Microfluidic-chip-based multiple-locus variable-number tandem-repeat fingerprinting with new primer sets for methicillin-resistant Staphylococcus aureus

Abstract

The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rand's coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.

Fig 1

Minimum spanning tree produced using 206 MRSA isolates by MLVA. A categorical coefficient and the priority rule using the highest number of single-locus changes were used for the clustering of MLVA profiles. Each circle represents a single MLVA profile, and the circle size indicates the number of isolates with that profile. Different clonal complexes are indicated by the different colors. These clonal complexes were assigned if 2 neighboring MLVA types did not differ in more than 1 VNTR locus. MLVA types and complexes are also indicated in characters, e.g., 5 denotes MLVA type 5 and MC5 represents MLVA clonal complex 5.

Fig 2

Population structure of the UMCG MRSA isolates after BURP analysis with a cost of ≤3. Clusters of linked spa types correspond to spa-CCs. The spa types that were defined as founders of particular clusters are indicated in blue, and subfounders are indicated in yellow.

Fig 3

MLVF dendrogram of the study isolates generated by the UPGMA algorithm. Isolate clusters were delineated with an 88% similarity cutoff value. For clarity, only the MLVF clusters are indicated. MLVF clusters are shown in characters, e.g., C1′ denotes MLVF cluster 1.