Dataset Information

Establishment of a Simple and Quick Method for Detecting Extended-Spectrum ?-Lactamase (ESBL) Genes in Bacteria.

ABSTRACT: Extended-spectrum ?-lactamase (ESBL) genes that render bacteria resistant to antibiotics are commonly detected using phenotype testing, which is time consuming and not sufficiently accurate. To establish a better method, we used phenotype testing to identify ESBL-positive bacterial strains and conducted PCR to screen for TEM (named after the patient Temoneira who provided the first sample), sulfhydryl reagent variable (SHV), cefotaxime (CTX)-M-1, and CTX-M-9, the 4 most common ESBL types and subtypes. We then performed multiplex PCR with 1 primer containing a biotin and hybridized the PCR products with gene-specific probes that were coupled with microbeads and coated with a specific fluorescence. The hybrids were linked to streptavidin-R-phycoerythrins (SA-PEs) and run through a flow cytometer, which sorted the fluorescently dyed microbeads and quantified the PEs. The results from single PCR, multiplex PCR, and cytometry were consistent with each other. We used this method to test 169 clinical specimens that had been determined for phenotypes and found 154 positive for genotypes, including 30 of the 45 samples that were negative for phenotypes. The CTX-M genotype tests alone, counting both positive and negative cases, showed 99.41% (168/169) consistency with the ESBL phenotype test. Thus, we have established a multiplex-PCR system as a simple and quick method that is high throughput and accurate for detecting 4 common ESBL types and subtypes.

SUBMITTER: Han ST

PROVIDER: S-EPMC5026501 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Establishment of a Simple and Quick Method for Detecting Extended-Spectrum β-Lactamase (ESBL) Genes in Bacteria.

Han Song-Tao ST Fei Ying Y Huang Jin-You JY Xu Mei M Chen Li-Chan LC Liao D Joshua DJ Tan Yu-Jie YJ

Journal of biomolecular techniques : JBT 20160916 4

Extended-spectrum β-lactamase (ESBL) genes that render bacteria resistant to antibiotics are commonly detected using phenotype testing, which is time consuming and not sufficiently accurate. To establish a better method, we used phenotype testing to identify ESBL-positive bacterial strains and conducted PCR to screen for TEM (named after the patient Temoneira who provided the first sample), sulfhydryl reagent variable (SHV), cefotaxime (CTX)-M-1, and CTX-M-9, the 4 most common ESBL types and sub ...[more]

PMID: 27672351

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Project description:BackgroundA. paniculata is widely known for its medicinal values and is traditionally used to treat a wide range of diseases such as cancer, diabetes, skin infections, influenza, diarrhoea, etc. The phytochemical constituents of this plant possess unique and interesting biological activities. The main focus of this study was to evaluate the antibacterial property of crude ethyl acetate (CEA) extract of A. paniculata against E. coli clinical isolates along with molecular docking of 10 different bioactive components from this plant with CTX-M-15.MethodsCEA extract was subjected to phytochemical and FTIR analysis. The E. coli isolates were tested for antibiotic susceptibility through disk-diffusion method to observe their resistance pattern towards different antibiotics. Antibacterial activity and biofilm assay were performed through broth microdilution using a 96-well microplate. CEA extract was further utilized to observe its effect on the expression of a gene encoding CTX-M-15. Finally, in-silico studies were performed where 10 different bioactive compounds from A. paniculata were molecularly docked with CTX-M-15.ResultsPhytochemical and FTIR analysis detected the presence of various secondary metabolites and functional groups in CEA extract respectively. Molecular docking provided the number of residues and bond lengths together with a positive docking score. Antibiotic susceptibility showed the multi-drug resistance of all the clinical strains of E. coli. The antibacterial and antibiofilm efficiency of CEA extract (25, 50 and 100 ?g/ml) was tested and 100 ?g/ml of the extract was more effective in all the strains of E. coli. All 3 ESBL producing strains of E. coli were subjected to gene expression analysis through PCR. Strains treated with 100 ?g/ml of the extract showed a downregulation of the gene encoding CTX-M-15 compared to untreated controls.ConclusionsThe utilization of CEA extract of A. paniculata proved an economical way of controlling the growth and biofilm formation of ESBL strains of E. coli. CEA extract was also able to downregulate the expression of a gene encoding CTX-M-15. Molecular docking of 10 different bioactive compounds from A. paniculata with CTX-M-15 provided the residues and bond lengths with a positive docking score.

Project description:Klebsiella oxytoca complex (KoC) species may overproduce their chromosomal class A OXY β-lactamases, conferring reduced susceptibility to piperacillin-tazobactam, expanded-spectrum cephalosporins and aztreonam. Moreover, since clavulanate maintains its ability to inhibit these enzymes, the resulting resistance phenotype may falsely resemble the production of acquired extended-spectrum β-lactamases (ESBLs). In this work, a collection of 44 KoC strains of human and animal origin was characterized with whole-genome sequencing (WGS) and broth microdilution (BMD) susceptibility testing. Comparison of ESBL producers (n = 11; including CTX-M-15 [n = 6] and CTX-M-1 [n = 5] producers) and hyperproducers of OXYs (n = 21) showed certain phenotypic differences: piperacillin-tazobactam (MIC90s: 16 versus >64 μg/mL), cefotaxime (MIC90s: 64 versus 4 μg/mL), ceftazidime (MIC90s: 32 versus 4 μg/mL), cefepime (MIC90s: 8 versus 4 μg/mL) and associated resistance to non-β-lactams (e.g., trimethoprim-sulfamethoxazole: 90.9% versus 14.3%, respectively). However, a clear phenotype-based distinction between the two groups was difficult. Therefore, we evaluated 10 different inhibitor-based confirmatory tests to allow such categorization. All tests showed a sensitivity of 100%. However, only combination disk tests (CDTs) with cefepime/cefepime-clavulanate and ceftazidime/ceftazidime-clavulanate or the double-disk synergy test (DDST) showed high specificity (100%, 95.5%, and 100%, respectively). All confirmatory tests in BMD or using the MIC gradient strip did not perform well (specificity, ≤87.5%). Of note, ceftazidime/ceftazidime-avibactam tests also exhibited low specificity (CDT, 87.5%; MIC gradient strip, 77.8%). Our results indicate that standard antimicrobial susceptibility profiles can raise some suspicion, but only the use of cefepime/cefepime-clavulanate CDT or DDST can guarantee distinction between ESBL-producing KoC strains and those hyperproducing OXY enzymes.

Dataset Information

Establishment of a Simple and Quick Method for Detecting Extended-Spectrum ?-Lactamase (ESBL) Genes in Bacteria.

Publications

Establishment of a Simple and Quick Method for Detecting Extended-Spectrum β-Lactamase (ESBL) Genes in Bacteria.

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