Phenotypic and Genomic Variability of Serial Peri-Lung Transplantation Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients

Cystic fibrosis (CF) represents one of the major genetic and chronic lung diseases affecting Caucasians of European descent. Patients with CF suffer from recurring infections that lead to further damage of the lungs. Pulmonary infection due to Pseudomonas aeruginosa is most prevalent, further increasing CF-related mortality. The present study describes the phenotypic and genotypic variations among 36 P. aeruginosa isolates obtained serially from a non-CF and five CF patients before, during and after lung transplantation (LTx). The classical and genomic investigation of these isolates revealed a common mucoid phenotype and only subtle differences in the genomes. Isolates originating from an individual patient shared ≥98.7% average nucleotide identity (ANI). However, when considering isolates from different patients, substantial variations in terms of sequence type (ST), virulence factors and antimicrobial resistance (AMR) genes were observed. Whole genome multi-locus sequence typing (MLST) confirmed the presence of unique STs per patient regardless of the time from LTx. It was supported by the monophyletic clustering found in the genome-wide phylogeny. The antibiogram shows that ≥91.6% of the isolates were susceptible to amikacin, colistin and tobramycin. For other antibiotics from the panel, isolates frequently showed resistance. Alternatively, a comparative analysis of the 36 P. aeruginosa isolates with 672 strains isolated from diverse ecologies demonstrated clustering of the CF isolates according to the LTx patients from whom they were isolated. We observed that despite LTx and associated measures, all patients remained persistently colonized with similar isolates. The present study shows how whole genome sequencing (WGS) along with phenotypic analysis can help us understand the evolution of P. aeruginosa over time especially its antibiotic resistance.

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Pseudomonas aeruginosa PA80 is a cystic fibrosis isolate deficient in RhlRI quorum sensing

Scientific Reports ◽

10.1038/s41598-021-85100-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Syed A. K. Shifat Ahmed ◽

Michelle Rudden ◽

Sabrina M. Elias ◽

Thomas J. Smyth ◽

Roger Marchant ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Genomic Islands ◽

Clinical Strain ◽

Whole Genome ◽

Synonymous Mutations ◽

Wide Range

AbstractPseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI’s) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

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1458. Uncharted territories: applying “precision medicine” to understand the treacherous landscape of extensively and multidrug resistant (XDR and MDR) Pseudomonas aeruginosa in a patient with cystic fibrosis and lung transplantation

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.1639 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S731-S731

Author(s):

Laura J Rojas ◽

Mohamad Yasmin ◽

Jacquelynn Benjamino ◽

Steven Marshall ◽

Kailynn DeRonde ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Lung Transplantation ◽

Precision Medicine ◽

Clinical Sample ◽

Phylogenetic Reconstruction ◽

Multidrug Resistant ◽

Population Diversity ◽

Before And After ◽

Research Grant

Abstract Background Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with cystic fibrosis (CF). Herein, we describe our experience managing a young woman suffering from CF with XDR P. aeruginosa who underwent lung transplantation. We highlight the contemporary difficulties reconciling the clinical, microbiological, and genetic information. Methods Mechanism-based-susceptibility disk diffusion synergy testing with double and triple antibiotic combinations aided in choosing tailored antimicrobial combinations to control the infection in the pre-transplant period, create an effective perioperative prophylaxis regimen, and manage recurrent infections in the post-transplant period. Thirty-six sequential XDR and PDR P. aeruginosa isolates obtained from the patient within a 17-month period, before and after a double-lung transplant were analyzed by whole genome sequencing (WGS) and RNAseq in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time Results Our phylogenetic reconstruction demonstrates that these isolates represent a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggests that a group of closely related strains was present in the patient prior to transplantation and continued to evolve throughout the course of treatment regardless of antibiotic usage.Our findings challenge antimicrobial stewardship programs that assist with the selection and duration of antibiotic regimens in critically ill and immunocompromised patients based on single-isolate laboratory-derived resistant profiles. We propose that an approach sampling the population of pathogens present in a clinical sample instead of single colonies be applied instead when dealing with XDR P. aeruginosa, especially in patients with CF. Conclusion In complex cases such as this, real-time combination testing and genomic/transcriptomic data could lead to the application of true “precision medicine” by helping clinicians choose the combination antimicrobial therapy most likely to be successful against a population of MDR pathogens present. Disclosures Federico Perez, MD, MS, Accelerate (Research Grant or Support)Merck (Research Grant or Support)Pfizer (Research Grant or Support) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

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EPS4.02 Porphyromonas, a candidate biomarker for detection of Pseudomonas aeruginosa pulmonary infection in cystic fibrosis

Journal of Cystic Fibrosis ◽

10.1016/s1569-1993(18)30254-6 ◽

2018 ◽

Vol 17 ◽

pp. S43 ◽

Cited By ~ 1

Author(s):

C.-A. Guilloux ◽

G. Marenne ◽

S. Mondot ◽

C. Lamoureux ◽

L. Billard ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Pulmonary Infection

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Effect of infection with transmissible strains of Pseudomonas aeruginosa on lung transplantation outcomes in patients with cystic fibrosis

The Journal of Heart and Lung Transplantation ◽

10.1016/j.healun.2014.09.040 ◽

2015 ◽

Vol 34 (4) ◽

pp. 588-593 ◽

Cited By ~ 8

Author(s):

Nadim Srour ◽

Cecilia Chaparro ◽

Katherine Vandemheen ◽

Lianne G. Singer ◽

Shaf Keshavjee ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Lung Transplantation ◽

Transplantation Outcomes

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Digging through the Obstruction: Insight into the Epithelial Cell Response to Respiratory Virus Infection in Patients with Cystic Fibrosis

Journal of Virology ◽

10.1128/jvi.01864-15 ◽

2016 ◽

Vol 90 (9) ◽

pp. 4258-4261 ◽

Cited By ~ 10

Author(s):

Matthew R. Hendricks ◽

Jennifer M. Bomberger

Keyword(s):

Cystic Fibrosis ◽

Lung Diseases ◽

Respiratory Virus ◽

Viral Infections ◽

Molecular Diagnostic ◽

Molecular Diagnostic Techniques ◽

Virus Infections ◽

Chronic Lung Diseases ◽

Respiratory Viral Infections ◽

Respiratory Virus Infections

Respiratory virus infections are common but generally self-limiting infections in healthy individuals. Although early clinical studies reported low detection rates, the development of molecular diagnostic techniques by PCR has led to an increased recognition that respiratory virus infections are associated with morbidity and acute exacerbations of chronic lung diseases, such as cystic fibrosis (CF). The airway epithelium is the first barrier encountered by respiratory viruses following inhalation and the primary site of respiratory viral replication. Here, we describe how the airway epithelial response to respiratory viral infections contributes to disease progression in patients with CF and other chronic lung diseases, including the role respiratory viral infections play in bacterial acquisition in the CF patient lung.

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Analysis and HPLC fractionation of urine from patients with cystic fibrosis, chronic lung diseases and normal controls

Clinical Biochemistry ◽

10.1016/s0009-9120(89)80036-0 ◽

1989 ◽

Vol 22 (5) ◽

pp. 377-383 ◽

Cited By ~ 3

Author(s):

Terese M. Guman-Wignot ◽

Jay Kaufman ◽

Douglas S. Holsclaw ◽

Irvin R. Schmoyer ◽

Jack Alhadeff

Keyword(s):

Cystic Fibrosis ◽

Lung Diseases ◽

Chronic Lung Diseases ◽

Hplc Fractionation ◽

Normal Controls

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Chronic infection sustained by a Pseudomonas aeruginosa High-Risk clone producing the VIM-1 metallo-β-lactamase in a cystic fibrosis patient after lung transplantation

Journal of Cystic Fibrosis ◽

10.1016/j.jcf.2018.01.007 ◽

2018 ◽

Vol 17 (4) ◽

pp. 470-474

Author(s):

Simona Pollini ◽

Claudia Mugnaioli ◽

Daniela Dolce ◽

Silvia Campana ◽

Anna Silvia Neri ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

High Risk ◽

Lung Transplantation ◽

Cystic Fibrosis Patient ◽

Chronic Infection

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Genome-wide association study of signature genetic alterations among pseudomonas aeruginosa cystic fibrosis isolates

PLoS Pathogens ◽

10.1371/journal.ppat.1009681 ◽

2021 ◽

Vol 17 (6) ◽

pp. e1009681

Author(s):

Wontae Hwang ◽

Ji Hyun Yong ◽

Kyung Bae Min ◽

Kang-Mu Lee ◽

Ben Pascoe ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Association Study ◽

Genome Wide Association Study ◽

Genetic Alterations ◽

Genome Wide Association ◽

Small Scale ◽

Outer Membrane Receptor ◽

Genome Wide ◽

A Genome

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes diverse human infections including chronic airway infection in patients with cystic fibrosis (CF). Comparing the genomes of CF and non-CF PA isolates has great potential to identify the genetic basis of pathogenicity. To gain a deeper understanding of PA adaptation in CF airways, we performed a genome-wide association study (GWAS) on 1,001 PA genomes. Genetic variations identified among CF isolates were categorized into (i) alterations in protein-coding regions, either large- or small-scale, and (ii) polymorphic variation in intergenic regions. We introduced each CF-associated genetic alteration into the genome of PAO1, a prototype PA strain, and validated the outcomes experimentally. Loci readily mutated among CF isolates included genes encoding a probable sulfatase, a probable TonB-dependent receptor (PA2332~PA2336), L-cystine transporter (YecS, PA0313), and a probable transcriptional regulator (PA5438). A promoter region of a heme/hemoglobin uptake outer membrane receptor (PhuR, PA4710) was also different between the CF and non-CF isolate groups. Our analysis highlights ways in which the PA genome evolves to survive and persist within the context of chronic CF infection.

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