Biology subjects

Paterson, D.

Publications and source records attributed to Paterson, D..

7 recordsLinked to original sources

bioRxiv2025

Emerging Resistance to Novel -βLactam β-Lactamase Inhibitor Combinations in Klebsiella pneumoniae bearing KPC Variants

BackgroundKlebsiella pneumoniae carbapenemase (KPC) variants, predominantly KPC-2 and KPC-3, are significant global resistance mechanisms. KPC-2 and KPC-3 confer resistance to a broad range of {beta}-lactams, including carbapenems, while remaining susceptible to ceftazidime-avibactam (CZA). Recently, new KPC variants have developed resistance to CZA through mutations, insertions, or deletions in regions such as the {Omega}-loop, 240-loop (237-243 aa), and 270-loop (266-275 aa). This study aimed to investigate the collateral resistance to cefiderocol (FDC) and cefepime/zidebactam (FPZ) among isolates with these mutations. MethodsFifteen clinical isolates of KPC-producing Klebsiella spp. were analyzed, representing 15 distinct variants. Antimicrobial susceptibility testing determined the MICs for CZA, carbapenems, FDC, FPZ, and other antibiotics. Synergy between CZA and FDC was assessed. Whole-genome sequencing (WGS) was used to identify mutations contributing to resistance. ResultsCZA resistance was confirmed in 12 of the 15 KPC variants. Collateral resistance to FDC was observed in eight isolates, with five exhibiting spontaneous resistant subpopulations. Six FDC-resistant strains had mutations in the 270-loop (266-275 aa). Collateral resistance to FPZ was seen in three KPC variants, especially those with mutations in the 270-loop (266-275 aa), though many {Omega}-loop and 240-loop (237-243 aa) mutants remained susceptible. WGS of FDC-resistant subpopulations revealed additional mutations in ompC, rpoC, dksA, and cirA. ConclusionsThis study demonstrates that emerging KPC variants showing resistance to CZA also exhibit resistance to FDC, with collateral resistance to FPZ observed to a lesser extent. Identifying mutations in blaKPC, cirA, and other genes is important to understand resistance mechanisms for effective therapies.

microbiology↗

bioRxiv2022

An optimised method for bacterial nucleic acid extraction from positive blood culture broths for whole genome sequencing, resistance phenotype prediction and downstream molecular applications

BackgroundA prerequisite to rapid molecular detection of pathogens causing bloodstream infections is an efficient, cost effective and robust DNA extraction solution. We describe methods for microbial DNA extraction direct from positive blood culture broths, suitable for metagenomic sequencing and the application of machine-learning based tools to predict antimicrobial susceptibility. MethodsProspectively collected culture-positive blood culture broths with Gram-negative bacteria, were directly extracted using various commercially available kits. We compared methods for efficient inhibitor removal, avoidance of DNA shearing or degradation, to achieve DNA of high quality and purity. Bacterial species identified via whole-genome metagenomic sequencing (Illumina, MiniSeq) from blood culture extracts were compared to conventional methods from cultured isolates (Vitek MS). A machine-learning algorithm (AREScloud) was used to predict susceptibility against commercially available antibiotics, compared to susceptibility testing (Vitek 2) and other commercially available rapid diagnostic instruments (Accelerate Pheno and BCID). ResultsA two-kit method using a modified MolYsis Basic kit (for host DNA depletion) and extraction using Qiagen DNeasy UltraClean microbial kits resulted in optimal extractions appropriate for multiple molecular applications, including PCR, short-read and long-read sequencing. DNA extracts from 40 blood culture broths were included. Taxonomic profiling by direct metagenomic sequencing matched species identification by conventional methods in 38/40 (95%) of samples, with two showing agreement to genus level. In two polymicrobial samples, a second organism was missed by sequencing. Whole genome sequencing antimicrobial susceptibility testing (WGS-AST) models were able to accurately infer profiles for 6 common pathogens against 17 antibiotics. Overall categorical agreement (CA) was 95%, with 11% very major errors (VME) and 3.9% major errors (ME). CA for WGS-AST was >95% for 5/6 of the most common pathogens (E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa and C. jejuni) while it was lower for K. oxytoca (66.7%), likely due to the presence of inducible cephalosporinases. Performance of WGS-AST was sub-optimal for uncommon pathogens (e.g. Elizabethkingia) and some combination antibiotic compounds (e.g. ticarcillin-clavulanate). Time to pathogen identification and resistance gene detection was fastest with BCID (1 h from blood culture positivity). Accelerate Pheno provided a rapid MIC result in approximately 8 h. While Illumina based direct metagenomic sequencing did not result in faster turn-around times compared conventional methods, use of real-time nanopore sequencing may allow faster data acquisition. ConclusionsThe application of direct metagenomic sequencing from positive blood culture broths is a feasible approach and solves some of the challenges of sequencing from low-bacterial load samples. Machine-learning based algorithms are also accurate for common pathogen / drug combinations, although additional work is required to optimise algorithms for uncommon species and more complex resistance genotypes, as well as streamlining methods to provide more rapid sequencing results.

microbiology↗

bioRxiv2022

Systematic benchmarking of all-in-one microbial SNP calling pipelines

Clinical and public health microbiology is increasingly utilising whole genome sequencing (WGS) technology and this has lead to the development of a myriad of analysis tools and bioinformatics pipelines. Single nucleotide polymorphism (SNP) analysis is an approach used for strain characterisation and determining isolate relatedness. However, in order to ensure the development of robust methodologies suitable for clinical application of this technology, accurate, reproducible, traceable and benchmarked analysis pipelines are necessary. To date, the approach to benchmarking of these has been largely ad-hoc with new pipelines benchmarked on their own datasets with limited comparisons to previously published pipelines. In this study, Snpdragon, a fast and accurate SNP calling pipeline is introduced. Written in Nextflow, Snpdragon is capable of handling small to very large and incrementally growing datasets. Snpdragon is benchmarked using previously published datasets against six other all-in-one microbial SNP calling pipelines, Lyveset, Lyveset2, Snippy, SPANDx, BactSNP and Nesoni. The effect of dataset choice on performance measures is demonstrated to highlight some of the issues associated with the current available benchmarking approaches. The establishment of an agreed upon gold-standard benchmarking process for microbial variant analysis is becoming increasingly important to aid in its robust application, improve transparency of pipeline performance under different settings and direct future improvements and development. Snpdragon is available at https://github.com/FordeGenomics/SNPdragon. Impact statementWhole-genome sequencing has become increasingly popular in infectious disease diagnostics and surveillance. The resolution provided by single nucleotide polymorphism (SNP) analyses provides the highest level of insight into strain characteristics and relatedness. Numerous approaches to SNP analysis have been developed but with no established gold-standard benchmarking approach, choice of bioinformatics pipeline tends to come down to laboratory or researcher preference. To support the clinical application of this technology, accurate, transparent, auditable, reproducible and benchmarked pipelines are necessary. Therefore, Snpdragon has been developed in Nextflow to allow transparency, auditability and reproducibility and has been benchmarked against six other all-in-one pipelines using a number of previously published benchmarking datasets. The variability of performance measures across different datasets is shown and illustrates the need for a robust, fair and uniform approach to benchmarking. Data SummaryO_LIPreviously sequenced reads for Escherichia coli O25b:H4-ST131 strain EC958 are available in BioProject PRJNA362676. BioSample accession numbers for the three benchmarking isolates are: O_LIEC958: SAMN06245884 C_LIO_LIMS6573: SAMN06245879 C_LIO_LIMS6574: SAMN06245880 C_LI C_LIO_LIAccession numbers for reference genomes against the E. coli O25b:H4-ST131 strain EC958 benchmark are detailed in table 2. C_LIO_LISimulated benchmarking data previously described by Yoshimura et al. is available at http://platanus.bio.titech.ac.jp/bactsnp (1). C_LIO_LISimulated datasets previously described by Bush et al. is available at http://dx.doi.org/10.5287/bodleian:AmNXrjYN8 (2). C_LIO_LIReal sequencing benchmarking datasets previously described by Bush et al. are available at http://dx.doi.org/10.5287/bodleian:nrmv8k5r8 (2). C_LI O_TBL View this table: org.highwire.dtl.DTLVardef@f2446corg.highwire.dtl.DTLVardef@16a299corg.highwire.dtl.DTLVardef@d1c7a5org.highwire.dtl.DTLVardef@8a3cbaorg.highwire.dtl.DTLVardef@198f455_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONReference genomes used in the EC958 benchmarking dataset and the percent identity against the three included samples. C_TABLECAPTION C_TBL

bioinformatics↗

bioRxiv2021

Evaluating mono and combination therapy of meropenem and amikacin against Pseudomonas aeruginosa bacteremia in the Hollow-Fiber Infection Model

Debate continues as to the role of combination antibiotic therapy for the management of Pseudomonas aeruginosa infections. We studied extent of bacterial killing and resistance emergence of meropenem and amikacin as monotherapy and as a combination therapy against susceptible and resistant P. aeruginosa isolates from bacteremic patients using the dynamic in vitro hollow-fiber infection model. Three P. aeruginosa isolates (meropenem MICs 0.125, 0.25 & 64 mg/L) were used simulating bacteremia with an initial inoculum ~1x105 CFU/mL and the expected pharmacokinetics of meropenem and amikacin in critically ill patients. For isolates susceptible to amikacin and meropenem (isolates 1 and 2), the rate of bacterial killing was increased with the combination regimen when compared with monotherapy of either antibiotic. Both the combination and meropenem monotherapy were able to sustain bacterial killing throughout the seven-day treatment course, whereas regrowth of bacteria occurred with amikacin monotherapy after 12 hours. For the meropenem-resistant P. aeruginosa isolate (isolate 3), only the combination regimen demonstrated bacterial killing. Given that tailored antibiotic regimens can maximize potential synergy against some isolates, future studies should explore the benefit of combination therapy against resistant P. aeruginosa.

pharmacology and toxicology↗

bioRxiv2020

Mechanistic insights into ventricular arrhythmogenesis of hydroxychloroquine and azithromycin for the treatment of COVID-19

Background: Recent reports on the use of hydroxychloroquine (HCQ) alone, or combined with azithromycin (AZM) in the management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have raised cardiac safety concerns. Currently, there is limited mechanistic data evaluating cardiac safety with HCQ and AZM therapy. Methods: Using comprehensive In Vitro ProArrhythmia Assay (CiPA) Schema IC50 paradigms, we examined the cardiac electrophysiological effects of HCQ and HCQ/AZM. Molecular modelling explored HCQ and AZM binding properties to hERG. Langendorff-perfused guinea-pig hearts were electrically and optically mapped by multi-electrode array and voltage (RH237) and Ca2+ (Rhod-2 AM) dyes. Human action potential and ion current reconstructions were performed in silico. Results: HCQ blocked IKr and IK1 with IC50 concentrations (10+-0.6 and 34+-5.0 microM) within the therapeutic range observed clinically. HCQ also blocked INa and ICaL but at higher IC50, whilst Ito and IKs were unaffected. Contrastingly, AZM produced minor inhibition of INa, ICaL, IKs, and IKr,, with no effect on IK1 and Ito. HCQ + AZM combined inhibited IKr and IK1 with IC50s of 7.7 +- 0.8 microM and 30.4 +- 3.0 microM, but spared INa, ICaL and Ito,. Molecular modelling confirmed potential HCQ binding to hERG. Cardiac mapping and ECG studies in isolated hearts demonstrated that HCQ slowed heart rate and ventricular conduction with associated prolongation of PR, QRS and QT intervals. Optical mapping demonstrated, and prolonged, more heterogeneous, action potential durations and intracellular Ca2+ transients. These effects were accentuated with combined HCQ+AZM treatment, which elicited electrical alternans, re-entrant circuits and wave breaks. Reconstruction in a human in-silico model demonstrated that this is attributable to the integrated action of HCQ and AZM reducing IKr, IKs and IK1. Conclusions: These data provide an electrophysiological basis for recent FDA guidelines cautioning against combined HCQ/AZM administration for the treatment of Covid-19 on the grounds of potential cardiac safety. We would strongly recommend monitoring of electrocardiographic QT interval with the use of this combination of medications.Competing Interest StatementThe authors have declared no competing interest.

pharmacology and toxicology↗

bioRxiv2019

Prospective evaluation of beta-lactamase detection in penicillin susceptible Staphylococcus aureus by interpretation of the penicillin disc edge

Penicillin susceptible Staphylococcus aureus (PSSA) may occasionally be encountered as a cause of complicated S. aureus infection, such as endocarditis or bloodstream infections. Clinicians may choose to treat these patients with penicillin over a semi-synthetic penicillin derivative, such as flucloxacillin or oxacillin, due to a favourable Pk/Pd profile. In this study, we prospectively evaluated the penicillin disc (1-IU) method for detection of blaZ, with interpretation of the penicillin edge according to EUCAST recommendations. 472 PSSA isolates were collected between September 2014 to December 2015 from three clinical microbiology laboratories in Queensland, Australia. Initial antimicrobial susceptibility testing was performed by the Vitek 2 system. Real-time PCR for blaZ was performed following phenotypic testing with the 1-IU penicillin disc and the PCR used as the gold standard for detection of penicillinase. The prevalence of blaZ amongst the isolates was 7%. The sensitivity, specificity, positive predictive value and negative predictive value of the penicillin disc method was 97%, 95%, 61% and 100% when compared to blaZ PCR. In summary, the penicillin disc zone size and edge interpretation is a reliable method for detection of blaZ in S. aureus isolates that otherwise test susceptible to penicillin by Vitek 2 AST.

microbiology↗

bioRxiv2017

Time-resolved analyses of elemental distribution and concentration in living plants: An example using manganese toxicity in cowpea leaves

O_LIKnowledge of elemental distribution and concentration within plant tissues is crucial in the understanding of almost every process that occurs within plants. However, analytical limitations have hindered the microscopic determination of changes over time in the location and concentration of nutrients and contaminants in living plant tissues.\nC_LIO_LIWe developed a novel method using synchrotron-based micro X-ray fluorescence (-XRF) that allows for laterally-resolved, multi-element, kinetic analyses of plant leaf tissues in vivo. To test the utility of this approach, we examined changes in the accumulation of Mn in unifoliate leaves of 7-d-old cowpea (Vigna unguiculata) plants grown for 48 h at 0.2 and 30 M Mn in solution.\nC_LIO_LIRepeated -XRF scanning did not damage leaf tissues demonstrating the validity of the method. Exposure to 30 M Mn for 48 h increased the initial number of small spots of localized high Mn and their concentration rose from 40 to 670 mg Mn kg-1 fresh mass. Extension of the two-dimensional -XRF scans to a three-dimensional geometry provided further assessment of Mn localization and concentration.\nC_LIO_LIThis method shows the value of synchrotron-based -XRF analyses for time-resolved in vivo analysis of elemental dynamics in plant sciences.\nC_LI

plant biology↗