Clonal expansion and microevolution of quinolone-resistant Salmonella enterica serotype typhi in Vietnam from 1996 to 2004

Abstract

Salmonella enterica serotype Typhi clinical isolates (n = 91) resistant to nalidixic acid (Nal(r)) were collected from sporadic cases and minor outbreaks throughout Vietnam between 1996 and 2004. These isolates were typed and compared by four methods: Vi phage typing, PstI ribotyping, XbaI and SpeI pulsed-field gel electrophoresis (PFGE), and single-nucleotide polymorphism (SNP) analysis. The results indicated that 65% of the isolates were not typeable by Vi phage typing. In contrast, the ribotyping and, with more accuracy, the SNP analysis methods indicated that all Nal(r) isolates belonged to a single clone (ribotype 3a, haplotype H58) that was found previously and that largely consisted of plasmid-encoded multidrug-resistant serotype Typhi isolates. PFGE demonstrated the occurrence of microevolution within this clone. We identified two major combined PFGE profiles: X1-S1 and X3-S6. X3-S6 predominated between 1996 and 2002 but was replaced by X1-S1 after 2002. Nevertheless, PFGE, with a Simpson's index of 0.78, was not considered an optimal discriminatory method for investigating typhoid fever outbreaks in Vietnam. The rate of quinolone resistance increased and the rate of multidrug resistance decreased during the study period. From 2002 to 2004, 80.6% of the isolates from South Vietnam were resistant only to Nal. The mechanism of Nal resistance in most of the isolates (94%) was a mutation in the quinolone resistance-determining chromosomal region of gyrA that led to the amino acid substitution Ser83Phe. No plasmid-located qnrA, qnrB, or qnrS was detected.

FIG. 1.

Representative PstI ribotypes obtained from a subset of 71 S. enterica serotype Typhi isolates under study. Ribotype numbering is indicated. The dendrograms generated by the BioNumerics software show the results of cluster analysis on the basis of PstI ribotyping. Similarity analysis was performed by using the Dice coefficient, and clustering was done by UPGMA. n, the number of isolates for each ribotype is indicated.

FIG. 2.

Representative XbaI and SpeI PFGE profiles obtained for the 91 S. enterica serotype Typhi isolates under study. The PFGE profile numbering is indicated. The dendrograms generated by the BioNumerics software show the results of cluster analysis on the basis of PFGE fingerprinting. Similarity analysis was performed by using the Dice coefficient, and clustering was done by UPGMA. n, the number of isolates for each PFGE profile is indicated.