High-Resolution Melting-Curve Analysis of Ligation-Mediated Real-Time PCR for Rapid Evaluation of an Epidemiological Outbreak of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli

Abstract

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n ‫؍‬ 15) and control isolates (n ‫؍‬ 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n ‫؍‬ 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.Antibiotic resistance among bacteria is an increasing problem in hospitals and other health care facilities. In order to prevent nosocomial infections, basic hygiene procedures must be strictly followed by all staff members. When outbreaks occur, it is crucial to identify and isolate patients as soon as possible (5). In order to identify potential nosocomial outbreaks, several molecular methods have been described (2,14,20). These epidemiological typing methods are based on both genomic and phenotypic principles. Many of the methods are time-consuming and expensive and require special equipment. When designing new typing methods, several factors need to be considered, depending on the purpose. These factors include stability, discriminatory power, reproducibility, speed, accessibility, cost-efficiency, and user efficiency (2,19,20). In addition, its appropriateness in a given situation (e.g., an outbreak situation) must be evaluated. As of today, pulsed-field gel electrophoresis (PFGE) is considered the gold standard for a large number of bacterial species because of its discriminatory power and high typeability (19,20). The drawbacks of PFGE are that it is laborious and time-consuming and that the interpretation can be complex and requires rigorous standardization and experienced personnel in order to achieve reproducible results that are comparable over time and place. Clear criteria to determine whether two or more isolates are identical during a restricted time and place have been developed (15,18). New genotyping methods are continuously being developed. One approach is repetiti...

Epidemiological characterization of a nosocomial outbreak of extended spectrum β‐lactamase Escherichia coli ST‐131 confirms the clinical value of core genome multilocus sequence typing

“…Our results contribute to the knowledge of molecular typing methods as it applies modern WGS-based methods on a nosocomial outbreak of clinical relevance where data on earlier generation molecular typing methods were available (7). Additionally, there is still a need to establish cgMLST as a tool in clinical practice and standardization of cgMLST is urgently needed.…”

“…Using cgMLST analysis from WGS data when investigating a clinically identified nosocomial outbreak of E. coli ST‐131, we were able to clearly identify two isolates (8 and 13) not belonging to the outbreak. In a previous analysis of the clinically defined epidemiological outbreak , PFGE and LMqPCR HRMA were able to differentiate between ST‐131 isolates and non‐ST‐131 isolates but had difficulties in judging on differences between isolates 8 and 13 and the rest of the ST‐131 suspected outbreak isolates. This prompted us to proceed to WGS followed by cgMLST and SNP analysis, where isolates 8 and 13 were clearly differentiated from the other ST‐131 isolates.…”

Evaluation of High-Resolution Melting Curve Analysis of Ligation-Mediated Real-Time PCR, a Rapid Method for Epidemiological Typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) Pathogens

“…This method has already been shown to identify a nosocomial outbreak of O25b-sequence type 131 (ST131)-associated extended-spectrum-␤-lactamase (ESBL)-producing Escherichia coli isolates with a resolution comparable to that found with PFGE (14). Hence, in the evaluation of two or more isolates from a suspected outbreak in the routine microbiological laboratory, this method can confirm whether such isolates are different and to a very high certainty if they are similar.…”

“…For Gram-positive bacteria, our results show that additional agarose electrophoresis might be of benefit, especially in cases where the isolates are similar by HRM analysis only. The species that gave the highest resolution in LMqPCR HRMA were K. pneumoniae, P. aeruginosa, E. coli (14), and E. cloacae. All of these species have GϩC contents between 51 and 66%, in contrast to E. faecium, S. aureus, and A. baumannii, which have GϩC contents between 33% and 39% (http://www .ncbi.nlm.nih.gov/genome).…”

“…Results comparable to those from PFGE can be achieved within 1 day in a single-tube system. (14) was used on the complete melting curves visualized in panels A and C, together with a subjective evaluation of the discrepancies between the melting curves for clustering analysis of the isolates. (B) Isolate E. faecium Ef01 is set as reference, plotting the difference of all other isolates to Ef01.…”