Controlled Performance Evaluation of the DiversiLab Repetitive-Sequence-Based Genotyping System for Typing Multidrug-Resistant Health Care-Associated Bacterial Pathogens

Abstract

Fast, reliable, and versatile typing tools are essential to differentiate among related bacterial strains for epidemiological investigation and surveillance of health care-associated infection with multidrug-resistant (MDR) pathogens. The DiversiLab (DL) system is a semiautomated repetitive-sequence-based PCR system designed for rapid genotyping. The DL system performance was assessed by comparing its reproducibility, typeability, discriminatory power, and concordance with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and by assessing its epidemiological concordance on well-characterized MDR bacterial strains (n ‫؍‬ 165). These included vanA Enterococcus faecium, extended-spectrum ␤-lactamase (ESBL)-producing strains of Klebsiella pneumoniae, Escherichia coli, and Acinetobacter baumannii, and ESBL-or metallo-␤-lactamase (MBL)-producing Pseudomonas aeruginosa strains. The DL system showed very good performance for E. faecium and K. pneumoniae and good performance for other species, except for a discrimination index of <95% for A. baumannii and E. coli (93.9% and 93.5%, respectively) and incomplete concordance with MLST for P. aeruginosa (78.6%) and E. coli (97.0%). Occasional violations of MLST assignment by DL types were noted for E. coli. Complete epidemiological concordance was observed for all pathogens, as all outbreak-associated strains clustered in identical DL types that were distinct from those of unrelated strains. In conclusion, the DL system showed good to excellent performance, making it a reliable typing tool for investigation of outbreaks caused by study pathogens, even though it was generally less discriminating than PFGE analysis. For E. coli and P. aeruginosa, MLST cannot be reliably inferred from DL type due to phylogenetic group violation or discordance.Rapid assessment of microbial clonal relationships enables the tracking of the spread of multidrug-resistant (MDR) pathogens and guides transmission control measures in the hospital setting. The ideal typing method should be rapid, easy to perform, high throughput, and applicable to a wide range of microorganisms and should meet performance criteria, such as full typeability, reproducibility, high discriminatory power, and concordance with validated typing methods as well as consistency with underlying subspecies genetic population structures (29, 31). In the hospital setting, identification of epidemic strains is usually based on fingerprinting methods, such as pulsed-field gel electrophoresis (PFGE), a labor-intensive method that requires 3 to 4 days to produce results. The DiversiLab (DL) system (bioMérieux, Marcy l'Etoile, France) is a repetitive-sequence-based PCR (rep-PCR) technology which offers semiautomated, easy-to-use, high-throughput, and rapid bacterial strain typing. It could be a suitable alternative to PFGE analysis for outbreak investigation of health care-associated pathogens. This method was recently validated for typing of Acinetobacter spp., Escherichia coli, and Klebsiella spp., but...

Overview of molecular typing methods for outbreak detection and epidemiological surveillance

“…The resulting data are automatically collected, normalised and analysed by the DiversiLab software. A number of studies have evaluated the usefulness of DiversiLab by comparing its performance with current standard typing methods using well-characterised collections of outbreak-related and epidemiologically unrelated bacterial isolates [24][25][26]. These studies have shown that the DiversiLab system is simple, easy to perform, rapid, reproducible, endowed with full typeability and applicable to a wide range of microorganisms.…”

Comparison of Molecular and Phenotypic Methods for the Detection and Characterization of Carbapenem Resistant Enterobacteriaceae

“…Briefl y, genomic DNA extraction and preparation was carried out using the Mo Bio UltraClean™ Microbial DNA Isolation Kit. As previously described, the rep-PCR amplifi cation was performed using the DiversiLab Klebsiella fi ngerprinting kit and detection done using the microfl uidic LabChip kits on Agilent 2100 bioanalyzer according to the manufacturer's instructions (bioMérieux) [14]. The amplifi ed rep-PCR DNA fragment patterns were analyzed with the DiversiLab analysis software using the modifi ed Kullback-Leibler statistical method.…”

Investigation of clonal relationships of K. pneumoniae isolates from neonatal intensive care units by PFGE and rep-PCR

“…It may be a suitable alternative to PFGE analysis for epidemic investigation of healthcare-associated pathogens such as methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and Klebsiella spp., and has been proven to be a rapid and reliable method for molecular analysis of nosocomial epidemics [26]. Nevertheless, although this method was recently validated for typing of Acinetobacter spp., Escherichia coli, and Klebsiella spp., it was found to be insufficiently discriminative for typing of S. aureus [30,31]. This method can be easily introduced into routine settings and requires less hands-on time than does PFGE.…”

“…It should also meet performance criteria, such as full typeability, reproducibility, high discriminatory power, and concordance with validated typing methods as well as consistency with underlying subspecies genetic population structures [30,32,33]. However, such an ideal molecular typing method does not yet exist.…”