High Throughput Multiple Locus Variable Number of Tandem Repeat Analysis (MLVA) of Staphylococcus aureus from Human, Animal and Food Sources

Animal and Food Sources. PLoS ONE 7(5): e33967. doi:10.1371/journal.pone.0033967 High Throughput Multiple Locus Variable Number of Tandem Repeat Analysis (MLVA) of Staphylococcus aureus from Human, Animal and Food Sources Daniel Sobral 0 Stefan Schwarz 0 Dominique Bergonier 0 Anne Brisabois 0 Andrea T. Feler 0 Florence B. Gilbert 0 Kristina Kadlec 0 Benoit Lebeau 0 Fabienne Loisy-Hamon 0 Michae l Treilles 0 Christine Pourcel 0 Gilles Vergnaud 0 Philip Supply, Institut Pasteur de Lille, France 0 1 Univ Paris-Sud, Institut de Ge ne tique et Microbiologie, UMR 8621, Orsay, France , 2 CNRS, Orsay , France , 3 Centre Europe en d'Expertise et de Recherche sur les Agents Microbiens (CEERAM), La Chapelle sur Erdre, France, 4 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI) , Neustadt-Mariensee, Germany, 5 INRA, UMR1225, IHAP, Toulouse, France, 6 Universite de Toulouse, INP, ENVT, UMR1225, IHAP, Toulouse, France, 7 ANSES , European Union Community Reference Laboratory for Coagulase Positive Staphylococci , Maisons-Alfort, France, 8 INRA , UR1282 Infectiologie Animale et Sante Publique (IASP) , Nouzilly , France , 9 Laboratoire de partemental d'analyses de la Manche , Saint-Lo , France , 10 DGA/MRIS- Mission pour la Recherche et l'Innovation Scientifique , Bagneux , France Staphylococcus aureus is a major human pathogen, a relevant pathogen in veterinary medicine, and a major cause of food poisoning. Epidemiological investigation tools are needed to establish surveillance of S. aureus strains in humans, animals and food. In this study, we investigated 145 S. aureus isolates recovered from various animal species, disease conditions, food products and food poisoning events. Multiple Locus Variable Number of Tandem Repeat (VNTR) analysis (MLVA), known to be highly efficient for the genotyping of human S. aureus isolates, was used and shown to be equally well suited for the typing of animal S. aureus isolates. MLVA was improved by using sixteen VNTR loci amplified in two multiplex PCRs and analyzed by capillary electrophoresis ensuring a high throughput and high discriminatory power. The isolates were assigned to twelve known clonal complexes (CCs) and -a few singletons. Half of the test collection belonged to four CCs (CC9, CC97, CC133, CC398) previously described as mostly associated with animals. The remaining eight CCs (CC1, CC5, CC8, CC15, CC25, CC30, CC45, CC51), representing 46% of the animal isolates, are common in humans. Interestingly, isolates responsible for food poisoning show a CC distribution signature typical of human isolates and strikingly different from animal isolates, suggesting a predominantly human origin. - Funding: Work by GV is part of the European Biodefense Laboratory Network (EBLN) project Database of B-agents supported by the European Defense Agency. The contribution of SS, ATF and KK is financially supported by the German Federal Ministry of Education and Research (BMBF) through the German Aerospace Center (DLR), grant number 01KI1014D (MedVet-Staph). This study also benefited from the support of the association Vaincre La Mucoviscidose (Grant Nu RC0630). The development of tools for the surveillance of bacterial pathogens is supported by the French Direction Generale de lArmement. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: DS, FLH, and BL are employees of Ceeram and hold stocks. Patent licensing arrangements (Patent number: 1054238; Patent name: Procede de genotypage de Staphylococcus aureus) exist with DS, FLH, BL, and CP. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. Staphylococcus aureus is a common commensal and frequent colonizer of humans and many animal species including companion animals as well as food-producing animals. In humans, the epithelium of the anterior nares is the primary ecological niche. S. aureus is also a major pathogen involved in a wide variety of diseases such as purulent skin and subcutaneous infections, pneumonia, endocarditis, abscesses and bacteremia. Moreover, S. aureus is an emerging issue in veterinary medicine and a cause of food poisoning by its ability to produce heat-stable enterotoxins [1]. The transfer of S. aureus isolates between humans and animals, especially in the case of livestock-associated MRSA ST398, has recently gained particular attention [2]. However, relatively little is known about the more global diversity of S. aureus isolates of animal origin [317]. This limits our ability to identify for example the origin of strains responsible for food poisoning. In order to implement control measures targeted at reservoirs and transmission routes, it is necessary to further improve current knowledge about animal-associated S. aureus. Essentially three techniques are currently used for the largescale analysis of the diversity of S. aureus isolates, namely multi locus sequence typing (MLST), spa typing, and multiple locus variable number of tandem repeats (VNTR) analysis (MLVA). In addition, pulsed field gel electrophoresis (PFGE) is still widely used and considered the gold-standard for typing S. aureus isolates. It has a high discriminatory power and it can be used for many bacterial pathogens. It is however not appropriate for routine interlaboratory comparisons [18]. MLST studies allowed the description of major clonal complexes (CC) underlying the S. aureus population structure [19,20]. MLST suffers from its relatively high costs and has a moderate discriminatory power. The spa typing is a widely used method in which variations in a highly variable tandem repeat are characterized by sequencing. The Ridom Spaserver http://spaserver.ridom.de allows the designation of spa types [21,22]. The spa typing is a very powerful tool, and is currently the most commonly used first line assay. However it may fail to identify new lineages due to inherent homoplasia and variable evolutionary rate of spa alleles and clustering based on spa data is complex. MLVA was developed more recently. Homoplasia at individual VNTR loci and potentially low variability of specific alleles are compensated at least partly by the use of multiple loci. An assay comprising as little as 8 VNTR loci (called MLVA-8Bilthoven in the present report) was highly congruent with MLST and able to assign a new isolate to the correct CC for much lower costs [23]. The 8 loci were amplified in two multiplex PCRs and analyzed by capillary electrophoresis. A MLVA assay with 14 loci (MLVA-14Orsay) providing higher discriminatory power was used in a survey of 309 isolates including clinical MRSA isolates, nasal carriage isolates and representatives of the main CCs present in humans [24]. Both schemes can be adapted to low resolution DNA sizing equipment (such as agarose gels) as well as to higher throughput systems (such as capillary electrophoresis-based devices). MLVA (...truncated)