Genomic diversity and antimicrobial resistance of Prevotella spp. isolated from chronic lung disease airways

Abstract

Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterised by increasingly frequent acute pulmonary exacerbations that reduce life quality and length. Human airways are home to a rich polymicrobial environment, including members of the obligately anaerobic genus, Prevotella. Despite their commonness, surprisingly little is known about the prevalence, role, genomic diversity, and antimicrobial resistance (AMR) potential of Prevotella species/strains in healthy and diseased airways. Here, we used comparative genomics to develop a real-time PCR assay to permit rapid Prevotella spp. quantification from cultures and clinical specimens. Assay specificity was validated across a panel of Prevotella and non-Prevotella species, followed by PCR screening of CF and COPD respiratory-derived cultures. Next, 35 PCR-positive isolates were subjected to whole-genome sequencing. Of eight identified species, P. histicola, P. melaninogenica, P. nanceiensis, P. salivae and P. denticola overlapped between participant cohorts. Phylogenomic analysis revealed considerable interhost but limited intrahost diversity, indicating patient-specific lineages rather than patient-to-patient transmission. Correlation of phenotypic AMR profiles with AMR gene presence identified excellent correlation between tetQ presence and decreased doxycycline susceptibility, and ermF presence and decreased azithromycin susceptibility and clindamycin resistance. AMR rates were higher in the CF isolates, reflecting greater antibiotic use in this cohort. All tested Prevotella isolates were tobramycin-resistant, providing a potential selection method to improve Prevotella culture retrieval rates. Our addition of 35 airway-derived Prevotella genomes to public databases will enhance ongoing efforts to unravel the role of this diverse and enigmatic genus in both chronic respiratory diseases and healthy lungs.

Express yourself: Quantitative real-time PCR assays for rapid chromosomal antimicrobial resistance detection in Pseudomonas aeruginosa

“…Testing towards 11 anti-pseudomonal antibiotics identified much higher rates of AMR (range: 0-9 antibiotics) and MDR (10/12 strains) in CF-derived isolates when compared with COPD-derived isolates (range: 0-2 antibiotics; 0/7 MDR strains) (Table 1). This was consistent with much greater antibiotic use in the CF cohort (Table 1) (40). The lowest susceptibility rates were towards cefepime (0% CF and 100% COPD) and piperacillin (33% CF and 100% COPD), whereas all 19 strains were susceptible to colistin and polymyxin B (Table 1).…”

“…Nine sputa from five CF participants, and six sputa from six COPD participants (40) (Table 1), all with high-load P. aeruginosa infections according to sputum culture onto MacConkey agar (Oxoid, Heidelberg West, VIC, Australia), were subjected to total RNA extraction to determine in vivo P. aeruginosa AMR gene expression profiles. Sputa were collected directly into RNA stabilisation reagent (10mM EDTA, 25mM sodium citrate, 700g/L ammonium sulphate; pH=5.2; Sigma-Aldrich, Castle Hill, NSW, Australia) to immediately halt transcriptional activity, and kept at 4°C until RNA extraction (up to ~1 month).…”

“…Nine sputa from five CF participants, and six sputa from six COPD participants (40) (Table 1), all with high-load P. aeruginosa infections according to sputum culture onto MacConkey agar (Oxoid, Heidelberg West, VIC, Australia), were subjected to total RNA extraction to determine in vivo P. aeruginosa AMR gene expression profiles. An additional six sputa from P. aeruginosa -infected COPD participants with low P. aeruginosa load were also examined, along with three COPD sputa and 1 COPD bronchial washing from P. aeruginosa culture-negative participants.…”

Rapid fluoroquinolone resistance detection inPseudomonas aeruginosausing mismatch amplification mutation assay-based real-time PCR

“…Eighty-four P. aeruginosa isolates from Southeast Queensland, Australia, were examined: 42 from sputum derived from adults with CF and chronic P. aeruginosa infection admitted to The Prince Charles Hospital between 2017 and 2019 (16); 35 bloodstream isolates retrieved from adults admitted to several public and private hospitals in Brisbane between 2008 and 2011 (17), three from COPD sputum, collected during in-home community nurse visits in the Sunshine Coast region (16); one from an adult with non-CF bronchiectasis collected in 2017, and one from an adult with urinary tract infection collected in 2018, both during admission to the Sunshine Coast University Hospital (Table 1). One ulcer and one ear infection isolate, both from Brisbane, were obtained from the 1,000 International P. aeruginosa Consortium collection (18).…”

“…One ulcer and one ear infection isolate, both from Brisbane, were obtained from the 1,000 International P. aeruginosa Consortium collection (18). Ethics approvals were obtained as previously described (16,17,19).…”