John Melbin Jose, J (2009) Molecular Characterisation and virulence Profiles of Group A Streptococci causing Human Infection in a South Indian community. Doctoral thesis, The Tamilnadu Dr. M.G.R. Medical University, Chennai.
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Abstract
1. A total of 698 GAS isolated from throat cultures of children with pharyngitis (PT, n =116), skin cultures of children with impetigo (SK, n = 276) and throat cultures from asymptomatic children (NT, n = 306) were included for the study. These isolates were selected from a total 769 GAS isolates that were subjected to emm typing. Same emm types of GAS isolated from same child in consecutive weeks were considered as the same strain and were therefore not included in this analysis. (Table 1). 2. Seventy seven emm types were identified among the 698 GAS isolates. This results show a high heterogeneity among GAS isolates circulating in and around Vellore (Table 2). To the best of our knowledge, this study has the largest series of GAS isolates subjected to emm typing from one community in a prospective manner. 3. The four most common types namely, emm 63 (4.6 %), emm 81(4.3 %), 28 (4.15 %) and 49 (3.9 %) accounted for 16.9 % of all isolates (Table 15, Fig. 26). Eleven types accounted for 272 isolates (38.96 %). This shows that many types predominate among this GAS population. Further, in an endemic country like India, no single or few types can be shown to predominate. 4. In this study, sixteen sequence types (st) identified among 698 GAS isolates accounted for 95 (13.6 %) isolates. Six of them namely, stKNB1 through stKNB6 were hitherto unrecognized new types (Table 2). The number of provisional types and new types identified in this study is much higher than many other reported literatures. 5. This shows that newer emm types tend to evolve through genetic mutation among strains that circulate and cause infections in this endemic area. Possibly, a highly susceptible population enhances the rate of mutation which in turn results in the development of new strains. The only non typable strain isolated in this study suggests that such strains are incidental and they may not be associated with pathogenesis. 6. Forty six emm types were identified among 116 PT GAS isolates shows a high heterogeneity among them. This probably results from a high rate of transmission of GAS among children who come from neighboring villages, but attend the same school. Types 28, 1 and 49 accounted for 17.2 % of the 116 isolates show predominance of certain types. Interestingly, type 1 which is a globally recognized invasive type accounted for only 2.6 % of 698 GAS isolates. Moreover, subtype 1.2-2 accounted for 14 of the 18 isolates of emm 1. (Fig. 5, Table 2). 7. Sixty two types were identified among 276 SK GAS isolates in the children of the same cohort again shows high heterogeneity among impetigo associated types (Fig 6). Type 122 was the most predominant (5.79 %) and seven types namely, 122, 81, 63, 44/61, 82, st854 and 85 accounted for 32.6 % of the 276 isolates. This shows that certain types do tend to predominate despite the high heterogeneity. 8. Sixty seven types were identified among 306 GAS isolates associated with normal throat colonization. The distribution pattern reflected the types that were predominant among PT and SK types. Types 81, 82 and st854 found in NT were associated with SK isolates while types 49, 63, 118, 42, st1731 and 77 were associated with both PT and SK isolates. This shows that colonization of normal throat with GAS can occur with strains associated with both pharyngitis and impetigo (Fig. 7). Moreover, the risk of GAS carriage and persistence of infecting strains varied from person to person which demonstrates the type specific immunity and immunity across emm types. 9. In this cohort of children, skin infection leading to throat infection or throat colonization was identified in 30 of the 140 students (Table 16). In contrast, throat infection or throat colonization prior to the development of skin lesion was not observed in any child. This shows that skin infection plays a major role in the transmission of GAS in this community. 10. Comparison of predominant emm types among PT, SK and NT isolates showed that three types namely 49, 63 and 44/61 were common to PT and SK isolates, while types 28, 1, 49, 63, 42, st1731 and st2147 were seen both among PT and NT isolates. Similarly, types 81, 63, 82, st854 and 49 were seen among SK and NT isolates. This again shows that impetigo associated types can colonize normal throat and may also cause pharyngitis or vice versa. (Fig. 8 & Table 3). 11. A total of 40 emm types common to PT and SK isolates were identified (Table 14). The tissue preference index was more for PT isolates in 24 types and in 16 emm types for SK isolates (Table 14). This comparison shows that some types do have predilection towards pharyngitis while others have a skin predilection. 12. Monthly distribution of emm types among PT isolates showed a tri-model clustering. Twenty seven types were restricted to first 8 months of survey and 11 types were restricted to the last 7 months while 9 types were seen scattered among different months of surveys. This probably is associated with the intensive treatment given to all symptomatic children as a result of which newer types appeared at later stages of survey (Fig 14). Similar dynamics of transmission of GAS strains were seen among SK and NT isolates. Since emm types associated with SK and NT isolates was far more heterogeneous, the survey-wise distribution of types was less pronounced (Figs. 15 & 16). 13. Seventy four GAS isolates representing 74 emm types were selected for determining the emm family pattern (Table 19). These included 40 from PT isolates, 29 from NT isolates and 5 from SK isolates. Family patterns could be established in 67 of the 74 types while it could not be determined in seven types. The latter included types 65, 87, 100, 122 as well as two new types, stKNB4 & stKNB5 and the lone nontypable isolate. Thirty nine (52.7 %) of the 74 emm types belonged to the pattern E while, 22 (29.7 %) of the 74 types belonged to pattern D. Thus, 67 (90.5 %) of the 74 types belonged to either of these types. This pattern distribution was similar among the three groups of isolates. Further, 12 (30 %) of the 40 types associated with pharyngitis belonged to pattern D which is reportedly skin specific and five of the six A-C patterns emm types were also found to be isolated from skin infection. Therefore, the emm family pattern may not be a satisfactory genotypic marker for skin or throat specificity. 14. In our study, 59.45 % of the 74 emm types tested were sof gene positive. Further 38 of the 39 types that belonged to family pattern E were sof gene positive while only 3 of the 22 types that belonged to pattern D were sof gene positive. Similarly only one of the six types that belonged to pattern A-C were sof gene positive. As it is known that all sof gene positive types belong to Class II M proteins which are believed to be non rheumatogenic. Thus 39 of the 74 types which were sof gene positive, belonged to M protein class II (Table 19). 15. A phylogenetic tree was developed based on whole emm gene sequences of 76 representative emm types (Fig. 17). This showed two distinct clusters with 53 and 23 emm types respectively. The patterns of clustering of the types show that there are probably two lineages among the 76 emm types. The types seen in the upper cluster are more congruent and therefore seem to have evolved from a common ancestor belonging to emm family pattern D, developed into E with very little genetic changes. In contrast, the lower cluster consists of more divergent types belonging to sub clusters of emm family pattern D and A-C which probably reflect more complicated genetic changes that might have occurred during evolution (Fig. 17). The sequential order of emm types arranged in the phylogenetic tree and the matrix analysis have shown that the pattern D is located in between pattern E and A-C, showing that pattern D has probably evolved from pattern A-C while pattern E has developed later from pattern D. 16. Analysis of the conserved region sequences of 76 emm types has shown that ten types including 18.12 indicate an intermediary position to Class II and I emm types (Fig 19). Only these two classes are known so far, but this result shows an intermediary group which has a combination of both the existing classes. This confirms the development of types of pattern E from those of pattern D. 17. The first 300 bp of the emm gene sequence of very closely related new types, stKNB1, 2, 3, 4, 5 and 6 were analyzed for the changes leading to antigenic variations (Fig. 20). They were clonally related and clustered together as shown by the phylogenetic analysis. 18. The presence of PAVF in 78.3 % of 218 GAS isolates had at least one PAVF while 149 (68.3 %) had two or less number of PAVF in them which indicates that there are many strains circulating in the community with minimal virulence genes. Forty five (20.8 %) had four or more PAVF in them. Only one isolate had seven PAVF in it while there was no isolate with eight or more PAVF. The existence of very few strains with large number of virulence genes mean that most of isolates are not capable of expressing virulence factors to cause complicated diseases. Presence of individual PAVF in these isolates varied from 0.5 % (sda) to 32.1 % (speH) which also supports the very low level of PAVF in these isolates (Fig. 21). Factors mf4 and sdn were not found in any of the isolates showing that some phages or some bacterial clones were not yet transmitted to this population. 19. All sequenced strains associated with severe invasive diseases harbor 5-7 virulence factors while the same emm type identified in the cohort of children had only one or two PAVF (Table 32). Also, no difference in the PAVF distribution was seen among isolates from uncomplicated infections, colonizing strains as well as those causing invasive GAS disease. Therefore it is difficult to prove the precise role of these virulence genes in causing GAS disease. 20. Distribution of chromosomally mediated toxin and fibronectin binding protein genes were studied in 140 GAS isolates. The most of the commonly known toxins like slo, ska, scp, speB, speF and speG are present in almost all the isolates except speZ and speJ which is present only in 37.4% and 16.5 % respectively (Fig. 24). Chromosomally encoded extra cellular products and fibronectin binding proteins are considered to be more stable and might have slowly altered in tune with natural selection and adaptation. Among the surface proteins, scl is present in all the isolates and a significant association of fba was identified with all the isolates of family patterns E and D. Thus gene fba appears to be a marker for skin and general specialist GAS isolates. The sic gene was identified in 15 (10.7 %) isolates, where all of which belonged to family pattern A-C except type 28 (n = 1) which belonged to pattern E. All the other factors are randomly present which raises the question as to whether they are associated with mobile genetic elements or phages. CONCLUSION: A highly sensitive molecular technique, emm typing was successfully standardized to identify a very high degree of heterogeneity among GAS isolates circulating and causing infections among south Indian rural school children. The findings of this study have shown a distinctly different epidemiology from those reported from western countries and Australia. However it is very similar to those reported from Africa, the indigenous population of Australia and Nepal. This study shows high diversity of genotypes among the GAS population that is encountered in different sites of the human body. Further, a comparison of emm types contained in the 26- valent hyper-variable region of M proteins with the types identified in this study shows that such a vaccine will not be effective in this population. It is also likely that the high heterogeneity of types encountered in this community will not permit the use of a multivalent M protein based vaccine. The distinction of throat and skin specific GAS becomes difficult due to the colonization of pharyngitis and impetigo associated types in the normal children combined with high heterogeneity and endemicity of GAS infections. Self transmission of skin isolates to throat plays a major role in the transmission of GAS in this community. Studies on month-wise distribution of types show dynamic changes in their distribution patterns. This is probably related to introduction of different strains by children attending the same school, but come from different villages in and around the school. There was no significant association between emm family pattern and the three groups of isolates, namely, PT, SK and NT isolates. However, with 53 % of the 74 emm types belonging to pattern E, a high percentage of the GAS population in this community are generalists; i.e. they can infect throat or skin or both. The sequential order of emm types arranged in the phylogenetic tree, matrix analysis and the conserved region have confirmed that pattern D is located in between pattern E and A-C and that pattern D has evolved from pattern A-C while pattern E has developed later from pattern D. The distribution of PAVF among GAS isolates from uncomplicated infections, colonizing strains as well as those causing invasive GAS disease did not show significance difference among them. However a relative lack of these factors were seen among these isolates probably indicating a lack of ability to express virulence factors that may be necessary for the development of disease complications. Therefore it is difficult to prove the precise role of these virulence genes in causing GAS disease.
| Item Type: | Thesis (Doctoral) |
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| Uncontrolled Keywords: | Molecular Characterisation, virulence Profiles, Group A Streptococci, Human Infection, South Indian Community. |
| Subjects: | Respiratory Medicine > Microbiology > Respiratory Medicine > Microbiology |
| Depositing User: | Subramani R |
| Date Deposited: | 19 Aug 2017 08:52 |
| Last Modified: | 27 Oct 2022 02:31 |
| URI: | http://repository-tnmgrmu.ac.in/id/eprint/2701 |
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