Article Figures & Data
Figures
- FIG. 1.
Unrooted neighbor-joining tree of 86 L. monocytogenes isolates based on the number of nucleotide differences in the six MVLST virulence gene fragments analyzed. Bootstrap values (1,000 replications) are shown at the interior branches. Lineage (i.e., I, II, and III) and serotype (i.e., 4b, 1/2a, and 1/2b) information for each isolate are included after the isolate identification number. Isolates described previously by Zhang et al. (60) were assigned new BL (Borland Laboratory) identification numbers and marked with asterisks in the figure.
Tables
- TABLE 1.
Sources, lineages, and subtypes of the 66 representative isolates sequenced in this study
Group and isolate Previous IDa Sourcea Lineagea Serotypea Ribotypea,e MVLSTb Epidemic clone I (30) 1981 Canada coleslaw outbreak* (WHOc, Health Canada) BL1301 FSL J1-108 Human I 4b DUP-1038B 20 BL1302 FSL J1-107 Human I 4d DUP-1038B 20 BL1303 FSL J1-003 Human I 4b DUP-1038B 20 BL1304 FSL N3-008 Food I 4b DUP-1038 20 1985 California soft cheese outbreak* (WHO) BL1201 FSL J1-002 Human I 4b DUP-1038B 20 BL1202 FSL J1-119 Human I 4b DUP-1038B 20 BL1203 FSL J1-110 Food I 4b DUP-1038B 20 1983-1987 Switzerland soft cheese outbreak* (WHO) BL1101 FSL J1-123 Human I 4b DUP-1038B 20 BL1102 FSL J1-126 Human I 4b DUP-1038B 20 BL1103 FSL N3-022 Food I 4b DUP-1038B 20 Epidemic clone II (30) 1998 Sara Lee hot dog outbreak (CDC) BL2101 H2444 NAd I 4b DUP-1044 19 BL2102 H3396 NA I 4b DUP-1044 19 BL2103 H6383 NA I 4b DUP-1044 19 2002 U.S. turkey deli outbreak (CDC) BL2201 J1838 NA I 4b DUP-1044A 19 BL2202 J2230 NA I 4b DUP-1044A 19 BL2203 J2685 NA I 4b DUP-1044A 19 BL2204 J3006 NA I 4b DUP-1044A 19 BL2205 J3033 NA I 4b DUP-1044A 19 BL2206 J3200 NA I 4b DUP-1044A 19 BL2207 J3238 NA I 4b DUP-1044A 19 Epidemic clone III (30) 2000 U.S. turkey deli outbreak* (CDC) BL3201 FSL R2-603 Food II 1/2a DUP-1053A 1 BL3202 FSL R2-499 Food II 1/2a DUP-1053A 1 1989 U.S. hot dog outbreak* (WHO) BL3101 FSL N3-031 Food II 1/2a DUP-1053A 1 BL3102 FSL J1-101 Human II 1/2a DUP-1053A 1 Epidemic clone IV (30) 1989 United Kingdom pate outbreak* (WHO) BL4101 FSL J1-129 Human I 4b DUP-1042B 21 BL4102 FSL J1-116 Human I 4b DUP-1042B 21 BL4103 FSL N3-013 Food I 4b DUP-1042B 21 1979 Boston vegetable outbreak BL4201 FSL J1-220 Human I 4b DUP-1042 21 Other outbreaks 1983 Boston milk outbreak BL9101 FSL J1-225 Human I 4b DUP-1042B 31 BL9102 FSL R2-578 Human I 4b DUP-1042B 31 BL9103 FSL R2-583 Human I 4b DUP-1042B 31 2000 North Carolina soft cheese outbreak* (CDC) BL9201 FSL R2-500 Food I 4b DUP-1042B 30 BL9202 FSL R2-501 Human I 4b DUP-1042B 30 1987 Pennsylvania ice cream outbreak BL9301 FSL J1-012 Human I 4b DUP-1038B 29 1981 United Kingdom Carlisle outbreak BL9501 FSL J1-105 Human II 1/2a DUP-1030 33 1994 Illinois chocolate milk outbreak* (WHO) BL9401 FSL R2-502 Food I 1/2b DUP-1051B 32 BL9402 FSL R2-503 Human I 1/2b DUP-1051B 32 Nonoutbreak isolates BL0002 FSL N1-014 Food II 1/2a NA 34 BL0003 FSL N1-011A Environmental I 1/2b NA 35 BL0006 3562 Food environment NA NA DUP-1041 36 BL0007 3563 Food environment NA NA DUP-1041 36 BL0026 FSL J2-044 Animal I 4b DUP-1042 37 BL0027 FSL N3-010 Food I 4b NA 38 BL0028 3564 Food environment NA NA DUP-1041 36 BL0029 FSL F2-239 Food I 1/2b DUP-1042C 39 BL0030 FSL F2-601 Human sporadic I 4b DUP-1042B 40 BL0031 FSL H1-030 Food environment NA NA DUP-1045B 41 BL0032 FSL F2-293 Food I 1/2b DUP-1031A 42 BL0033 FSL S4-436 Environment NA NA DUP-1025A 43 BL0034 FSL H1-051 Food environment NA NA DUP-1039C 44 BL0035 FSL L3-151 Food environment NA NA DUP-1039A 45 BL0036 FSL F2-032 Food II 1/2a DUP-1045B 46 BL0037 FSL F2-373 Food II 1/2a DUP-1039C 47 BL0038 FSL N4-588 Environment NA NA DUP-1045B 48 BL0039 FSL R2-219 Food environment NA NA DUP-1062A 49 BL0040 FSL F3-831 Food environment NA NA DUP-1045A 50 BL0041 FSL S6-016 Environment NA NA NA 51 BL0042 FSL F2-525 Human sporadic III 4b DUP-1061A 52 BL0043 FSL F2-663 Human sporadic II 1/2a DUP-1054C 53 BL0044 FSL C1-387 Food II 1/2a DUP-1039B 45 BL0045 FSL F2-695 Human sporadic III 4a DUP-1061A 54 BL0046 FSL F2-655 Human sporadic III NA DUP-1061A 55 BL0047 3569 Food environment NA NA DUP-1030 56 BL0048 3571 Food environment NA NA DUP-1039 45 BL0049 3580 Food environment NA NA DUP-1062 57 BL0050 3583 Food environment NA NA DUP-1023 58 ↵a Isolates and information (sources, serotypes, ribotypes, and pulsotypes) were obtained from the Cornell Food Safety Laboratory, the U.S. CDC, and a meat-processing plant. ID, identification.
↵b Sequence types were assigned according to methods reported previously by Zhang et al. (60).
↵c Health agencies that characterized the outbreaks are included in parentheses following the name of the outbreaks. Isolates from the outbreaks followed by an asterisk were designated by the ILSI North America as a standard set of isolates for validating molecular subtyping methods.
↵d NA, not available.
↵e Ribotyping pattern designations followed by a capital letter indicate subribotype (i.e., DUP-1053A is a subribotype of ribogroup DUP-1053).
- TABLE 2.
Primer sequences, size of fragment, and percent coverage of complete coding sequence for each gene analyzed in this studya
Gene Size of fragment (bp) Coverage of complete CDS (%) PCR and sequencing primers (5′-3′) Reference or source MVLST genes prfA 469 65.69 AACGGGATAAAACCAAAACCA (F) 60 TGCGATGCCACTTGAATATC (R) inlB 433 22.87 CATGGGAGAGTAACCCAACC (F) 60 GCGGTAACCCCTTTGTCATA (R) inlC 418 46.91 CGGGAATGCAATTTTTCACTA (F) 60 AACCATCTACATAACTCCCACCA (R) dal 441 41.10 GGTTTCTGCGTAGCCATTTT (F) 60 GGAAGGGGTCAATCCATACA (R) clpP 419 70.18 CCAACAGTAATTGAACAAACTAGCC (F) 60 GATCTGTATCGCGAGCAATG (R) lisR 448 65.70 CGGGGTAGAAGTTTGTCGTC (F) 60 ACGCATCACATACCCTGTCC (R) Additional virulence genes inlA 458 19.01 GCTTTCAGCTGGGCATAAC (F) This study ATTCATTTAGTTCCGCCTGT (R) actA 582 32.01 AAGAGGTAAATGCTTCGGACT (F) This study ATTCCATTTAGTTCCGCCTGT (R) ↵a CDS, coding sequence; F, forward; R, reverse.
- TABLE 3.
Nucleotide substitutions at the 28 nucleotide polymorphic sites in the six MVLST genes which were capable of differentiating all epidemiologically unrelated isolates analyzed in this study
Isolate SNP locations in the six virulence genes prfAa inlB inlC dal clpP lisR * * * * * * * * * * * * * * * * 1 3 3 2 3 3 3 4 4 1 2 4 1 1 3 4 3 3 3 3 4 8 2 6 5 9 2 1 7 7 2 2 1 4 7 6 4 1 9 5 5 0 4 8 1 2 6 8 3 3 1 3 4 0 3 8 0 2 0 1 7 7 5 7 2 3 4 5 5 0 9 5 3 8 7 7 3 ECIb C C C G C G A G T T C C G G C T A G C A G C C G C T G C ECII . . . . . . . . . . . . T A . C . . . . . T . . . . . . ECIII . T . . . . . . G C . T . . . . . T . G . T . . . . . T ECIV . . . . . . T A . . . . . . . . . . . . . . . . . . . . Boston milkb . . . . . . T A . . . . . . . . . . . . . . T . . . . . NC soft cheese T . . . T . . . . . . . T A . C . . . . . T . . . . . . PA ice cream . . . . . . . . . C . . T A . C . . . . . . . . . . . . IL milk . . . . T . . . G C . . T A . C . . . . . . . . . . . . United Kingdom Carlisle . . . A . A . . A C . . . . . . . T . G . T . . . . . . BL0002 . T . . . . . . G C . . T A . C . T . G . T . A . . . T BL0003 . . . . T . . . G . . . T A . C . . . . . . . . . G . T BL0004 . T . . . . . . G . . . . . T . . . . . . . . A T . . . BL0005 . . . . . . T A . . . . T A . C . . . . . T . . . . . . BL0008 . T . A . . . . A C . . . . . . . T . G . T . . . . . . BL0009 . T . A T . . . A C . T . . . . C T . G . T . . . . . T BL0010 . T . . . . T A . . . . . . . C . . . . A . . A T . . T BL0011 . . . . T . . . G C . . . A . C . . . . . T . . . . . T BL0012 . T . A T . . . A C . T . . . . . T . . . T . . . . . . BL0013 . T . A T . . . A C . . . . . . . T . G . T . . . . . . BL0014 . T . . . . . A . . . . . . . C C . . . . . . A T . . . BL0015 . . T . T . . . G C . . T A . C . . . . . T . . . . . . BL0016 . . . . T . . . G C . . . . . C . . . . . T . . . . . . BL0017 . T . . . A . . A C . . . . . . . . . G . T . . . . . . BL0018 . T . A . . . . G C . T . . T . . T . G . T . . . . . . BL0019 . T . A . A . . A C . T . . T . . T . G . T . . . . . . BL0020 . . T . T . . . G C . . . A . C . . . . . T . . . . . . BL0021 . T . . . . . . . . . . . . . C . . T . . . . A T . . . BL0022 . T . . . . . . G . . . . . . C . . . . . . . A T . . . BL0023 . T . . . . . A . . . T . . T . . . . . A . . A T . . . BL0024 . . . . . . . . . . . . . A . C . . . . . . . . . . . . BL0025 . . . . T . . . . . . . T A . C . . . . A T . . . . . . BL0026 . . . . . . T A . . T . . . . . . . . . . T . . . . . . BL0027 . . . . . . T A . . T . . . . . . . . . . . . . . . . . BL0028 . T . A T . . . A C . T . . . . . T . G . T . . . . . T BL0029 . . . . T . . . G . . . T A . C . . . . . . . . . . . . BL0030 . . . . T . . . . . . . T A . C . . T . . T . . . . . T BL0031 . T . A A . . A C . T . . T . . T . G . T . . T . . T BL0032 . . . . T . . . G C . . . . . . . . . . T . . . . . . BL0033 . . . . T . . . G C . . . . . . . . . G . T . . . . . . BL0034 . T . A . A . . G C . T . . T . . T . G . T . . T . . T BL0035 . T . A . . . . A C . T . . T . . T . G . T . . . . . T BL0036 . T . A . . . . G C . T . . . . . T . G . T . . . . . T BL0037 . T . A T . . . A C . T . . . . . T . G . T . . . . C T BL0038 . T . . . . . . G C . T . . . . . T . G . T . A T . . T BL0039 . . . . T . . . G C . . . . . . . . . . . T . . . . . T BL0040 . T . . . . . . G C . T . . T . . T . G . T . . . . . T BL0041 . T . A . . . . A C . T . . . . . T . G . T . . . . . T BL0042 . T . . . . . A . . . . . . . C C . . . A . . . T . . T BL0043 . . A . . . . A C . T . . T . . T . G . T . . . . . T BL0044 . T . A . . . . A C . T . . T . . T . G . T . . . . . T BL0045 . T . . . . . A . . . . . . . C C . . . A . . A T . . T BL0046 . T . . . . T A . . . . . . . C C . . . A . . A T . . T BL0047 . . A . A . . A C . T . . T . . T . G . T . . . . . T BL0048 . T . A . . . . A C . T . . T . . T . G . T . . . . . T BL0049 . T . A . A . . A C . T . . T . . T . G . T . . . . . T BL0050 . T . A . . . . A C . T . . T . . C . G . T . . . . . T ↵a Locations of the variable nucleotide sites in the virulence genes are shown by the numbers above. Position numbers that are in boldface and start with an asterisk represent the set of 16 nucleotide substitutions capable of differentiating all epidemic clones and outbreaks. Identical nucleotides at each polymorphic site are indicated by periods.
↵b All isolates within each epidemic clone or outbreak.
- TABLE 4.
Description analysis of nucleotide sequence data in all isolates, lineage I isolates, and outbreak isolates of serotype 4b
Isolate Gene No. of polymorphic sites % of polymorphic sites No. of synonymous mutations No. of nonsynonymous mutations dN/dS ratioa π/siteb All isolates prfA 38 8.10 33 6 0.015 0.017 inlB 53 12.24 39 15 0.080 0.039 inlC 36 8.31 22 14 0.115 0.020 dal 84 19.05 70 14 0.063 0.067 clpP 40 9.55 38 5 0.020 0.028 lisR 63 14.1 57 7 0.004 0.031 Lineage I isolates prfA 11 1.71 10 1 0.017 0.0033 inlB 25 5.77 20 5 0.139 0.023 inlC 3 0.72 1 2 0.605 0.004 dal 7 1.36 6 1 0.074 0.048 clpP 15 3.58 13 2 0.067 0.0005 lisR 8 1.79 5 3 0.064 0.004 Outbreak isolates of serotype 4b prfA 3 0.64 2 1 0.058 0.0015 inlB 8 1.84 5 3 0.377 0.0036 inlC 3 0.72 1 2 0.572 0.0017 dal 5 1.13 4 1 0.074 0.0027 clpP 0 0 0 0 0 lisR 3 0.67 3 0 0.0028
