Detection of the tetM resistance determinant among phenotypically sensitive Ureaplasma species by a novel real-time PCR method

Kotrotsiou, Tzimoula; Exindari, Maria; Diza, Eudoxia; Melidou, Angeliki; Malisiovas, Nikolaos

doi:10.1016/j.diagmicrobio.2014.10.013

Cited by 11 publications

(4 citation statements)

References 23 publications

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“…Real-time polymerase chain reaction (real-time PCR) combines amplification and detection of gene targets in a single assay. Advantages include rapid detection, the ability to be implemented in high throughput settings [25], and cost effectiveness [26]. e porA pseudogene is a N. gonorrhoeae species-specific marker [27][28][29] and is highly conserved across a diverse range of N. gonorrhoeae strains making it a useful target for identification [27,28,30,31].…”

Section: Introductionmentioning

confidence: 99%

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens

Mitchev

Singh

Ramsuran

et al. 2022

International Journal of Microbiology

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Background. Antimicrobial resistance is limiting treatment options for Neisseria gonorrhoeae infections. To aid or replace culture and the syndromic management approach, molecular assays are required for antimicrobial susceptibility testing to guide appropriate and rapid treatment. Objective. We aimed to detect single-nucleotide polymorphisms and plasmids associated with antimicrobial resistance from N. gonorrhoeae isolates from a clinic population in South Africa, using real-time PCR as a rapid test for AMR detection. Methods. N. gonorrhoeae isolates, from female and male patients presenting for care at a sexually transmitted infections clinic in Durban, South Africa, were analysed using phenotypic and genotypic methods for identification and antibiotic susceptibility testing (AST). Real-time PCR and high-resolution melting analysis were used to detect porA pseudogene (species-specific marker) and resistance-associated targets. Whole-genome sequencing was used as the gold standard for the presence of point mutations. Results. The real-time porA pseudogene assay identified all N. gonorrhoeae-positive isolates and specimens. Concordance between molecular detection (real-time PCR and HRM) and resistance phenotype was ≥92% for blaTEM (HLR penicillin), rpsJ_V57M (tetracycline), tetM (tetracycline), and gyrA_S91F (ciprofloxacin). Resistance determinants 16SrRNA_C1192U (spectinomycin), mtrR_G45D (azithromycin), and penA_D545S, penA_mosaic (cefixime/ceftriaxone) correlated with the WHO control isolates. Conclusions. Eight resistance-associated targets correlated with phenotypic culture results. The porA pseudogene reliably detected N. gonorrhoeae. Larger cohorts are required to validate the utility of these targets as a convenient culture-free diagnostic tool, to guide STI management in a South African population.

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Section: Introductionmentioning

confidence: 99%

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens

Mitchev

Singh

Ramsuran

et al. 2022

International Journal of Microbiology

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“…In recent years, a method combining culture and qPCR has been reported for the AST (Hunfeld et al, 2004;Beuving et al, 2011;Tzimoula et al, 2015;Rolain et al, 2004). In this method, the nucleic acid change of the bacteria during culture was determined by qPCR detection of a specific gene fragment of the bacteria.…”

Section: Introductionmentioning

confidence: 99%

Parallel susceptibility testing of bacteria through culture-quantitative PCR in 96-well plates

Jun

Yang

et al. 2018

International Journal of Infectious Diseases

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“…The tet (M) gene is widely distributed among both gram-positive and gram-negative bacteria . Moreover, the tet (M) determinant is considered to motivate the sole tetracycline resistance mechanism in mycoplasmas, which protects the bacterial ribosome from the effects of antibiotics. , The most common method for detecting antibiotic resistance genes uses traditional nucleic acid amplification such as polymerase chain reaction (PCR), which requires multi-step reactions, well-trained personnel, and many expensive reagents with a two-hour assay time. , These shortcomings have impeded application in nonlaboratory and limited-resource settings. To overcome such limitations of traditional nucleic acid detection methods, it is crucial to develop new diagnostic tools for screening and detecting antibiotic resistance genes such as simple and rapid point-of-care testing.…”

mentioning

confidence: 99%

“… 2 Moreover, the tet (M) determinant is considered to motivate the sole tetracycline resistance mechanism in mycoplasmas, which protects the bacterial ribosome from the effects of antibiotics. 4 , 5 The most common method for detecting antibiotic resistance genes uses traditional nucleic acid amplification such as polymerase chain reaction (PCR), which requires multi-step reactions, well-trained personnel, and many expensive reagents with a two-hour assay time. 3 , 6 These shortcomings have impeded application in nonlaboratory and limited-resource settings.…”

mentioning

confidence: 99%

Rapid and Sensitive Detection of Antibiotic Resistance Genes by Utilizing TALEs as a Diagnostic Probe with 2D-Nanosheet Graphene Oxide

2023

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As antibiotic resistance has risen as one of the major health concerns associated with infectious diseases due to the reduced efficacy of antibiotics, rapid and sensitive detection of antibiotic resistance genes is critical for more effective and faster treatment of infectious diseases. A class of programmable DNA-binding domains called transcriptional activator-like effectors (TALEs) provides a novel scaffold for designing versatile DNA-binding proteins due to their modularity and predictability. Here, we developed a simple, rapid, and sensitive system for detecting antibiotic resistance genes by exploring the potential of TALE proteins for the creation of a sequence-specific DNA diagnostic along with 2D-nanosheet graphene oxide (GO). TALEs were engineered to directly recognize the specific double-stranded (ds) DNA sequences present in the tetracycline resistance gene (tetM), avoiding the need for dsDNA denaturation and renaturation. We take advantage of the GO as an effective signal quencher to quantum dot (QD)-labeled TALEs for creating a turn-on strategy. QD-labeled TALEs are adsorbed on the GO surface, which will bring QDs in close proximity to GO. Due to the fluorescence quenching ability of GO, QDs are expected to be quenched by GO via fluorescence resonance energy transfer (FRET). QDlabeled TALE binding to the target dsDNA would lead to the conformational change, which would result in dissociation from the GO surface, thereby restoring the fluorescence signal. Our sensing system was able to detect low concentrations of dsDNA sequences in the tetM gene after only 10-minute incubation with the DNA, providing a limit of detection as low as 1 fM of Staphylococcus aureus genomic DNA. This study demonstrated that our approach of utilizing TALEs as a new diagnostic probe along with GO as a sensing platform can provide a highly sensitive and rapid method for direct detection of the antibiotic resistance gene without requiring DNA amplification or labeling.

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Detection of the tetM resistance determinant among phenotypically sensitive Ureaplasma species by a novel real-time PCR method

Cited by 11 publications

References 23 publications

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens

Parallel susceptibility testing of bacteria through culture-quantitative PCR in 96-well plates

Rapid and Sensitive Detection of Antibiotic Resistance Genes by Utilizing TALEs as a Diagnostic Probe with 2D-Nanosheet Graphene Oxide

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