Molecular typing and epidemiological study of Salmonella enterica serotype Typhimurium isolates from cattle by fluorescent amplified-fragment length polymorphism fingerprinting and pulsed-field gel electrophoresis

Abstract

One hundred twenty Salmonella enterica serotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.

FIG. 1

Dendrogram showing the results of cluster analysis on the basis of FAFLP fingerprintings of 120 serotype Typhimurium strains. The dendrogram was constructed by using UPGMA clustering on a matrix based on the Dice coefficient.

FIG. 2

GeneScan 2.1 software-derived electropherograms showing examples of areas of polymorphism within FAFLP profiles for EcoRI plus A and MseI plus T amplifications of serotype Typhimurium genomes. Segments of FAFLP profiles obtained from serotype Typhimurium H6 (profile A4), N59 (profile B5), N54 (profile C3), and #2 (profile D2) are represented. The fragment size scale (base pairs) is indicated above each segment. The solid arrowheads and peaks indicate a fragment characteristic of that profile (sizes are indicated in base pairs).

FIG. 3

(A) PFGE analysis of XbaI-digested genomic DNA from serotype Typhimurium strains. Lanes (with designated PFGE profiles in parentheses [Table 1]): M, lambda 48.5-kbp ladder; 1, KT43 (Ia); 2, NET2 (Ib); 3, N36 (Ic); 4, N77 (IIa); 5, L719 (IIb); 6, NET20 (IIc); 7, L767 (IId); 8, #2 (IIe); 9, KT1 (IIf); 10, NCTC9324 (IIg); 11, 478 (IIh); 12, NET25 (IIi); 13, LT2 (IIj); 14, NET55 (IIk); 15, NET31 (III); 16, N59 (IIm); 17, NET37 (IIn); 18, N78 (IIo); 19, NET30 (IIIa); 20, NET40 (IIIb); 21, N50 (IIIc); 22, N54 (IIId); 23, KT6 (IIIe); 24, N68 (IIIf); 25, KT3 (IIIg). (B) Dendrogram and schematic representation of PFGE fragments following XbaI macrorestriction of serotype Typhimurium genomic DNA. Similarity analysis was performed using the Dice coefficient, and clustering was by UPGMA.

FIG. 4

Sequence analysis of cluster-specific fragments. (A) Sequence similarity between the 132-bp fragment (bp 14 to 120) and traG (bp 3981 to 4087; GenBank accession no. M5976). (B) Sequence similarity between the 142-bp fragment (bp 14 to 130) and P22 phage (bp 4868 to 4984; GenBank accession no. L06296). Asterisks represent identity to the corresponding nucleotides, and dashes represent missing nucleotides.

FIG. 5

Hybridization of 132-bp fragment to plasmids in serotype Typhimurium. (Top) Visualization of Salmonella plasmids by agarose gel electrophoresis. Lanes (with designated FAFLP profiles in parentheses [Table 1]): M, molecular weight standards (lambda DNA digested with HindIII); 1, NET57 (A1); 2, NET2 (A2); 3, NET8 (A3); 4, H6 (A4); 5, NET25 (B1); 6, NET20 (B2); 7, N49 (B3); 8, 478 (B4); 9, N81 (B5); 10, NET52 (B6); 11, NET30 (C1); 12, NET21 (C2); 13, N54 (C3); 14, N57 (C4); 15, N48 (C5); 16, L767 (D1); 17, #2 (D2). Chromosomal DNA bands (arrow) are seen in each lane. (Bottom) Southern blot analysis of the plasmid in serotype Typhimurium, using a 132-bp fragment probe.

FIG. 6

Hybridization of 142-bp fragment to HindIII digest of serotype Typhimurium chromosomal DNA. A Southern blot was made with HindIII and hybridized with the 142-bp fragment. Lanes (with designated FAFLP profiles in parentheses [Table 1]): M, molecular weight standards (lambda DNA digested with HindIII); 1, NET57 (A1); 2, NET2 (A2); 3, NET8 (A3); 4, H6 (A4); 5, NET25 (B1); 6, NET20 (B2); 7, N49 (B3); 8, 478 (B4); 9, N81 (B5); 10, NET52 (B6); 11, NET30 (C1); 12, NET21 (C2); 13, N54 (C3); 14, N57 (C4); 15, N48 (C5); 16, L767 (D1); 17, #2 (D2); 18, U1 (A2); 19, U2 (A2); 20, U3 (A2); 21, U4 (A2); 22, U5 (A2); 23, U6 (A1); 24, U7 (A2); 25, U8 (A2); 26, U9 (A1); 27, U17 (A2); 28, U18 (A1); 29, U20 (A1).