E. coli and K. pneumoniae isolates resistant to beta-lactam, aminoglycoside and fluoroquinolone antibiotics.
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ABSTRACT:
PROVIDER: PRJEB44242 | ENA |
REPOSITORIES: ENA
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Project description:New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In the current study we developed and evaluated a targeted LC-MS/MS assay for the detection of beta-lactam, aminoglycoside and fluoroquinolone resistance mechanisms in blood cultures positive for E. coli or K. pneumoniae. Selected targets were the beta-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal E. coli AmpC, OXA-48-like, NDM, VIM and KPC, the aminoglycoside modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6’)-Ib, ANT(2”)-I and APH(3’)-VI, the 16S-RMTases ArmA, RmtB, RmtC and RmtF, the quinolone resistance mechanisms QnrA, QnrB, AAC(6’)-Ib-cr, the wildtype QRDR of GyrA, and for E. coli, the porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, 100 negative blood cultures inoculated with isolates that were previously collected from blood cultures, and 48 isolates inoculated with isolates carrying genes of less prevalent resistance mechanisms.
2022-07-01 | PXD025365 | Pride
Project description:Co-occurrence of aminoglycoside and beta-lactam resistance in E.coli
| PRJEB36139 | ENA
Project description:Aminoglycoside antibiotics target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal aminoglycosides induce not only single errors, but also clusters of errors in full-length proteins in vivo with as much as four amino acid substitutions in a row. The downstream errorsin a cluster are up to 10,000-fold more frequent than the first error and independent of the intracellular aminoglycoside concentration. The prevalence, length, and composition of error clusters dependsnot only on the misreading propensity of a given aminoglycoside, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a new class of misreading events in vivo that may provide the predominant source of proteotoxic stress at low aminoglycoside concentration, which is particularly important for the autocatalytic uptake of the drugs.
2021-04-06 | PXD022098 | Pride
Project description:Beta-lactam resistant isolates Genome sequencing
| PRJNA543210 | ENA
Project description:Background: Antimicrobial resistance is generally studied using a combination of growth inhibition measurements, sometimes in combination with DNA detection methods. However, the actual proteins that cause resistance such as enzymes, efflux pumps and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, these three methods are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Objectives: We aimed to analyze which currently known antimicrobial resistance genes are detected at the protein level using liquid chromatography-mass spectrometry (LC-MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described and to correlate the abundance of different porins to resistance. Methods: Whole genome sequencing, high-resolution LC-MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Results & Conclusion: Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC-MS/MS. The detected mechanisms could explain phenotypic resistance in most of the studied isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC-MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.
2021-07-09 | PXD023736 | Pride
Project description:Background: Antimicrobial resistance is generally studied using a combination of growth inhibition measurements, sometimes in combination with DNA detection methods. However, the actual proteins that cause resistance such as enzymes, efflux pumps and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, these three methods are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Objectives: We aimed to analyze which currently known antimicrobial resistance genes are detected at the protein level using liquid chromatography-mass spectrometry (LC-MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described and to correlate the abundance of different porins to resistance. Methods: Whole genome sequencing, high-resolution LC-MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Results & Conclusion: Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC-MS/MS. The detected mechanisms could explain phenotypic resistance in most of the studied isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC-MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.
2021-07-09 | PXD023739 | Pride
Project description:We purified Atoh1-GFP positive hair cells from organotypic cultures of P1 cochlea 3 hours after 0.5mM gentamicin treatment and performed RNA sequencing to profile the early transcriptional response of hair cells to aminoglycoside antibiotics.
2015-04-30 | GSE66775 | GEO
Project description:We purified Atoh1-GFP positive hair cells from organotypic cultures of P1 cochlea 3 hours after 0.5mM gentamicin treatment and performed RNA sequencing to profile the early transcriptional response of hair cells to aminoglycoside antibiotics. Levels of mRNA in gentamicin-treated hair cells (three replicates) were compared to untreated hair cells (three replicates). GFP negative, non-hair cells populations from treated organs were compared to those from untreated organs (three replicates for each condition).
2015-04-30 | E-GEOD-66775 | biostudies-arrayexpress
Project description:Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10M-BM-5g/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended. 12 samples
2014-06-04 | E-GEOD-58213 | biostudies-arrayexpress
Project description:Aminoglycoside antibiotics (AGAs) target the ribosome and induce mistranslation, yet which translation errors induce bacterial cell death is unclear. The analysis of cellular proteins by quantitative mass spectrometry shows that bactericidal AGAs induce not only single translation errors, but also clusters of errors in full-length proteins in vivo with as much as four amino acid substitutions in a row. The downstream errors in a cluster are much more frequent than the first error (0.4 vs. 10-3, respectively) and independent of the intracellular AGA concentration. The prevalence, length, and composition of error clusters depends not only on the misreading propensity of a given AGA, but also on its ability to inhibit ribosome translocation along the mRNA. Error clusters constitute a new class of misreading events in vivo that may constitute the predominant source of proteotoxic stress at low AGA concentration, which is particularly important for the autocatalytic uptake of the drugs.
2021-04-06 | PXD019188 | Pride