Molecular epidemiology of shigellosis in Andaman

A study on molecular epidemiology of shigellosis was initiated with the objective of understanding the clonal relatedness of shigellae isolates from Andaman and Nicobar Islands and elsewhere in India. Genetic characterization of shigella isolates was carried out by studying the plasmid profile, RAPD finger prints and Pused Field Gel Electrophoresis patterns. A total of 89 shigellae strains were included in the study. These isolates were recovered from patients attending the hospitals in South Andaman since 1994 as well as during a community based surveillance study in a village near Port Blair. Forty among these were Shigella dysenteriae, 34 S. flexneri, 25 S. sonnei and 4 Shigella boydii. Besides 14 strains (6 S. dysenteriae type 1 and 8 S. flexneri type 2 a) obtained from Kolkata, Manipal and Lucknow were also included.

Plasmids were extracted and separated in 1% agarose gel by horizontal electrophoresis. In PFGE analysis, high molecular weight chromosomal DNA in agarose plugs were prepared and restriction enzyme (Xba I) digestion was carried out using standard procedure. The enzyme digested genomic DNA was separated using CHEF Mapper XA chiller system (Bio-Rad). Clonal relationship was analysed using neighbour joining method with percentage of matched bands summarizing the degree of similarity.

Fig 1 shows the plasmid profile of 16 Shigella isolates obtained from Andaman during the period 1994—2001. The distribution of plasmids ranged between 1-55 Kb with 0-11 different fragments. Each of the four species showed different patterns. Within each species, minor variations were observed. There were some differences in the plasmid profiles of isolates obtained at different periods of time. Among Shigella dysenteriae type 1 two isolates recovered in 1994 and 1997 respectively (Lanes 6 & 8) showed identical patterns.

Fig. 2 shows the plasmid profiles of S. dysenteriae isolated from Andamans and Kolkata. The Andaman isolates had seven different patterns, whereas the Kolkata isolates showed a single pattern. Number of plasmids in Andaman isolates ranged from three to seven and the size of the plasmids ranged between 1.5 kb to 220 kb. All the strains showed a plasmid of 23 kb in size.

Fig 3 Plasmid profiles of Shigella flexneri

type 2 a isolated from Andamans (A) and other parts of India (B)

Fig 4 Plasmid profiles of Shigella sonnei isolated during 2000—01 (A) and those isolated earlier from Andamans (B).

Fig 3 shows the plasmid profile of S. flexneri type 2 a isolated from Andamans and elsewhere. Eight patterns with 5—9 plasmids were observed. The profile of Andaman isolates and isolates from other parts of the country were similar.

Fig. 4  shows the plasmid profile of Shigella sonnei isolated during 2001—02  and earlier from Andamans. The strains isolated during 2001-02 show identical pattern and this pattern is slightly different from the pattern shown by the isolates prior to 2001. The isolates after 2001 were nalidixic acid resistant, whereas the isolates were nalidixic acid sensitive till 2001.

Fig 5 RAPD patterns of Shigella sonnei

isolated during 2001—02 (A) and earlier (B) from Andamans

Fig 6. NotI PFGE profiles of Shigella sonnei isolated during 2001—02 (A) and earlier (B) from Andamans

Fig 5 shows the RAPD profile of Shigella sonnei isolates during 2001—02 and earlier. Seven of the nine isolates during 2001—02 show identical patterns. Three isolates prior to 2001 (lanes 2, 15 and 16) also show closely related pattern. Other isolates prior to 2001 show 45% to 87% similarity.

Fig 6 shows NotI PFGE profile of Shigella sonnei isolates. All the isolates except one isolated prior to 2001 (lane 11) showed 100% homology. The molecular characterization of Shigella sonnei using various techniques indicate that a nalidixic acid resistant strain of S. sonnei is emerging as the predominant shigella strain in these islands.