Characteristics of Listeria monocytogenes isolates representing 1 novel outbreak strain and 2 newly defined epidemic clones, ECVI and ECVII, United States, 2011*
Figure 1 Four AscI/ApaI pulsed-field gel electrophoresis (PFGE) profiles (identified at the time the research was performed) displayed by Listeria monocytogenes clinical isolates (L2624, L2625, L2626, and L2676) and isolates from food or environmental samples (more ...)
Isolates were grown overnight in tryptic soy broth with yeast extract at 37°C, and DNA was extracted by using the Ultra Clean Microbial DNA Isolation Kit (Mo Bio Laboratories, Solana Beach, CA, USA) for isolates from CDC and USDA and the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) for isolates from FDA. Sequence types (STs) identified by using MLST were assigned as described (6
) on the basis of whole genome sequence data (C. Tarr, Y. Chen, unpub. data) and compared with those publicly available (www.pasteur.fr/mlst
). MVLST data were obtained as described (3
) or extracted from whole genome sequences (Y. Chen, unpub. data). Sequences were compared with those on the MVLST database available in the laboratory of S.K. (3,4
) and analyzed by using MEGA5.0 (10
). New virulence types (VTs) were assigned to USDA isolates: VT60 (isolates 239, 441, 442, 458, 541, 565, 577); VT68 (350, 470); VT69 (247); VT70 (502); VT71 (450); VT72 (342), and VT73 (267). comK
prophage JFs were sequenced as described (5
). Prophage types (PTs) were assigned by comparing JF sequences with those available from previous reports (4,5
prophage JF sequences were submitted to GenBank for isolate L2676 (accession nos. JQ407079 and JQ407080) and 3 USDA isolates (accession nos. JQ750615–JQ750618).
Isolates L2624, LIS0075, and LIS0078 (PFGE profile 1) belonged to the globally disseminated ST5 (6
) and had the same VT (VT63) as 5 other 1/2b isolates in the database: isolates 10-0810 and 10-0811, from an imitation crabmeat–borne outbreak in Canada during 1996 (4,11
); and isolates 98-0041, 233, and 466 (; ). Because VT63 isolates were associated with multiple outbreaks, they should be considered part of a novel EC (ECVI). ECVI isolates from cantaloupe and USDA isolate 233 showed no amplification of comK
prophage JFs (). PT11/11 was identified during the 1996 imitation crabmeat–associated outbreak in Canada (4
) and in USDA isolate 466 (). Further research is needed to determine why comK
PTs were identical during different years and in different geographic locations and food processing plants.
Figure 2 Unrooted neighbor-joining tree computed in MEGA 5.0 (10) for multivirulence locus sequence typing data based on sequencing of 6 virulence genes, prfA, inlB, inlC, dal, clpP, and lisR (3) obtained for the 93 Listeria monocytogenes isolates compared in (more ...)
Isolate L2625 (VT74, PFGE profile 2) from cantaloupe differed by 1 single nucleotide polymorphism in inlC
from 3 other serotype 1/2a VT61 isolates (10-4758, 10-4754, and 06-6956) associated with the 2002 cheese-associated listeriosis outbreak in Canada (4,12
) (; ). L2625 was assigned to ST29, an infrequent sequence type in the Institut Pasteur MLST database that differs from the ST (ST405) assigned to the isolates from cheese in the 2002 outbreak in Canada. No amplification of comK
prophage JFs was observed, consistent with the PTs in the 2002 cheese-associated outbreak in Canada (4
). Given this evidence, isolate L2625 does not represent a novel EC but should be considered a novel outbreak strain.
Isolates L2626 and LIS0077 (PFGE profile 3, ST7) and L2676, LIS0072, and LIS0087 (PFGE profile 4, ST561) from cantaloupe samples shared the same VT (VT56) as isolates 10-0813 and 10-0812 associated with a listeriosis outbreak related to whipping cream during 2000 in Canada (4,12
) and isolates 06-6909, BL0047, 261, and 498 (Table; ). These Listeria
isolates from cantaloupe displayed 2 highly similar PFGE profiles and STs, and the same serotype, Apa
I PFGE pattern, and VT (; ). Isolates L2626 and LIS0077 showed no amplification of comK
prophage JFs, which was also consistent with the upstream PT in the outbreak associated with whipping cream in Canada (). The JF sequences in isolates L2676, LIS0072, and LIS0087 were identical to those in USDA isolate 261 (). These isolates matched those from the whipping cream–associated outbreak in Canada in terms of VT56 and downstream PT (PT13) (). However, the upstream JF could not be amplified in the strain identified in whipping cream (4
), possibly because of extensive recombination within the comK
), especially in the upstream JF (5
). These STs and VTs were also found in clinical isolates over extended periods (6
). Therefore, by definition (2,3
), these isolates also represent a novel EC (ECVII).