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Abstract
Shigella causes Shigellosis or bacillary dysentery which is a major diarrheal disease throughout the world especially in developing countries. The annual incidence of Shigella infections was estimated to be 164.7 million with 1.1 million deaths, with the majority of the cases and deaths involving children under 5 years old. Humans are the only host of Shigella. Among the four species of Shigella, S. flexneri is the most predominant species affecting economically poor populations. S. flexneri is further divided into different serotypes based on the structure of the O-antigen. Up to now, at least 15 serotypes have been recognized. All except serotype F6 belongs to the same evolutionary cluster based on housekeeping genes. Thus understanding the molecular epidemiology of S. flexneri will contribute significantly to the development of new strategies for disease prevention and control. This study aimed to identify highly VNTR loci to develop a MLVA typing scheme for S. flexneri and to study further the molecular evolution of S. flexneri.
This study used a new approach to identify VNTRs. Through searching two VNTR databases using a range of parameters this study identified 52 potential VNTRs. Variable VNTRs were further refined from comparison of three available S. flexneri genomes. This reduced the number of VNTRs to 13 for initial screening. However, while this project was ongoing, an MLVA study of S. flexneri was published which tested 36 potential VNTRs. The 13 most variable VNTRs from that study, 10 of which overlapped with our initial screening, were adopted for MLVA typing in this project.
The 13 VNTRs were used to type 51 S. flexneri isolates. The average number of isolates that can be typed for a VNTR was 95%. The VNTR SF17 has the lowest typability. Only 67% of the isolates can be typed. Only one VNTR, O157-11, had a typability of 100%. MLVA-13 (all VNTRs) and MLVA-8 (the eight most variable VNTRs) had a discriminatory power of 0.998 while MLVA-4 (the 4 most variable VNTRs) had a slightly lower power of 0.994.
MLVA typing schemes developed in this project and by other studies were tested in 55 isolates. The level of differentiation of the 55 isolates into MLVA profiles depends on the number of VNTR loci used. The scheme using all 13 VNTR loci divided the 32 of the 55 isolates that can fully be typed into 31 MLVA profiles. Using the MLVA-8 scheme, 49 of the 55 isolates had a complete VNTR data and can be assigned into 47 MLVA types. Further reducing to the 4 most variable VNTRs (the MLVA-4 scheme), 52 of the 55 isolates had a complete VNTR data and were differentiated into 46 MLVA profiles. Phylogenetic analysis of MLVA data showed the isolates can be divided into 4 clusters with the most prevalent serotype 2a being mostly located in cluster I.
This study applied MLST-7 to Australian isolates. The 15 Australian isolates were typed into four different STs, ST245, ST626, ST651 and ST1025 with the latter being a new ST. Together with 36 isolates which has been previously typed, this study showed that the majority of the isolates belonged to ST245. This ST appears to be a globally distributed clone. The isolates of ST245 were further separated by inclusion of two genes from MLST-15 scheme.. MLST-15 ST91 comprised 16 of the 34 ST245 isolates. For the first time this study showed that the ST91 which was first reported in China as a multidrug resistant clone is present in other Asian countries and Australia although the isolates in this study have not been tested for multidrug resistance.
This study provided another dataset to measure the variability of these 13 VNTRs and provided data for the general applicability of VNTRs for typing S. flexneri. The data from this project confirmed the proposed two MLVA schemes using four and eight VNTRs for different levels of resolution were applicable to our strains. The combination of MLST-7 and MLST-15 schemes revealed predominant S. flexneri STs circulating globally, demonstrating the usefulness and limitations of MLST for global epidemiology. There is significant consistency between MLVA clustering and MLST sequence types. This study has significantly increased our understanding of epidemiology and evolution of S. flexneri.