. was isolated from the lungs of 7 CF patients undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P.a. within airway epithelial cells in 3 out of the 7 patients analyzed, at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. CONCLUSION: This is the first study describing the presence of intracellular P.a. in CF lung tissues. While intracellular P.a. in airway epithelial cells are likely relatively rare events, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

Siderophores promote cooperative interspecies and intraspecies cross-protection against antibiotics in vitro.

The antibiotic cefiderocol hijacks iron transporters to facilitate its uptake and resists ß-lactamase degradation. While effective, resistance has been detected clinically with unknown mechanisms. Here, using experimental evolution, we identified cefiderocol resistance mutations in Pseudomonas aeruginosa. Resistance was multifactorial in host-mimicking growth media, led to multidrug resistance and paid fitness costs in cefiderocol-free environments. However, kin selection drove some resistant populations to cross-protect susceptible individuals from killing by increasing pyoverdine secretion via a two-component sensor mutation. While pyochelin sensitized P. aeruginosa to cefiderocol killing, pyoverdine and the enterobacteria siderophore enterobactin displaced iron from cefiderocol, preventing uptake by susceptible cells. Among 113 P. aeruginosa intensive care unit clinical isolates, pyoverdine production directly correlated with cefiderocol tolerance, and high pyoverdine producing isolates cross-protected susceptible P. aeruginosa and other Gram-negative bacteria. These in vitro data show that antibiotic cross-protection can occur via degradation-independent mechanisms and siderophores can serve unexpected protective cooperative roles in polymicrobial communities.

Determining Susceptibility and Potential Mediators of Resistance for the Novel Polymyxin Derivative, SPR206, in Acinetobacter baumannii.

With the increase in carbapenem-resistant A. baumannii (CRAB) infections, there has been a resurgence in the use of polymyxins, specifically colistin (COL). Since the reintroduction of COL-based regimens in treating CRAB infections, several COL-resistant A. baumannii isolates have been identified, with the mechanism of resistance heavily linked with the loss of the lipopolysaccharide (LPS) layer of the bacterial outer membrane through mutations in lpxACD genes or the pmrCAB operon. SPR206, a novel polymyxin derivative, has exhibited robust activity against multidrug-resistant (MDR) A. baumannii. However, there is a dearth of knowledge regarding its efficacy in comparison with other A. baumannii-active therapeutics and whether traditional polymyxin (COL) mediators of A. baumannii resistance also translate to reduced SPR206 activity. Here, we conducted susceptibility testing using broth microdilution on 30 A. baumannii isolates (17 COL-resistant and 27 CRAB), selected 14 COL-resistant isolates for genomic sequencing analysis, and performed time-kill analyses on four COL-resistant isolates. In susceptibility testing, SPR206 demonstrated a lower range of minimum inhibitory concentrations (MICs) compared with COL, with a four-fold difference observed in MIC50 values. Mutations in lpxACD and/or pmrA and pmrB genes were detected in each of the 14 COL-resistant isolates; however, SPR206 maintained MICs ≤ 2 mg/L for 9/14 (64%) of the isolates. Finally, SPR206-based combination regimens exhibited increased synergistic and bactericidal activity compared with COL-based combination regimens irrespective of the multiple resistance genes detected. The results of this study highlight the potential utility of SPR206 in the treatment of COL-resistant A. baumannii infections.

Evolved bacterial siderophore-mediated antibiotic cross-protection.

Antibiotic cross-protection enables resistant bacteria to protect other bacteria that would be otherwise susceptible to the drug. Cefiderocol is the first siderophore cephalosporin antibiotic approved for treating Gram-negative bacterial infections, including carbapenem-resistant Pseudomonas aeruginosa strains. While highly effective, CFDC resistance has been detected clinically, and mechanisms of resistance and cross-protection are not completely understood. In this study, we used experimental evolution and whole genome sequencing to identify cefiderocol resistance mechanisms and evaluated the trade-offs of evolving resistance. We found some cefiderocol-resistant populations evolved cross-protective social behavior, preventing cefiderocol killing of susceptible siblings. Notably, cross-protection was driven by increased secretion of bacterial iron-binding siderophores, which is unique from previously described antibiotic degradation mediated cross-protection. While concerning, we also showed that resistance can be selected against in drug-free environments. Deciphering the costs associated with antibiotic resistance might aid the development of evolution-informed therapeutic approaches to delay the evolution of antibiotic resistance.

A compensatory RNase E variation increases Iron Piracy and Virulence in multidrug-resistant Pseudomonas aeruginosa during Macrophage infection.

During chronic cystic fibrosis (CF) infections, evolved Pseudomonas aeruginosa antibiotic resistance is linked to increased pulmonary exacerbations, decreased lung function, and hospitalizations. However, the virulence mechanisms underlying worse outcomes caused by antibiotic resistant infections are poorly understood. Here, we investigated evolved aztreonam resistant P. aeruginosa virulence mechanisms. Using a macrophage infection model combined with genomic and transcriptomic analyses, we show that a compensatory mutation in the rne gene, encoding RNase E, increased pyoverdine and pyochelin siderophore gene expression, causing macrophage ferroptosis and lysis. We show that iron-bound pyochelin was sufficient to cause macrophage ferroptosis and lysis, however, apo-pyochelin, iron-bound pyoverdine, or apo-pyoverdine were insufficient to kill macrophages. Macrophage killing could be eliminated by treatment with the iron mimetic gallium. RNase E variants were abundant in clinical isolates, and CF sputum gene expression data show that clinical isolates phenocopied RNase E variant functions during macrophage infection. Together these data show how P. aeruginosa RNase E variants can cause host damage via increased siderophore production and host cell ferroptosis but may also be targets for gallium precision therapy.

Pharmacologic improvement of CFTR function rapidly decreases sputum pathogen density, but lung infections generally persist.

BackgroundLung infections are among the most consequential manifestations of cystic fibrosis (CF) and are associated with reduced lung function and shortened survival. Drugs called CF transmembrane conductance regulator (CFTR) modulators improve activity of dysfunctional CFTR channels, which is the physiological defect causing CF. However, it is unclear how improved CFTR activity affects CF lung infections.MethodsWe performed a prospective, multicenter, observational study to measure the effect of the newest and most effective CFTR modulator, elexacaftor/tezacaftor/ivacaftor (ETI), on CF lung infections. We studied sputum from 236 people with CF during their first 6 months of ETI using bacterial cultures, PCR, and sequencing.ResultsMean sputum densities of Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Achromobacter spp., and Burkholderia spp. decreased by 2-3 log10 CFU/mL after 1 month of ETI. However, most participants remained culture positive for the pathogens cultured from their sputum before starting ETI. In those becoming culture negative after ETI, the pathogens present before treatment were often still detectable by PCR months after sputum converted to culture negative. Sequence-based analyses confirmed large reductions in CF pathogen genera, but other bacteria detected in sputum were largely unchanged. ETI treatment increased average sputum bacterial diversity and produced consistent shifts in sputum bacterial composition. However, these changes were caused by ETI-mediated decreases in CF pathogen abundance rather than changes in other bacteria.ConclusionsTreatment with the most effective CFTR modulator currently available produced large and rapid reductions in traditional CF pathogens in sputum, but most participants remain infected with the pathogens present before modulator treatment.Trial RegistrationClinicalTrials.gov NCT04038047.FundingThe Cystic Fibrosis Foundation and the NIH.

In-Human Multiyear Evolution of Carbapenem-Resistant Klebsiella pneumoniae Causing Chronic Colonization and Intermittent Urinary Tract Infections: A Case Study.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a frequent pathogen of the urinary tract, but how CRKP adapts in vivo over time is unclear. We examined 10 CRKP strains from a patient who experienced chronic colonization and recurrent urinary tract infections over a period of 4.5 years. We performed whole-genome sequencing and phenotypic assays to compare isolates that had evolved relative to the first isolate collected and to correlate genetic and phenotypic changes over time with the meropenem-containing regimen received. Phylogenetic analysis indicated that all 10 strains originated from the same sequence type 258 (ST258) clone and that three sublineages (SL) evolved over time; strains from two dominant sublineages were selected for detailed analysis. Up to 60 new mutations were acquired progressively in genes related to antibiotic resistance, cell metabolism, and biofilm production over time. Doubling of meropenem MICs, increases in biofilm production and blaKPC expression, and altered carbon metabolism occurred in the latter strains from the last sublineage compared to the initial strain. Subinhibitory meropenem exposure in vitro significantly induced or maintained high levels of biofilm production in colonizing isolates, but isolates causing infection were unaffected. Despite acquiring different mutations that affect carbon metabolism, overall carbon utilization was maintained across different strains. Together, these data showed that isolated urinary CRKP evolved through multiple adaptations affecting carbon metabolism, carbapenem resistance, and biofilm production to support chronic colonization and intermittent urinary tract infections. Our findings highlight the pliability of CRKP in adapting to repeated antibiotic exposure and should be considered when developing novel therapeutic and stewardship strategies. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) can cause a variety of infections such as recurrent urinary tract infections (rUTI) with the ability to change with the host environment over time. However, it is unclear how CRKP adapts to the urinary tract during chronic infections and colonization. Here, we studied the evolution of CRKP strains from a patient who experienced chronic colonization and recurrent UTIs over a period of 4.5 years despite multiple treatment courses with meropenem-containing regimens. Our findings show the flexibility of CRKP strains in developing changes in carbapenem resistance, biofilm production, and carbon metabolism over time, which could facilitate their persistence in the human body for long periods of time in spite of repeated antibiotic therapy.

A population-level strain genotyping method to study pathogen strain dynamics in human infections.

A hallmark of chronic bacterial infections is the long-term persistence of 1 or more pathogen species at the compromised site. Repeated detection of the same bacterial species can suggest that a single strain or lineage is continually present. However, infection with multiple strains of a given species, strain acquisition and loss, and changes in strain relative abundance can occur. Detecting strain-level changes and their effects on disease is challenging because most methods require labor-intensive isolate-by-isolate analyses, and thus, only a few cells from large infecting populations can be examined. Here, we present a population-level method for enumerating and measuring the relative abundance of strains called population multi-locus sequence typing (PopMLST). The method exploits PCR amplification of strain-identifying polymorphic loci, next-generation sequencing to measure allelic variants, and informatic methods to determine whether variants arise from sequencing errors or low-abundance strains. These features enable PopMLST to simultaneously interrogate hundreds of bacterial cells that are cultured en masse from patient samples or are present in DNA directly extracted from clinical specimens without ex vivo culture. This method could be used to detect epidemic or super-infecting strains, facilitate understanding of strain dynamics during chronic infections, and enable studies that link strain changes to clinical outcomes.

Pseudomonas aeruginosa mexR and mexEF Antibiotic Efflux Pump Variants Exhibit Increased Virulence.

Antibiotic-resistant Pseudomonas aeruginosa infections are the primary cause of mortality in people with cystic fibrosis (CF). Yet, it has only recently become appreciated that resistance mutations can also increase P. aeruginosa virulence, even in the absence of antibiotics. Moreover, the mechanisms by which resistance mutations increase virulence are poorly understood. In this study we tested the hypothesis that mutations affecting efflux pumps can directly increase P. aeruginosa virulence. Using genetics, physiological assays, and model infections, we show that efflux pump mutations can increase virulence. Mutations of the mexEF efflux pump system increased swarming, rhamnolipid production, and lethality in a mouse infection model, while mutations in mexR that increased expression of the mexAB-oprM efflux system increased virulence during an acute murine lung infection without affecting swarming or rhamnolipid gene expression. Finally, we show that an efflux pump inhibitor, which represents a proposed novel treatment approach for P. aeruginosa, increased rhamnolipid gene expression in a dose-dependent manner. This finding is important because rhamnolipids are key virulence factors involved in dissemination through epithelial barriers and cause neutrophil necrosis. Together, these data show how current and proposed future anti-Pseudomonal treatments may unintentionally make infections worse by increasing virulence. Therefore, treatments that target efflux should be pursued with caution.

Evaluation of Antimicrobial Susceptibility Testing Methods for Burkholderia cenocepacia and Burkholderia multivorans Isolates from Cystic Fibrosis Patients.

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

Elastase Activity From Pseudomonas aeruginosa Respiratory Isolates and ICU Mortality.

BACKGROUND: Pseudomonas aeruginosa (PA) is a common cause of respiratory infection and morbidity. Pseudomonas elastase is an important virulence factor regulated by the lasR gene. Whether PA elastase activity is associated with worse clinical outcomes in ICU patients is unknown. RESEARCH QUESTION: Is there an association between PA elastase activity and worse host outcomes in a cohort of ICU patients? METHODS: PA respiratory isolates from 238 unique ICU patients from two tertiary-care centers within the University of Pittsburgh Medical Center health system were prospectively collected and screened for total protease and elastase activity, biofilm production, antimicrobial resistance, and polymicrobial status. The association between pathogen characteristics and 30-day and 90-day mortality was calculated using logistic regression. For subgroup analysis, two patterns of early (≤72 h) and late sample (>72 h) collection from the index ICU admission were distinguished using a finite mixture model. Lung inflammation and injury was evaluated in a mouse model using a PA high elastase vs low elastase producer. RESULTS: PA elastase activity was common in ICU respiratory isolates representing 75% of samples and was associated with increased 30-day mortality (adjusted OR [95% CI]: 1.39 [1.05-1.83]). Subgroup analysis demonstrated that elastase activity was a risk factor for 30- and 90-day mortality in the early sample group, whereas antimicrobial resistance was a risk factor for 90-day mortality in the late sample group. Whole genome sequencing of high and low elastase producers showed that predicted loss-of-function lasR genotypes were less common among high elastase producers. Mice infected with a high elastase producer showed increased lung bacterial burden and inflammatory profile compared with mice infected with a low elastase producer. INTERPRETATION: Elastase activity is associated with 30-day ICU mortality. A high elastase producing clinical isolate confers increased lung tissue inflammation compared with a low elastase producer in vivo.

The anti-sigma factor MucA is required for viability in Pseudomonas aeruginosa.

During decades-long infections in the cystic fibrosis (CF) airway, Pseudomonas aeruginosa undergoes selection. One bacterial genetic adaptation often observed in CF isolates is mucA mutations. MucA inhibits the sigma factor AlgU. Mutations in mucA lead to AlgU misregulation, resulting in a mucoid phenotype that is associated with poor CF disease outcomes. Due to its ability to be mutated, mucA is assumed to be dispensable for bacterial viability. Here we show that, paradoxically, a portion of mucA is essential in P. aeruginosa. We demonstrate that mucA is no longer required in a strain lacking algU, that mucA alleles encoding for proteins that do not bind to AlgU are insufficient for viability, and that mucA is no longer essential in mutant strains containing AlgU variants with reduced sigma factor activity. Furthermore, we found that overexpression of algU prevents cell growth in the absence of MucA, and that this phenotype can be rescued by the overproduction of RpoD, the housekeeping sigma factor. Together, these results suggest that in the absence of MucA, the inability to regulate AlgU activity results in the loss of bacterial viability. Finally, we speculate that the essentiality of anti-sigma factors that regulate envelope function may be a widespread phenomenon in bacteria.

The Unrecognized Threat of Secondary Bacterial Infections with COVID-19.

Coronavirus disease 2019 (COVID-19) is the greatest pandemic of our generation, with 16 million people affected and 650,000 deaths worldwide so far. One of the risk factors associated with COVID-19 is secondary bacterial pneumonia. In recent studies on COVID-19 patients, secondary bacterial infections were significantly associated with worse outcomes and death despite antimicrobial therapies. In the past, the intensive use of antibiotics during the severe acute respiratory syndrome coronavirus (SARS-CoV) pandemic led to increases in the prevalence of multidrug-resistant bacteria. The rising number of antibiotic-resistant bacteria and our decreasing capacity to eradicate them not only render us more vulnerable to bacterial infections but also weaken us during viral pandemics. The COVID-19 pandemic reminds us of the great health challenges we are facing, especially regarding antibiotic-resistant bacteria.

Variable Release of Lipoteichoic Acid From Staphylococcus aureus Bloodstream Isolates Relates to Distinct Clinical Phenotypes, Strain Background, and Antibiotic Exposure.

BACKGROUND: Staphylococcus aureus is a leading cause of bacterial bloodstream infections. The heterogeneity in patient outcomes in S. aureus bacteremia (SAB) can be attributed in part to strain characteristics, which may influence host response to infection. We specifically examined the relationship between lipoteichoic acid (LTA) release from S. aureus and disease phenotype, strain background, and antibiotic exposure. METHODS: Seven strains of S. aureus causing different clinical phenotypes of bacteremia and two reference strains (LAC USA 300 and Mu3) were analyzed for LTA release at baseline and following exposure to antibiotics from different pharmacologic classes (vancomycin, ceftaroline, and tedizolid). LTA release was quantified by LTA-specific ELISA. Whole genome sequencing was performed on the clinical strains and analyzed using open-source bioinformatics tools. RESULTS: Lipoteichoic acid release varied by 4-fold amongst the clinical strains and appeared to be related to duration of bacteremia, independent of MLST type. Low LTA releasing strains were isolated from patients who had prolonged duration of bacteremia and died. Antibiotic-mediated differences in LTA release appeared to be associated with MLST type, as ST8 strains released maximal LTA in response to tedizolid while other non-ST8 strains demonstrated high LTA release with vancomycin. Genetic variations related to the LTA biosynthesis pathway were detected in all non-ST8 strains, though ST8 strains showed no variations despite demonstrating differential LTA release. CONCLUSION: Our findings provide the basis for future studies to evaluate the relationship between LTA release-mediated host immune response and clinical outcomes as well as the potential for antibiotic modulation of LTA release as a therapeutic strategy and deserve confirmation with larger number of strains with known clinical phenotypes.

Quantitative Visualization of Gene Expression in Mucoid and Nonmucoid Pseudomonas aeruginosa Aggregates Reveals Localized Peak Expression of Alginate in the Hypoxic Zone.

It is well appreciated that oxygen- and other nutrient-limiting gradients characterize microenvironments within chronic infections that foster bacterial tolerance to treatment and the immune response. However, determining how bacteria respond to these microenvironments has been limited by a lack of tools to study bacterial functions at the relevant spatial scales in situ Here, we report the application of the hybridization chain reaction (HCR) v3.0 to provide analog mRNA relative quantitation of Pseudomonas aeruginosa single cells as a step toward this end. To assess the potential for this method to be applied to bacterial populations, we visualized the expression of genes needed for the production of alginate (algD) and the dissimilatory nitrate reductase (narG) at single-cell resolution within laboratory-grown aggregates. After validating new HCR probes, we quantified algD and narG expression across microenvironmental gradients within both single aggregates and aggregate populations using the agar block biofilm assay (ABBA). For mucoid and nonmucoid ABBA populations, narG was expressed in hypoxic and anoxic regions, while alginate expression was restricted to the hypoxic zone (∼40 to 200 µM O2). Within individual aggregates, surface-adjacent cells expressed alginate genes at higher levels than interior cells, revealing that alginate expression is not constitutive in mucoid P. aeruginosa but instead varies with oxygen availability. These results establish HCR v3.0 as a versatile and robust tool to resolve subtle differences in gene expression at spatial scales relevant to microbial assemblages. This advance has the potential to enable quantitative studies of microbial gene expression in diverse contexts, including pathogen activities during infections.IMPORTANCE A goal for microbial ecophysiological research is to reveal microbial activities in natural environments, including sediments, soils, or infected human tissues. Here, we report the application of the hybridization chain reaction (HCR) v3.0 to quantitatively measure microbial gene expression in situ at single-cell resolution in bacterial aggregates. Using quantitative image analysis of thousands of Pseudomonas aeruginosa cells, we validated new P. aeruginosa HCR probes. Within in vitroP. aeruginosa aggregates, we found that bacteria just below the aggregate surface are the primary cells expressing genes that protect the population against antibiotics and the immune system. This observation suggests that therapies targeting bacteria growing with small amounts of oxygen may be most effective against these hard-to-treat infections. More generally, this proof-of-concept study demonstrates that HCR v3.0 has the potential to identify microbial activities in situ at small spatial scales in diverse contexts.

Incorporation of a chiral gem-disubstituted nitrogen heterocycle yields an oxazolidinone antibiotic with reduced mitochondrial toxicity.

gem-Disubstituted N-heterocycles are rarely found in drugs, despite their potential to improve the drug-like properties of small molecule pharmaceuticals. Linezolid, a morpholine heterocycle-containing oxazolidinone antibiotic, exhibits significant side effects associated with human mitochondrial protein synthesis inhibition. We synthesized a gem-disubstituted linezolid analogue that when compared to linezolid, maintains comparable (albeit slightly diminished) activity against bacteria, comparable in vitro physicochemical properties, and a decrease in undesired mitochondrial protein synthesis (MPS) inhibition. This research contributes to the structure-activity-relationship data surrounding oxazolidinone MPS inhibition, and may inspire investigations into the utility of gem-disubstituted N-heterocycles in medicinal chemistry.

Direct Lung Sampling Indicates That Established Pathogens Dominate Early Infections in Children with Cystic Fibrosis.

Culture and sequencing have produced divergent hypotheses about cystic fibrosis (CF) lung infections. Culturing suggests that CF lungs are uninfected before colonization by a limited group of CF pathogens. Sequencing suggests diverse communities of mostly oral bacteria inhabit lungs early on and diversity decreases as disease progresses. We studied the lung microbiota of CF children using bronchoscopy and sequencing, with measures to reduce contamination. We found no evidence for oral bacterial communities in lung lavages that lacked CF pathogens. Lavage microbial diversity varied widely, but decreases in diversity appeared to be driven by increased CF pathogen abundance, which reduced the signal from contaminants. Streptococcus, Prevotella, and Veillonella DNA was detected in some lavages containing CF pathogens, but DNA from these organisms was vastly exceeded by CF pathogen DNA and was not associated with inflammation. These findings support the hypothesis that established CF pathogens are primarily responsible for CF lung infections.

Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in Pseudomonas aeruginosa.

While much attention has been focused on acquired antibiotic resistance genes, chromosomal mutations may be most important in chronic infections where isolated, persistently infecting lineages experience repeated antibiotic exposure. Here, we used experimental evolution and whole-genome sequencing to investigate chromosomally encoded mutations causing aztreonam resistance in Pseudomonas aeruginosa and characterized the secondary consequences of resistance development. We identified 19 recurrently mutated genes associated with aztreonam resistance. The most frequently observed mutations affected negative transcriptional regulators of the mexAB-oprM efflux system and the target of aztreonam, ftsI While individual mutations conferred modest resistance gains, high-level resistance (1,024 µg/ml) was achieved through the accumulation of multiple variants. Despite being largely stable when strains were passaged in the absence of antibiotics, aztreonam resistance was associated with decreased in vitro growth rates, indicating an associated fitness cost. In some instances, evolved aztreonam-resistant strains exhibited increased resistance to structurally unrelated antipseudomonal antibiotics. Surprisingly, strains carrying evolved mutations which affected negative regulators of mexAB-oprM (mexR and nalD) demonstrated enhanced virulence in a murine pneumonia infection model. Mutations in these genes, and other genes that we associated with aztreonam resistance, were common in P. aeruginosa isolates from chronically infected patients with cystic fibrosis. These findings illuminate mechanisms of P. aeruginosa aztreonam resistance and raise the possibility that antibiotic treatment could inadvertently select for hypervirulence phenotypes.IMPORTANCE Inhaled aztreonam is a relatively new antibiotic which is being increasingly used to treat cystic fibrosis patients with Pseudomonas aeruginosa airway infections. As for all antimicrobial agents, bacteria can evolve resistance that decreases the effectiveness of the drug; however, the mechanisms and consequences of aztreonam resistance are incompletely understood. Here, using experimental evolution, we have cataloged spontaneous mutations conferring aztreonam resistance and have explored their effects. We found that a diverse collection of genes contributes to aztreonam resistance, each with a small but cumulative effect. Surprisingly, we found that selection for aztreonam resistance mutations could confer increased resistance to other antibiotics and promote hypervirulence in a mouse infection model. Our study reveals inherent mechanisms of aztreonam resistance and indicates that aztreonam exposure can have unintended secondary effects.

Restoring Cystic Fibrosis Transmembrane Conductance Regulator Function Reduces Airway Bacteria and Inflammation in People with Cystic Fibrosis and Chronic Lung Infections.

RATIONALE: Previous work indicates that ivacaftor improves cystic fibrosis transmembrane conductance regulator (CFTR) activity and lung function in people with cystic fibrosis and G551D-CFTR mutations but does not reduce density of bacteria or markers of inflammation in the airway. These findings raise the possibility that infection and inflammation may progress independently of CFTR activity once cystic fibrosis lung disease is established. OBJECTIVES: To better understand the relationship between CFTR activity, airway microbiology and inflammation, and lung function in subjects with cystic fibrosis and chronic airway infections. METHODS: We studied 12 subjects with G551D-CFTR mutations and chronic airway infections before and after ivacaftor. We measured lung function, sputum bacterial content, and inflammation, and obtained chest computed tomography scans. MEASUREMENTS AND MAIN RESULTS: Ivacaftor produced rapid decreases in sputum Pseudomonas aeruginosa density that began within 48 hours and continued in the first year of treatment. However, no subject eradicated their infecting P. aeruginosa strain, and after the first year P. aeruginosa densities rebounded. Sputum total bacterial concentrations also decreased, but less than P. aeruginosa. Sputum inflammatory measures decreased significantly in the first week of treatment and continued to decline over 2 years. Computed tomography scans obtained before and 1 year after ivacaftor treatment revealed that ivacaftor decreased airway mucous plugging. CONCLUSIONS: Ivacaftor caused marked reductions in sputum P. aeruginosa density and airway inflammation and produced modest improvements in radiographic lung disease in subjects with G551D-CFTR mutations. However, P. aeruginosa airway infection persisted. Thus, measures that control infection may be required to realize the full benefits of CFTR-targeting treatments.