Vi antigen expression in Salmonella enterica serovar Typhi clinical isolates from Pakistan

Abstract

The accurate identification of Salmonella enterica subsp. enterica serovar Typhi variants that fail to express the capsular polysaccharide, Vi, is an important and much discussed issue for medical microbiology. We have tested a multiplex PCR method which shows the presence or absence of the genetic locus required for Vi expression. Of 2,222 Salmonella serovar Typhi clinical isolates collected from patients' blood over a 4-year period in a region of Pakistan where typhoid is endemic, 12 tested negative for Vi expression by serological agglutination. However, only 1 of these 12 was Vi negative by the multiplex PCR method. This result was confirmed by immunofluorescence, the most sensitive method for Vi characterization in Salmonella serovar Typhi. The multiplex PCR described therefore represents a simple and accurate method for surveillance for Vi-negative variants of Salmonella serovar Typhi in Pakistan. Testing of clinical isolates of Salmonella serovar Typhi, before subculture, from other regions where Vi-negative Salmonella serovar Typhi has been described should be carried out so that the impact of vaccination with purified Vi antigen on the levels of Vi-negative Salmonella serovar Typhi in bacterial populations can be assessed.

FIG. 1.

Schematic representation of the multiplex PCR for the detection of SPI-7 and tviB.

FIG. 2.

Multiplex PCR for detection of SPI-7 and tviB. The presence of the tviB gene in the viaB locus is shown by a band at about 0.8 kb, and the absence of the SPI-7 region is shown by a band at about 1.5 kb. The aroC products are at 0.6 kb. Lanes: Vi-positive controls Ty2 (lane 1), SARB63 (lane 16), and CT18 (lane 24); Vi-positive clinical isolates BL13807, BL15339, and BL13510 (lanes 2 to 4, respectively); Vi agglutination-negative Salmonella isolates BL9282 (lane 5), BL14907 (lane 6), BL13843 (lane 7), BL9283 (lane 8), BL12035 (lane 9), BL3081 (lane 10), BA788 (lane 11), BL4426 (lane 12), BL6886 (lane 13), BL6397 (lane 14), BL6583 (lane 15), BL2934 (lane 22), BL13994 (lane 17), BL9737 (lane 18), BL1838 (lane 20), and BL2580 (lane 21); Vi negative controls Salmonella serovar Paratyphi A BL11161 (lane 19) and SARB64 (lane 23).

FIG. 3.

Sequence alignment of PCR product from the aroC gene of Vi agglutination-negative S. enterica. The sequences were compared to that of aroC of strain CT18. Boxed nucleotides represent variations from the consensus sequence.

FIG. 4.

Sequence alignment of PCR product across the pheU tRNA gene of Vi agglutination-negative S. enterica. The sequence from Salmonella serovar Typhi CT18 with SPI-7 removed is presented for comparison. Boxed nucleotides represent variations from the consensus sequence.

FIG. 5.

Immunofluorescent staining of Vi agglutination-negative Salmonella serovar Typhi. Strain BL6397 is the Vi-positive control, and strain BL11161 is the Vi-negative control. First column, bacteria labeled with anti-Salmonella antibody (green channel); second column, bacteria labeled with anti-Vi antibody (red channel); third column, merged images of the first two columns. Original magnification, ×1,000.