An optimized MLVA assay and its complementarity with PFGE and MLST for Listeria monocytogenes clone identification and surveillance

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An optimized MLVA assay and its complementarity with PFGE and MLST for Listeria monocytogenes clone identification and surveillance
  Page 1 1 An optimized MLVA assay and its complementarity with PFGE and 1 MLST for Listeria   monocytogenes clone identification and 2 surveillance 3 4 Viviane Chenal-Francisque 1,2,3 , Laure Diancourt 4 , Thomas Cantinelli 1,2,3 , Virginie 5 Passet 4,5 , Coralie Tran-Hykes 4 , Hélène Bracq-Dieye 1,2,3 , Alexandre Leclercq 1,2,3 , 6 Christine Pourcel 5,6 , Marc Lecuit 1,2,3,7,8 *  and Sylvain Brisse 4,9 *   7 8 1 Institut Pasteur, National Reference Centre and World Health Organisation 9 Collaborating Centre for Listeria   10 2 Institut Pasteur, Biology of Infection Unit, Paris, France 11 3 Inserm U1117, Paris, France 12 4 Institut Pasteur, Genotyping of Pathogens and Public Health, Paris, France 13 5 Univ Paris-Sud, Institut de Génétique et Microbiologie, UMR 8621, Orsay, France 14 6 CNRS, Orsay, France 15 7 Paris Descartes University, Sorbonne Paris Cité, Institut Imagine, Paris, France 16 8  Necker-Enfants Malades University Hospital, APHP, Division of Infectious Diseases 17 and Tropical Medicine, Paris, France 18 9  Institut Pasteur, Microbial Evolutionary Genomics, Paris, France 19 * Corresponding authors 20 S. Brisse. Microbial Evolutionary Genomics Unit, Institut Pasteur, 28 rue du Dr Roux, 21 F-75724 Paris, France. E-mail:; Phone +33 1 40 61 36 58 22 M. Lecuit. Biology of Infection Unit, Institut Pasteur, 25 rue du Dr Roux, F-75724 23 Paris, France. E-mail:; Phone +33 1 40 61 30 29 24 25 Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00606-13 JCM Accepts, published online ahead of print on 10 April 2013  Page 2 2 Abstract   26 Populations of the foodborne pathogen Listeria monocytogenes  are genetically 27 structured into a small number of major clonal groups, some of which have been 28 implicated in multiple outbreaks. The goal of this study was to develop and evaluate 29 an optimized multilocus variable number of tandem repeat (VNTR) analysis (MLVA) 30 subtyping scheme for strain discrimination and clonal group identification. We 31 evaluated 18 VNTR loci and combined the 11 best ones into two multiplexed PCR 32 assays (MLVA-11). A collection of 255 isolates representing the diversity of clonal 33 groups within phylogenetic lineages 1 and 2, including representatives of epidemic 34 clones, were analyzed by MLVA-11, multilocus sequence typing (MLST) and pulsed 35 field gel electrophoresis (PFGE). MLVA-11 was less discriminatory than PFGE, 36 except for some clones, and was unable to distinguish some epidemiologically 37 unrelated isolates. Yet it distinguished all major MLST clones and therefore 38 constitutes a rapid method to identify epidemiologically relevant clonal groups. Given 39 its high reproducibility and high-throughput, MLVA represents a very attractive first- 40 line screening method to alleviate PFGE workload in outbreak investigations and 41 listeriosis surveillance. 42 43  Page 3 3 Introduction 44 45 Listeriosis is a foodborne infection caused by the bacterium Listeria monocytogenes . 46 Invasive forms of human listeriosis include septicaemia, meningitis, and maternal- 47 fetal infections (31). Listeriosis is associated with high hospitalization and fatality 48 rates (almost 100% and 25-30%, respectively). Populations at risk include pregnant 49 women, immunocompromised individuals and the elderly. L. monocytogenes  is 50 widely present in the environment, including soil, water,   vegetation and silage, as well 51 as in animals and animal-derived food, and can contaminate food in processing 52 plants and retail establishments. L. monocytogenes  is recognized as a public health 53 issue and a serious challenge for the food industry, and this has led to the 54 establishment of national surveillance system in several countries. L. monocytogenes   55 also stands out as a model system in the fields of microbiology, cell biology and 56 immunology and for the study of host-pathogens interactions (9, 18, 27, 50). 57 L. monocytogenes  strain characterization based on serotyping and molecular 58 typing methods is used for surveillance, epidemiological tracking and outbreak 59 investigation purposes (24, 44). Genetic variants of L. monocytogenes  have 60 diversified into four major phylogenetic lineages, with lineages 1 and 2 each 61 containing multiple clonal groups of public health importance (23, 36-39, 47, 48). As 62 these groups appear to differ in virulence and epidemic potentials (21, 24), it will be 63 interesting to better define their epidemiological, clinical and microbiological 64 specificities. For this purposes, easy identification tools of clonal groups are needed 65 to recognize such groups and determine their presence in a large variety of sources. 66 Several typing methods are currently available  for L. monocytogenes  strains. 67 Conventional serotyping (42) and its molecular proxy PCR-serogrouping (12) 68  Page 4 4 discriminate major categories of strains that correlate strongly (albeit not totally) with 69 lineages and clones (38, 39, 48), but these methods do not have the necessary 70 discriminatory power in the context of outbreak investigations. Pulsed field gel 71 electrophoresis (PFGE) is established as the gold standard for L. monocytogenes   72 strain subtyping and is widely used for surveillance of listeriosis and outbreak 73 investigation (16). Yet, PFGE presents several practical disadvantages, as it is time- 74 consuming and requires stringent standardization for inter-laboratory data 75 comparison. Multilocus sequence typing (MLST) is a well-established reference 76 method for global epidemiology and population biology (13, 33), as it renders inter- 77 laboratory genotype comparisons easy and unambiguous, and as sequence data can 78 be used to infer useful population genetics information such as amounts of genetic 79 diversity, recombination rates and strain phylogeny. MLST also provides backward 80 compatibility with genome sequencing (22). However, MLST is neither rapid nor 81 cheap and has limited discriminatory power within L. monocytogenes  (11, 39). Given 82 the current limitations of available methods for L. monocytogenes  strain typing, a 83 potentially useful complementary approach is multilocus variable number of tandem 84 repeats (VNTR) analysis (MLVA). This method is largely used for epidemiological 85 tracking of bacterial pathogens (29, 46) because it is relatively easy and cheap to 86 implement and because it has remarkable discriminatory power in many, although 87 not all, bacterial species. MLVA relies on the study of the variability of the number of 88 tandem repeats at specific loci in bacterial genomes. MLVA schemes are constructed 89 based on an open choice of several VNTR chromosomal loci, and five different 90 MLVA schemes have been developed almost simultaneously for L. monocytogenes   91 strain typing (26, 30, 34, 35, 43). Subsequent use of MLVA in outbreak investigations 92  Page 5 5 and strain diversity studies has relied on these different schemes or combinations 93 thereof (2, 5, 19, 28, 32). 94 The five previously proposed MLVA schemes differ in the number of VNTR 95 loci that were included, ranging from only 3 (34) to 8 (43) and even 10 (26). These 96 studies also differed in the number, diversity and inclusion criteria of 97 L. monocytogenes  isolates used to evaluate strain typeability and discrimination. As 98 strains of both lineage 1 and lineage 2 are frequent among sporadic human 99 infections and can cause outbreaks (6, 23, 38, 48), a MLVA scheme should ideally 100 be applicable to all strains of both lineages. Many of the primer pairs defined so far to 101 amplify VNTR loci did not amplify a number of strains, which limits their 102 discriminatory power and complicates the interpretation of differences among strains. 103 Besides, it is important to calibrate the MLVA method against PFGE on the one 104 hand, and MLST on the other hand. Indeed, PFGE is the standard for 105 epidemiological investigations, whereas MLST is well established as a population 106 biology tool and provides standard operational definition of clones as clonal 107 complexes (8, 39). Only one of the five proposed MLVA schemes was compared with 108 PFGE data obtained following the current standard protocol of dual restriction 109 enzyme use,  ApaI   and  AscI   (43), while comparison with MLST was performed only in 110 one study so far (34). The added value of MLVA relative to PFGE and MLST thus 111 clearly remains to be precisely defined. 112 The aims of our study were ( i  ) to identify novel VNTR loci and evaluate them 113 as well as all previously described loci for typeability of isolates representative of a 114 broad range of genotypes of lineages 1 and 2, including all major clones defined by 115 MLST; ( ii  ) to evaluate the ability of MLVA to identify MLST-defined clonal groups; and 116
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