ORCID Profile
0000-0002-6682-9351
Current Organisation
University of Queensland
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Publisher: Cold Spring Harbor Laboratory
Date: 07-06-2019
DOI: 10.1101/663674
Abstract: Patient-derived isolates of the opportunistic pathogen Pseudomonas aeruginosa are frequently resistant to antibiotics due to the presence of sequence variants in resistance-associated genes. However, the frequency of antibiotic resistance and of resistance-associated sequence variants in environmental isolates of P. aeruginosa has not been well studied. Antimicrobial susceptibility testing (ciprofloxacin, ceftazidime, meropenem, tobramycin) of environmental (n=50) and cystic fibrosis (n=42) P. aeruginosa isolates was carried out. Following whole genome sequencing of all isolates, twenty-five resistance-associated genes were analysed for the presence of likely function-altering sequence variants. Environmental isolates were susceptible to all antibiotics with one exception, whereas patient-derived isolates had significant frequencies of resistance to each antibiotic and a greater number of likely resistance-associated genetic variants. These findings indicate that the natural environment does not act as a reservoir of antibiotic-resistant P. aeruginosa , supporting a model in which antibiotic susceptible environmental bacteria infect patients and develop resistance during infection.
Publisher: Cold Spring Harbor Laboratory
Date: 04-02-2022
DOI: 10.1101/2022.02.03.22270419
Abstract: The rise of antimicrobial-resistant (AMR) bacteria is a global health emergency. One critical facet in tackling this epidemic is more rapid AMR diagnosis in serious multi-drug resistant pathogens like Pseudomonas aeruginosa . Here, we designed and then validated two multiplex quantitative real-time PCR (qPCR) assays to simultaneously detect differential expression of the resistance-nodulation- ision efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM, the AmpC β-lactamase, and the porin OprD, which are commonly associated with chromosomally-encoded AMR. Next, qPCRs were tested on 15 sputa from 11 participants with P. aeruginosa respiratory infections to determine AMR profiles in vivo . We confirm multiplex qPCR testing feasibility directly on sputa, representing a key advancement in in vivo AMR diagnosis. Notably, comparison of sputa with their derived isolates grown in Luria-Bertani broth (±2.5% NaCl) or a 5-antibiotic cocktail showed marked expression differences, illustrating the difficulty in replicating in vivo expression profiles in vitro . Cystic fibrosis sputa showed significantly reduced mexE and mexY expression when compared with chronic obstructive pulmonary disease sputa, despite harbouring fluoroquinolone- and aminoglycoside-resistant strains, indicating that these loci are not contributing to AMR in vivo. oprD was also significantly downregulated in cystic fibrosis sputa, even in the absence of contemporaneous carbapenem use, suggesting a common adaptive trait in chronic infections that may affect carbapenem efficacy. Sputum C expression was highest in participants receiving carbapenems (6.7-15x), some of whom were simultaneously receiving cephalosporins, the latter of which would be rendered ineffective by the upregulated C . Our qPCR assays provide valuable insights into the P. aeruginosa resistome, and their use on clinical specimens will permit timely treatment alterations that will improve patient outcomes and antimicrobial stewardship measures.
Publisher: Microbiology Society
Date: 11-2019
DOI: 10.1099/JMM.0.001085
Abstract: Patient-derived isolates of the opportunistic pathogen
Publisher: Wiley
Date: 27-03-2019
DOI: 10.1111/RESP.13544
Abstract: Aerosol transmission of Pseudomonas aeruginosa has been suggested as a possible mode of respiratory infection spread in patients with cystic fibrosis (CF) however, whether this occurs in other suppurative lung diseases is unknown. Therefore, we aimed to determine if (i) patients with bronchiectasis (unrelated to CF) or chronic obstructive pulmonary disease (COPD) can aerosolize P. aeruginosa during coughing and (ii) if genetically indistinguishable (shared) P. aeruginosa strains are present in these disease cohorts. People with bronchiectasis or COPD and P. aeruginosa respiratory infection were recruited for two studies. Aerosol study: Participants (n = 20) underwent cough testing using validated cough rigs to determine the survival of P. aeruginosa aerosols in the air over distance and duration. Genotyping study: P. aeruginosa sputum isolates (n = 95) were genotyped using the iPLEX20SNP platform, with a subset subjected to the enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) assay to ascertain their genetic relatedness. Aerosol study: Overall, 7 of 20 (35%) participants released P. aeruginosa cough aerosols during at least one of the cough aerosol tests. These cough aerosols remained viable for 4 m from the source and for 15 min after coughing. The mean total aerosol count of P. aeruginosa at 2 m was two colony-forming units. Typing study: No shared P. aeruginosa strains were identified. Low viable count of P. aeruginosa cough aerosols and a lack of shared P. aeruginosa strains observed suggest that aerosol transmission of P. aeruginosa is an unlikely mode of respiratory infection spread in patients with bronchiectasis and COPD.
Publisher: Microbiology Society
Date: 03-02-2022
Abstract: Cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterized by increasingly frequent acute pulmonary exacerbations that reduce life quality and length. Human airways are home to a rich polymicrobial environment, which includes members of the obligately anaerobic genus Prevotella . Despite their commonness, surprisingly little is known about the prevalence, role, genomic ersity and antimicrobial resistance (AMR) potential of Prevotella species and strains in healthy and diseased airways. Here, we used comparative genomics to develop a real-time PCR assay to permit rapid Prevotella species identification and 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 Prevotella species, P. histicola , P. melaninogenica , P. nanceiensis , P. salivae and P. denticola overlapped between participant cohorts. Phylogenomic analysis revealed considerable interhost but limited intrahost ersity, suggesting patient-specific lineages in the lower airways, probably from oral cavity aspirations. Correlation of phenotypic AMR profiles with AMR genes 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 erse and enigmatic genus in both diseased and healthy lungs.
Publisher: Elsevier BV
Date: 10-2017
Publisher: Cold Spring Harbor Laboratory
Date: 31-08-2021
DOI: 10.1101/2021.08.30.21262864
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 ersity, 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 ersity, suggesting patient-specific lineages in the lower airways, probably from oral cavity aspirations. 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 erse and enigmatic genus in both chronic respiratory diseases and healthy lungs. Thirty-five Prevotella spp . genomes generated in this study are available in the Sequence Read Archive (SRA) and GenBank databases under BioProject accession PRJNA742126.
Publisher: Cold Spring Harbor Laboratory
Date: 19-12-2021
DOI: 10.1101/2021.12.12.21266792
Abstract: Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug resistant ESKAPE pathogen, Pseudomonas aeruginosa . We aimed to develop two SYBR Green-based mismatch lification mutation assays (SYBR-MAMAs) targeting GyrA T83I ( gyrA 248), and GyrA D87N, D87Y, and D87H ( gyrA 259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa . Following assay validation, the gyrA 248 and gyrA 259 SYBR-MAMAs were tested on 84 clinical P. aeruginosa isolates from Queensland, Australia, 45 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (84%) of isolates with intermediate/full FQ resistance. Importantly, all FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100% concordance with SYBR-MAMA genotypes. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa . An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91%. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.
Publisher: Elsevier BV
Date: 2022
Publisher: Public Library of Science (PLoS)
Date: 16-05-2023
DOI: 10.1371/JOURNAL.PONE.0285856
Abstract: Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of in idual mutations and mutation combinations. Thirty-five mutants with reduced susceptibility to ceftazidime were evolved from two antibiotic-sensitive P . aeruginosa reference strains PAO1 and PA14. Mutations were identified by whole genome sequencing. The evolved mutants tolerated ceftazidime at concentrations between 4 and 1000 times that of the parental bacteria, with most mutants being ceftazidime resistant (minimum inhibitory concentration [MIC] ≥ 32 mg/L). Many mutants were also resistant to meropenem, a carbapenem antibiotic. Twenty-eight genes were mutated in multiple mutants, with dacB and mpl being the most frequently mutated. Mutations in six key genes were engineered into the genome of strain PAO1 in idually and in combinations. A dacB mutation by itself increased the ceftazidime MIC by 16-fold although the mutant bacteria remained ceftazidime sensitive (MIC 32 mg/L). Mutations in C , mexR , nalC or nalD increased the MIC by 2- to 4-fold. The MIC of a dacB mutant was increased when combined with a mutation in C , rendering the bacteria resistant, whereas other mutation combinations did not increase the MIC above those of single mutants. To determine the clinical relevance of mutations identified through experimental evolution, 173 ceftazidime-resistant and 166 sensitive clinical isolates were analysed for the presence of sequence variants that likely alter function of resistance-associated genes. dacB and C sequence variants occur most frequently in both resistant and sensitive clinical isolates. Our findings quantify the in idual and combinatorial effects of mutations in different genes on ceftazidime susceptibility and demonstrate that the genetic basis of ceftazidime resistance is complex and multifactorial.
Publisher: Microbiology Society
Date: 27-10-2022
DOI: 10.1099/JMM.0.001593
Abstract: Background. Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, Pseudomonas aeruginosa . Objectives. We aimed to develop two SYBR Green-based mismatch lification mutation assays (SYBR-MAMAs) targeting GyrA T83I ( gyrA 248) and GyrA D87N, D87Y and D87H ( gyrA 259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa . Methods. Following assay validation, the gyrA 248 and gyrA 259 SYBR-MAMAs were tested on 84 Australian clinical P. aeruginosa isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing. Results. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes. Conclusions. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa . An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.
Publisher: American Society for Microbiology
Date: 17-05-2022
DOI: 10.1128/AAC.00204-22
Abstract: The rise of antimicrobial-resistant (AMR) bacteria is a global health emergency. One critical facet of tackling this epidemic is more rapid AMR diagnosis in serious multidrug-resistant pathogens like Pseudomonas aeruginosa .
No related grants have been discovered for Kay Ramsay.