High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli

Harrison, Lucas; Hanson, Nancy D.

doi:10.1128/aac.00265-17

Cited by 16 publications

(16 citation statements)

References 30 publications

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“…; Figs and respectively. These results indicate that the use of a suitable primer with appropriate length is effective at the specific Tm, which is consistent with some studies (Harrison and Hanson ; Dehbashi et al ). The PCR amplification curves from a representative experiment for the detection of P. aeruginosa using the 16srRNA primer set shown in Fig.…”

Section: Resultssupporting

confidence: 92%

New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

Tahmasebi

Dehbashi

Arabestani

2020

Lett Appl Microbiol

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Colistin‐resistant Pseudomonas aeruginosa (CRPA), as a health care system threat, is increasing globally. This study aimed was to determine CRPA by high‐resolution melting curve (HRM) analysis method. The HRM method was done on standard strains of P. aeruginosa and CRPA strains. Ninefold serial dilutions of known genomic DNA (gDNA) and plasmid DNA (pDNA) concentrations, extracted from P. aeruginosa NCTC13715 and P. aeruginosa NCTC10728 were prepared and tested by melting curve analysis and HRM assay. Data analysis was performed using the Step‐One Plus Software v2.3 and hrm Software v3.0.1. The results of the melt curve analysis and HRM showed a very similar melt peak for all positive controls with a melt temperature that ranged from 88·1°C to 88·6°C for the 16srRNA, 90·0°C to 90·05°C for the mcr‐1 and 91·2°C to 91·7°C for the pmrA gene. Furthermore, the data indicated that the HRM approach has the sensitivity to detect 100 CFU per ml for the 16srRNA gene, 101 CFU per ml for the pmrA gene, and 103 CFU per ml for the mcr‐1 gene. According to our findings, it was concluded that the HRM method could detect 100 to 103 CFU per ml of P. aeruginosa gDNA and pDNA. Therefore, CRPA strains can be identified with high sensitivity and specificity by HRM assay. Significance and Impact of the Study The highlight of our research is about the detection of bacteria resistance genes to antibiotics by advanced molecular methods, which means high‐resolution melting curve (HRM) analysis. We determined that the HRM method in identifying colistin‐resistant P. aeruginosa has high accuracy and speed, and with high sensitivity and specificity.

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Section: Resultssupporting

confidence: 92%

New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

Tahmasebi

Dehbashi

Arabestani

2020

Lett Appl Microbiol

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“… 29 Other applications of HRMA in this area include detection of rifaximin-resistance in Clostridium difficile , 30 identification of nosocomial ESBL-producing E coli including ST131, 31 detection of antimicrobial resistance in Neisseria gonorrhoeae , 32 and detection of sequence type 131 E coli . 33 These PCR-HRMA approaches have the potential to be used as tests to determine efficiently and rapidly some antibiotic resistance genes in pathogens and therefore could facilitate the timely administration of appropriate antimicrobial therapy for the infected patient.…”

Section: Discussionmentioning

confidence: 99%

A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci

Ozbak

2018

Annals of Saudi Medicine

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BACKGROUNDVancomycin-resistant enterococci (VRE) are resistant to most classes of antibiotics. Diagnosis of VRE using standard methods takes 2 to 5 days. Development of a rapid PCR-assay that detects and identifies resistant genes in bacteria would provide time-critical information on the presence of VRE in clinical samples allowing early treatment and management of infected patients.OBJECTIVESInvestigate the use of high resolution melting analysis (HRMA) and 16S-rRNA-PCR approach for rapid and cost-effective identification of VRE.DESIGNDescriptive antibiotic susceptibility studies.SETTINGManchester Academic Health Sciences Centre and School of Translational Medicine, University of Manchester, UK, and Department of Clinical Laboratory Sciences, Taibah University, Saudi Arabia.MATERIALS AND METHODSPCR-HRMA using 16S-rRNA V1-primers was used to detect and identify VRE. DNA from different strains of vancomycin-resistant and -sensitive Enterococcus faecalis (VSE) and Enterococcus faecium were amplified using V1-primer followed by HRMA in a single run. Differentiation of VRE from VSE was based on curve shapes generated against reference organisms (Bacteroides fragilis).MAIN OUTCOMES MEASURESAmplification curves and difference plots for VRE and VSE.RESULTSDifference plots were generated for all vancomycin-resistant and -sensitive E faecalis and E faecium strains by subtracting their fluorescence melting profile from that of a reference-species B fragilis. A characteristic curve shape was produced by vancomycin-sensitive E faecalis and E faecium. However, vancomycin-resistant strains of these bacteria were associated with a markedly different curve shape facilitating a clear differentiation.CONCLUSIONThe 16S-PCR-HRMA approach has the potential for detecting vancomycin-resistant E faecium and E faecalis. Data with VRE provide the basis for combining VRE identification with pathogens speciation in a rapid, cheap assay able to identify a pathogen as an Enterococcus and whether it is vancomycin-sensitive or -resistant E faecium or E faecalis in a single PCR and HRMA run.LIMITATIONSTested on specific, but not all, reference Enterococcus species and clinical isolates.

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“…In this study, the visualization of the differences between the profiles was facilitated by the use of BioNumerics software. The identification of the ST131 clone can be determined by standard MLST, which requires Sanger sequencing; by multiplex PCR assay; and by high-resolution melting analysis (36)(37)(38). Besides showing a higher discriminatory power than MLST, MLVA could determine the serogroup of an ST131 E. coli strain.…”

Section: Discussionmentioning

confidence: 99%

Rapid and Simple Universal Escherichia coli Genotyping Method Based on Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Single-Tube Multiplex PCR and Standard Gel Electrophoresis

Caméléna

Birgy

Smail

et al. 2019

Appl Environ Microbiol

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We developed a multiplex PCR method based on multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) that was designed for the rapid typing of Escherichia coli and Shigella isolates. The method amplifies seven VNTRs and does not require a sequencing capillary or fluorescent dyes. The amplification products are simply loaded on a standard agarose gel for electrophoresis, and the banding patterns are analyzed visually. We evaluated the method on 220 strains belonging to different collections: the E. coli reference (ECOR) collection (n = 72), O1:K1 isolates causing neonatal meningitis (n = 38), extended-spectrum beta-lactamase-producing fecal isolates belonging to the worldwide sequence type 131 (ST131) clone (n = 38), Shiga toxin-producing E. coli (STEC) isolates of serogroups O157:H7 (n = 21) and O26 (n = 16, 8 of which belonged to an outbreak), 27 Shigella isolates (22 Shigella sonnei isolates, including 5 epidemic strains), and 8 reference strains. The performances were compared to those of multilocus sequence typing (MLST), the DiversiLab automated repetitive element palindromic PCR (REP-PCR), pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). We found 66 different profiles among the isolates in the ECOR collection. Among the clonal group O1:K1 isolates, 14 different profiles were identified. For the 37 STEC isolates, we found 23 profiles, with 1 corresponding to the 8 epidemic strains. We found 19 profiles among the 27 Shigella isolates, with 1 corresponding to the epidemic strain. The method was able to recognize strains of the ST131 clone and to distinguish the O16 and O25b serogroups and identified 15 different MLVA types among them. This method allows the simple, fast, and inexpensive typing of E. coli/Shigella isolates that can be carried out in any laboratory equipped for molecular biology and has a discriminatory power superior to that of MLST and DiversiLab REP-PCR but slightly lower than that of PFGE. IMPORTANCE Fast typing methods that can easily and accurately distinguish clonal groups and unrelated isolates are of particular interest for microbiologists confronted with outbreaks or performing epidemiological studies. Highly discriminatory universal methods, like PFGE, optical mapping, or WGS, are expensive and/or time-consuming. MLST is useful for phylogeny but is less discriminatory and requires sequencing facilities. PCR methods, which are fast and easy to perform, also have drawbacks. Random PCRs and REP-PCR are universal but lack reproducibility. Other PCR methods may lack the discriminatory power to differentiate isolates during outbreaks. MLVA combines the advantages of PCR methods with a high discriminatory power but in its standard form requires sequencing capillary electrophoresis. The method that we have developed combines the advantages of standard PCR (simple, fast, and inexpensive) with the high discriminatory power of MLVA and permits the typing of all E. coli isolates (either intestinal or extraintestinal pathogenic isolates as well as commensal isolates).

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High-Resolution Melting Analysis for Rapid Detection of Sequence Type 131 Escherichia coli

Cited by 16 publications

References 30 publications

New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci

Rapid and Simple Universal Escherichia coli Genotyping Method Based on Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Single-Tube Multiplex PCR and Standard Gel Electrophoresis

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