Multilocus sequence typing for characterization of clinical and environmental salmonella strains

Abstract

Multilocus sequence typing (MLST) based on the 16S RNA, pduF, glnA, and manB genes was developed for Salmonella, and its discriminatory ability was compared to those of pulsed-field gel electrophoresis (PFGE) and serotyping. PFGE differentiated several strains undifferentiable by serotyping, and 78 distinct PFGE types were identified among 231 Salmonella isolates grouped into 22 serotypes and 12 strains of undetermined serotype. The strains of several PFGE types were further differentiated by MLST, which suggests that the discriminatory ability of MLST for the typing of Salmonella is better than that of serotyping and/or PFGE typing. manB-based sequence typing identified two distinct genetic clusters containing 32 of 54 (59%) clinical isolates whose manB gene sequences were analyzed. The G+C contents and Splitstree analysis of the manB, glnA, and pduF genes of Salmonella indicated that the genes differ in their evolutionary origins and that recombination played a significant role in their evolution.

FIG. 1.

PFGE patterns of XbaI-digested DNA of Salmonella strains. Lane A, bacteriophage λ ladder PFG marker; lane B, CDC standard strain Salmonella serotype Newport 01144 (XbaI digest); lane C, low-range PFG marker; lanes 1 through 26, representative Salmonella PFGE types P1 through P26, respectively.

FIG. 2.

Dendrogram portraying the genetic diversity of various Salmonella strains, constructed on the basis of the PFGE patterns of XbaI-digested Salmonella DNA. The values in the Strains column indicate the number of clinical isolates/number of environmental isolates.

FIG. 3.

Neighbor-joining tree of Salmonella isolates, constructed by the maximum parsimony method by using the sequences of the manB gene fragments. The designations at the branches indicate the following: serotype, strain number (all hyphenated strains are clinical isolates), and PFGE type (PX, strains not typeable by PFGE). The following serotype designations are used: Sag, serotype Agona; San, serotype Anatum; Sbr, serotype Branderberg; Sca, serotype Cambridge; Sen, serotype Enteritidis; Sht, serotype Haardt; Sha, serotype Hadar; She, serotype Heidelberg; Sjo, serotype Johannesburg; Ske, serotype Kentucky; Smb, serotype Mbandaka; Snp, serotype Newport; Sre, serotype Reading; Sse, serotype Senftenberg; Sty, serotype Typhimurium; Ssc, serotype Schwarzengrund; Swo, serotype Worthington; and UND, undetermined serotype. For example, the branch labeled Snp36-1 P26, at the top of the figure indicates Salmonella serotype Newport, strain 36-1, PFGE type P26. Horizontal length represents genetic distance, and vertical lengths are not meaningful. For the sake of space, the figure contains a short version of the dendrogram, containing 127 strains from among a total of 229 isolates whose manB gene sequences were analyzed.

FIG. 4.

Split graphs based on bootstrap (100 replicates) analyses of the glnA, pduF, and manB gene sequences. The designations at the branches are described in the legend to Fig. 3. A includes the following isolates: San96, Sca93, Sen4, Sen5, Sen23-1, Sen29-1, Sen30-1, Sen34, She49-1, Sha56, Sha60, Sha122, Sha130, Sha135, Sha138, Sha150, Sha151, Sha153, Sha157, Sha158, Sha161, Sha164, Sha165, Sha173, Sha175, Sha176, Sha183, Sha186, Sht20, Smb27, Snp34-1, Snp41-1, Snp128, Sse28, Sse94, Sse95, Sty15, Sty62, Sty86, Sty87, Sty90, Swo44, UND3, UND8, UND64, UND79, UND80, and UND109. B includes the following isolates: Sag75, San37, Sen24, Sha71, Sha124, Sha141, Sha146, Sha148, Sha160, Sha182, She7-1, She9, Ske69, Smb17, Snp33-1, Ste41, Snp40-1, Sty2, Sty6, Sty13, Sty19-1, Sty20-1, Sty31, Sty54-1, and Sty61. C includes the following isolates: Sag129, Sha49, Sha50, Sha131, Sha154, She3-1, Sty52, Sty85, and Sty89. D includes the following isolates: Sag75, Sag129, San37, San96, Sbr68, Sca93, Sen5, Sen23-1, Sen24, Sen24-1, Sen29-1, Sen30-1, Sen34, Sen57-1, Sha48, Sha49, Sha56, Sha60, Sha71, Sha77, Sha102, Sha122, Sha124, Sha130, Sha131, Sha138, Sha139, Sha141, Sha146, Sha147, Sha148, Sha149, Sha150, Sha151, Sha153, Sha154, Sha157, Sha158, Sha160, Sha161,Sha164, Sha165, Sha169, Sha173, Sha176, Sha177, Sha182, Sha186, Sht20, Ske69, Smb27, Snp34-1, Snp39-1, Snp40-1, Snp41-1, Snp76, Sre115, Ssc40, Sse18, Sse94, Sse95, Ste41, Sty2, Sty6, Sty13, Sty13-1, Sty15, Sty15-1, Sty19-1, Sty20-1, Sty31, Sty54, Sty54-1, Sty61, Sty62, Sty85, Sty86, Sty89, Sty90, Swo44, UND3, UND64, UND79, UND80, UND109, and UND110. E includes the following isolates: Sag129, Sen4, Sha48, Sha49, Sha102, Sha131, Sha165, Sha175, Sha182, Sha183, She7-1, Ske69, Snp33-1, Ssc40, Sse28, Sty6, Sty15, Sty15-1, Sty52, Sty89, Sty92, UND109, and UND110. F includes the following isolates: San96, Sca93, Sen23-1, Sen24, Sen24-1, Sen29-1, Sen30-1, Sen34, Sha50, Sha56, Sha77, Sha122, Sha130, Sha138, Sha150, Sha153, Sha157, Sha164, Sha173, Sha176, Sha177, Sha186, She3-1, Sht20, Snp76, Sse95, Ste41, StyI3-1, Sty86, Swo44, UND3, and UND80. G includes the following isolates: Sen5, Sha58, Sha71, Sha141, Sha160, She9, Sty2, Sty13, Sty20-1, Sty31, Sty54, Sty54-1, Sty87, Snp40-1, Snp41-1, and Sse18.