Core Genome Multilocus Sequence Typing for Identification of Globally Distributed Clonal Groups and Differentiation of Outbreak Strains of Listeria monocytogenes.

Errol A. Strain,Thomas S. Hammack,E. G. Dudley,Yi Chen,Marc W. Allard,Eric W. Brown,Narjol Gonzalez-Escalona

doi:10.1128/aem.01532-16

Abstract

ABSTRACTMany listeriosis outbreaks are caused by a few globally distributed clonal groups, designated clonal complexes or epidemic clones, of Listeria monocytogenes, several of which have been defined by classic multilocus sequence typing (MLST) schemes targeting 6 to 8 housekeeping or virulence genes. We have developed and evaluated core genome MLST (cgMLST) schemes and applied them to isolates from multiple clonal groups, including those associated with 39 listeriosis outbreaks. The cgMLST clusters were congruent with MLST-defined clonal groups, which had various degrees of diversity at the whole-genome level. Notably, cgMLST could distinguish among outbreak strains and epidemiologically unrelated strains of the same clonal group, which could not be achieved using classic MLST schemes. The precise selection of cgMLST gene targets may not be critical for the general identification of clonal groups and outbreak strains. cgMLST analyses further identified outbreak strains, including those associated with recent outbreaks linked to contaminated French-style cheese, Hispanic-style cheese, stone fruit, caramel apple, ice cream, and packaged leafy green salad, as belonging to major clonal groups. We further developed lineage-specific cgMLST schemes, which can include accessory genes when core genomes do not possess sufficient diversity, and this provided additional resolution over species-specific cgMLST. Analyses of isolates from different common-source listeriosis outbreaks revealed various degrees of diversity, indicating that the numbers of allelic differences should always be combined with cgMLST clustering and epidemiological evidence to define a listeriosis outbreak. IMPORTANCE Classic multilocus sequence typing (MLST) schemes targeting internal fragments of 6 to 8 genes that define clonal complexes or epidemic clones have been widely employed to study L. monocytogenes biodiversity and its relation to pathogenicity potential and epidemiology. We demonstrated that core genome MLST schemes can be used for the simultaneous identification of clonal groups and the differentiation of individual outbreak strains and epidemiologically unrelated strains of the same clonal group. We further developed lineage-specific cgMLST schemes that targeted more genomic regions than the species-specific cgMLST schemes. Our data revealed the genome-level diversity of clonal groups defined by classic MLST schemes. Our identification of U.S. and international outbreaks caused by major clonal groups can contribute to further understanding of the global epidemiology of L. monocytogenes.

Highlights

Many listeriosis outbreaks are caused by a few globally distributed clonal groups, designated clonal complexes or epidemic clones, of Listeria monocytogenes, several of which have been defined by classic multilocus sequence typing (MLST) schemes targeting 6 to 8 housekeeping or virulence genes
We performed in silico MLST and multi-virulence-locus sequence tying (MVLST) to determine their lineage information and performed in silico PCR serogrouping using the tool built in the Pasteur MLST L. monocytogenes database for isolates suspected to be serotype 4b variants
The combination of 51 genomes sequenced in this study and 207 genomes obtained from National Center for Biotechnology Information (NCBI) and the European Nucleotide Archive (ENA) represented three lineages, 12 serotypes, 10 clonal complex (CC) that are epidemic clone (EC), and an additional 43 CCs or singletons

Summary

MATERIALS AND METHODS

The other genome (SLCC2482) was annotated as a complete genome but contained an unclosed gap (15) and was not used to define core genes These genomes represented lineages I, II, and III (including lineage IIIB, which was redefined as lineage IV) and 11 serotypes. This resulted in a species-specific scheme, the L. monocytogenes cgMLST (Lm-cgMLST) scheme containing 1,827 core genes (hereinafter referred to as 1,827-cgMLST). To develop an L. monocytogenes lineage I-specific scheme (LmLI-cgMLST), 31 complete genomes were used with J1776 To develop an L. monocytogenes lineage II-specific scheme (LmLII-cgMLST), 29 complete genomes were used with EGD-e To develop an L. monocytogenes lineage III-specific scheme (LmLIII-cgMLST), 7 complete genomes were used with HCC23 Food NAd Pre-1961 UK, poultry Chicken 2011 China, sheep 1987 Pennsylvania outbreak, clinical 1921 France, clinical 1976 France Anjou outbreak, clinical 1983–1987 Switzerland cheese outbreak, clinical 1983–1987 Switzerland cheese outbreak, clinical 1985 California cheese outbreak, clinical 1985 California cheese outbreak, clinical 1981 Canada coleslaw outbreak, clinical 1981 Canada coleslaw outbreak, clinical 1981 Canada coleslaw outbreak, food 2008 Austria jellied pork outbreak, food 2008 Austria jellied pork outbreak, food 2008 Austria jellied pork outbreak, clinical 2008 Austria jellied pork outbreak, clinical 2014–2015 U.S multistate caramel apple outbreak, food 2014–2015 U.S multistate caramel apple outbreak, food 2014–2015 U.S multistate caramel apple outbreak, food 2014–2015 U.S multistate caramel apple outbreak, food 2014–2015 U.S multistate caramel apple outbreak, clinical France, clinical France, clinical France, clinical 1983 Boston, MA, milk outbreak, clinical 1983 Boston, MA, milk outbreak, clinical 1983 Boston, MA, milk outbreak, clinical 1983 Boston, MA, cheese outbreak 1979 Boston, MA, vegetable outbreak NA Pre-1961 United States, sheep 1987–1989 UK pâté outbreak, clinical 1987–1989 UK pâté outbreak, clinical 1987–1989 UK pâté outbreak, food 1987–1989 UK pâté outbreak, food 2012 Italy, cheese facility 1997 Italy corn gastroenteritis outbreak, clinical 1993–1994 Texas frozen vegetable outbreak, clinical 1993–1994 Texas frozen vegetable outbreak, clinical 1993–1994 Texas frozen vegetable outbreak, clinical 1993–1994 Texas frozen vegetable outbreak, clinical 1993–1994 Texas frozen vegetable outbreak, clinical 1993–1994 Texas frozen vegetable outbreak, clinical 2014 U.S multistate cheese outbreak, environment 2014 U.S multistate cheese outbreak, environment 2014 U.S multistate cheese outbreak, food 2014 U.S multistate cheese outbreak, food 2006 Brazil, dairy product 1994 Illinois chocolate milk gastroenteritis outbreak 1994 Illinois chocolate milk gastroenteritis outbreak 1999 United States, food Environment 1967, chinchilla 1966, clinical 1987 Pennsylvania outbreak, clinical 1998, food France, clinical 1999–2000 France pork rillettes outbreak, clinical Massachusetts sporadic case, clinical

VIII SRR1814375

SRR3644928

RESULTS AND DISCUSSION