DNA SEQUENCE-BASED SUBTYPING OF EPIDEMIC CLONES AND OUTBREAK CLONES OF LISTERIA MONOCYTOGENES

Open Access
Author:
Chen, Yi
Graduate Program:
Food Science
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
June 15, 2007
Committee Members:
  • Stephen John Knabel, Committee Chair
  • Catherine Nettles Cutter, Committee Member
  • Robert F Roberts, Committee Member
  • Chitrita Debroy, Committee Member
Keywords:
  • LISTERIA MONOCYTOGENES
  • EPIDEMIC CLONES
  • SEQUENCE TYPING
Abstract:
Listeria monocytogenes is a Gram-positive intracellular foodborne pathogen that can cause the often fatal disease listeriosis. Various molecular subtyping strategies have been developed to recognize an outbreak, match case isolates with food and environmental isolates and discriminate between outbreak isolates and isolates that are not epidemiologically associated with the outbreak. A recently developed multi-virulence-locus sequence typing strategy (MVLST) was applied to study two recent U.S. multistate outbreaks, the 1998 hot dog outbreak and the 2002 turkey deli outbreak. MVLST clarified the epidemiology of these two outbreaks when combined with Epidemic Clone II (ECII) PCR. MVLST was then used to further analyze a set of representative isolates from fourteen listeriosis outbreaks and isolates not associated with any of the outbreaks. The evolutionary clusters identified by MVLST agreed with known lineages and serotypes of L. monocytogenes. MVLST accurately identified four known epidemic clones of L. monocytogenes: epidemic clone I (ECI), Epidemic Clone II (ECII), epidemic clone III (ECIII) and epidemic clone IV (ECIV). MVLST revealed a population structure of L. monocytogenes similar to those suggested in previous studies. Virulence gene sequences appear to be excellent molecular markers for identifying epidemic clones and clarifying the epidemiology of L. monocytogenes. Sequence analysis of MVLST data identified a minimum of twenty eight single nucleotide polymorphisms (SNPs) capable of differentiating all isolates, including all four epidemic clones. Sequence analysis of Genbank data identified three SNPs that can separate outbreak clones within ECI and ECIII. A combination of pulsed field gel electrophoresis (PFGE) and ligation-mediated (LM) PCR identified a SNP in bacteriophage A118 in L. monocytogenes that can separate outbreak clones within ECII. SNPs in prophage regions that could separate outbreak clones within ECIII and ECIV were subsequently identified. To simplify molecular typing of L. monocytogenes, a multiplex PCR scheme was developed for simultaneous identification of Listeria spp., L. monocytogenes, serotype 4b and 1/2a, and ECI, ECII and ECIII. Molecular markers specific for epidemic clones I (17B), II (LMOh7858_0487-0498) and III (LMOf6854_2467-2472); for serotypes 4b (ORF2110) and 1/2a (lmo0737); and for L. monocytogenes (lmo2234) and Listeria spp. (iap) were targeted in this Multiplex PCR scheme. This multiplex PCR scheme can be used for screening and subgrouping L. monocytogenes before more advanced subtyping methods are performed.