Prediction of fluoroquinolone susceptibility directly from whole genome sequence data using liquid chromatography-tandem mass spectrometry to identify mutant genotypes

Ismah, Wan Ahmad Kamil Wan Nur; Takebayashi, Yuiko; Findlay, Jacqueline; Heesom, Kate J; Jiménez-Castellanos, Juan-Carlos; Zhang, Jay; Graham, Lee H.; Bowker, Karen E.; Williams, O. Martin; MacGowan, Alasdair; Avison, Matthew B.

doi:10.1101/138248

Cited by 12 publications

(26 citation statements)

References 37 publications

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“…LC-MS/MS data was collected as previously described. 17 The raw data files were processed and quantified using Proteome Discoverer software v1.4 (Thermo Scientific) and searched against bacterial genome and horizontally acquired resistance genes as described previously. 18 Whole genome sequencing and analyses WGS was performed by MicrobesNG (https://microbesng.uk/) on a HiSeq 2500 instrument (Illumina, San Diego, CA, USA) using 2x250 bp paired end reads.…”

Section: Bacterial Isolates Identification and Susceptibility Testingmentioning

confidence: 99%

Characterization of AmpC-hyperproducing Escherichia coli from humans and dairy farms collected in parallel in the same geographical region

Alzayn

Findlay

Schubert

et al. 2020

Journal of Antimicrobial Chemotherapy

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Objectives To characterize putative AmpC-hyperproducing third-generation cephalosporin-resistant E. coli from dairy farms and their phylogenetic relationships; to identify risk factors for their presence; and to assess evidence for their zoonotic transmission into the local human population. Methods Proteomics was used to explain differences in antimicrobial susceptibility. WGS allowed phylogenetic analysis. Multilevel, multivariable logistic regression modelling was used to identify risk factors. Results Increased use of amoxicillin/clavulanate was associated with an increased risk of finding AmpC hyperproducers on farms. Expansion of cephalosporin resistance in AmpC hyperproducers was seen in farm isolates with marR mutations (conferring cefoperazone resistance) or when AmpC was mutated (conferring fourth-generation cephalosporin and cefoperazone resistance). Phylogenetic analysis confirmed the dominance of ST88 amongst farm AmpC hyperproducers but there was no evidence for acquisition of farm isolates by members of the local human population. Conclusions Clear evidence was found for recent farm-to-farm transmission of AmpC-hyperproducing E. coli and of adaptive mutations to expand resistance. Whilst there was no evidence of isolates entering the local human population, efforts to reduce third-generation cephalosporin resistance on dairy farms must address the high prevalence of AmpC hyperproducers. The finding that amoxicillin/clavulanate use was associated with an increased risk of finding AmpC hyperproducers is important because this is not currently categorized as a highest-priority critically important antimicrobial and so is not currently targeted for specific usage restrictions in the UK.

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Section: Bacterial Isolates Identification and Susceptibility Testingmentioning

confidence: 99%

Characterization of AmpC-hyperproducing Escherichia coli from humans and dairy farms collected in parallel in the same geographical region

Alzayn

Findlay

Schubert

et al. 2020

Journal of Antimicrobial Chemotherapy

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“…Genomes were sequenced by MicrobesNG on a HiSeq 2500 instrument (Illumina, San Diego, CA, USA) as previously described. 35 Insertion Sequences (IS) were identified using ISFinder. 36…”

Section: Whole Genome Sequencing (Wgs)mentioning

confidence: 99%

“…Total cell extractions of the clinical isolates (in three biological replicates) were prepared and 1 µg of each sample was analysed using an Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) and quantified using Proteome Discoverer software v1.4 (Thermo Fisher Scientific) as outlined previously. 35 The raw data files were searched against the UniProt A. baumannii ACIBA database (67,615 protein entries) and an in-house mobile resistance determinant database. 37 Abundance values of each protein were converted to ratios relative to the average abundance of 30S and 50S ribosomal proteins, for ease of comparison between isolates.…”

Section: Proteome Analysis Via Orbitrap Lc-ms/msmentioning

confidence: 99%

Variability in carbapenemase activity of intrinsic OxaAb (OXA-51-like) β-lactamase enzymes inAcinetobacter baumannii

Takebayashi

Findlay

Heesom

et al. 2020

Journal of Antimicrobial Chemotherapy

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Objectives To measure the variability in carbapenem susceptibility conferred by different OxaAb variants, characterize the molecular evolution of oxaAb and elucidate the contribution of OxaAb and other possible carbapenem resistance factors in the clinical isolates using WGS and LC–MS/MS. Methods Antimicrobial susceptibility tests were performed on 10 clinical Acinetobacter baumannii isolates. Carbapenem MICs were evaluated for all oxaAb variants cloned into A. baumannii CIP70.10 and BM4547, with and without their natural promoters. Molecular evolution analysis of the oxaAb variants was performed using FastTree and SplitsTree4. Resistance determinants were studied in the clinical isolates using WGS and LC–MS/MS. Results Only the OxaAb variants with I129L and L167V substitutions, OxaAb(82), OxaAb(83), OxaAb(107) and OxaAb(110) increased carbapenem MICs when expressed in susceptible A. baumannii backgrounds without an upstream IS element. Carbapenem resistance was conferred with the addition of their natural upstream ISAba1 promoter. LC–MS/MS analysis on the original clinical isolates confirmed overexpression of the four I129L and L167V variants. No other differences in expression levels of proteins commonly associated with carbapenem resistance were detected. Conclusions Elevated carbapenem MICs were observed by expression of OxaAb variants carrying clinically prevalent substitutions I129L and L167V. To drive carbapenem resistance, these variants required overexpression by their upstream ISAba1 promoter. This study clearly demonstrates that a combination of IS-driven overexpression of oxaAb and the presence of particular amino acid substitutions in the active site to improve carbapenem capture is key in conferring carbapenem resistance in A. baumannii and other mechanisms are not required.

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“…Chemicals were from Sigma and growth media from Oxoid, unless otherwise stated. Strains used were K. pneumoniae Ecl8 (34), plus the clinical isolate KP47 (35). To select colistin resistant mutants, one hundred microlitre aliquots of overnight cultures of the parent strain grown in Cation Adjusted Muller-Hinton Broth (CAMHB) were spread onto Mueller-Hinton Agar containing 32 µg.mL -1 colistin, which were then incubated for 24 h. Insertional inactivation of phoP, pmrD, or pmrA was performed using the pKNOCK suicide plasmid (36).…”

Section: Materials Bacterial Isolates Selection and Generation Of Mmentioning

confidence: 99%

Proteomic Investigation of the Signal Transduction Pathways Controlling Colistin Resistance in Klebsiella pneumoniae

Cheung

Dulyayangkul

Heesom

et al. 2020

Antimicrob Agents Chemother

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Colistin resistance in Klebsiella pneumoniae is predominantly caused by mutations that increase expression of the arn (also known as pbg or pmrF) operon. Expression is activated by the PhoPQ and PmrAB two-component systems. Constitutive PhoPQ activation occurs directly by mutation or following loss of MgrB. PhoPQ may also cross-activate PmrAB via the linker protein PmrD. Using proteomics, we show that MgrB loss causes a wider proteomic effect than direct PhoPQ activation, suggesting additional targets for MgrB. Different mgrB mutations cause different amounts of Arn protein production, which correlated with colistin MICs. Disruption of phoP in an mgrB mutant had a reciprocal effect to direct activation of PhoQ in a wild-type background, but the regulated proteins showed almost total overlap. Disruption of pmrD or pmrA slightly reduced Arn protein production in an mgrB mutant, but production was still high enough to confer colistin resistance; disruption of phoP conferred wild-type Arn production and colistin MIC. Activation of PhoPQ directly or through mgrB mutation did not significantly activate PmrAB or PmrC production, but direct activation of PmrAB by mutation was able to do this, and also activated Arn production and conferred colistin resistance. There was little overlap between the PmrAB and PhoPQ regulons. We conclude that under the conditions used for colistin susceptibility testing, PhoPQ-PmrD-PmrAB cross-regulation is not significant and that independent activation of PhoPQ or PmrAB is the main reason that Arn protein production increases above the threshold required for colistin resistance.

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Prediction of fluoroquinolone susceptibility directly from whole genome sequence data using liquid chromatography-tandem mass spectrometry to identify mutant genotypes

Cited by 12 publications

References 37 publications

Characterization of AmpC-hyperproducing Escherichia coli from humans and dairy farms collected in parallel in the same geographical region

Characterization of AmpC-hyperproducing Escherichia coli from humans and dairy farms collected in parallel in the same geographical region

Variability in carbapenemase activity of intrinsic OxaAb (OXA-51-like) β-lactamase enzymes inAcinetobacter baumannii

Proteomic Investigation of the Signal Transduction Pathways Controlling Colistin Resistance in Klebsiella pneumoniae

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