Microbiol. Immunol. Vol. 36 (5), 539-543, 1992
Genetic
Variation
as Detected
among
by Ribosomal
Tikki
PANG, 1, * Martin Chong
Lek
KoH,3
Malaysian
Isolates
of Salmonella typhi
RNA Gene Restriction ALTWEGG,2 and
Savithri
Gladys
Patterns
MARTINETTI,2
PUTHUCHEARY4
1Institute
for AdvancedStudies, Universityof Malaya, Kuala Lumpur, Malaysia, of Medical Microbiology,Universityof Zurich, Zurich, Switzerland, 3Departmentof Geneticsand Cellular Biology, and 4Departmentof Medical Microbiology,Universityof Malaya, Kuala Lumpur, Malaysia
2Department
(Accepted for publication, February 1, 1992) Abstract Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.
Typhoid fever remains an important public health problem in many parts of the tropical world (3). Endemic regions often experience periodic outbreaks which emphasize the need to have better markers to identify and monitor isolates of epidemiologic importance (4). In addition, efforts to effectively control this disease have been hampered by the unavailability of a universally effective vaccine. One currently used formulation appears to be showing variable effectiveness in different parts of the world (10), with lowest efficacy in highly endemic areas with high attack rates. This observed variability could be due to many reasons, including the possibility of genetic variation in strains prevalent in various geographical regions. Studies to determine genetic variation among S. typhi by multilocus enzyme electrophoresis indicated limited diversity in that all strains analysed had identical isoenzyme patterns suggesting that they belong to a single clone (11). However, analysis of ribosomal RNA (rRNA) gene restriction patterns indicated considerable variation at the DNA level (1). Heterogeneity has also been noted with lipopolysaccharide (LPS) phenotypes among S. typhi strains (7). It would seem to be of interest to apply molecular genetic analysis to S. typhi strains from different parts of the world, especially in endemic areas which periodically experience 'epidemics' of typhoid fever. We report here the results of a study to determine rRNA gene (rDNA) restriction patterns among S. typhi strains isolated in Malaysia. Isolates of S. typhi from either blood or stool cultures were used in this study. These strains were isolated using standard methods (2) from patients admitted to 539
540
T. PANG
ET AL
the University Hospital, Kuala Lumpur. Strains 2-11 were isolated between January- September, 1987, and strains 103-113 between January- September, 1990, a period of increased disease activity. Susceptibility testing was performed on Mueller-Hinton agar (BBL Co., U.S.A.) by the disc-diffusion method. All strains were sensitive to ampicillin, chloramphenicol, and co-trimoxazole. Phage typing of the strains was performed according to standard procedures by the Salmonella Reference Centre at the Institute for Medical Research, Kuala Lumpur. Anlysis of rRNA gene restriction patterns was performed according to previously published procedures (9). Briefly, chromosomal DNA digested by restriction endonucleases Smal or Pstl was separated by agarose gel electrophoresis, transferred to a nylon membrane and then probed with biotinylated plasmid pKK3535 DNA. pKK3535
Table
1.
Phage
type and rDNA gene restriction
patterns
of S. typhi isolates
NOTES
541
is a pBR322-derived plasmid containing one copy each of the genes coding for 5S RNA, 16S RNA, 23S RNA and tRNAgiu from E. coli. Of the 20 strains analyzed, 18 strains belonged to 7 different phage types and two were untypeable (Table 1). Considerable variability among strains was detected when rRNA gene restriction patterns (Fig. 1) were analyzed with eight different pattern combinations present (Table 1). The variability of the patterns also depended upon the restriction endonuclease used; Pstl produced more different patterns than did Smal (Table 1). Interestingly, pattern combinations 6, 7, and 8 were found only in strains 103 to 113 while pattern combination 2 was found only in strains 2 to 11 (Table 1). Our results clearly show that during both periods, typhoid fever was not carried by a single strain but rather by a multitude of different clones. The suspected clusters were, therefore, not epidemics in the strictest sense and the increase in observed cases, seen during the time of isolation of strains 103 to 113, was not due to single strains of increased virulence. However, the factor(s) responsible for the higher incidence of this disease during certain periods remain to he elucidated. In light of continuing disease activity in many parts of the developing world,
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Fig. 1. rDNA gene restriction patterns of S. typhi (St) isolates following digestion with PstI and hybridization with plasmid pKK3535. Lane 1 =Serratia fonticola, 2=St111, 3=St109, 4=St105, 5=St104, 6=St11, 7=St110, 8=St7, 9=St10, 10=St5, 11=St3, 12=St113, 13=St112, 14=St108, 15=St107, 16=St103, 17=St9, 18=St8,19=St6,20=St4, 21 = St2, 22 =lambda HindII I markers.
542
T. PANG
ET AL
documentation of the extent of genetic variation among strains of S. typhi is of utmost importance. In addition to the obviously important implications for the design of more effective vaccines, there has also been recent interest in correlating bacterial population genetics with bacterial pathogenesis (6). With regards to typhoid fever it might be possible that disease severity is associated with particular genotypes although such a correlation has not been documented so far (5). The findings in the present study, with strains isolated in an endemic area, confirmed those published previously which indicated considerable genetic variation among S. typhi strains (1) and suggest that rRNA gene restriction patterns are of value in differentiating individual strains and may complement phage typing for epidemiological purposes. These two approaches may help to determine if epidemics are due to a single clone or to the simultaneous activity of several different clones. Clonal diversity based on rRNA gene restriction patterns has also been observed with Vibrio cholerae(8). However, it is not clear at this stage what these genetic variations mean at the phenotype level and whether the variations are in any way related to virulence of individual strains. The collection of information pertaining to genetic variation among S. typhi strains globally may ultimately provide an invaluable database for comparison, epidemiological surveillance, and effective implementation of vaccination strategies. The above research project was funded by grants from the Working Group on Biotechnology Ministry of Science, Technology & Environment, Malaysia, the University of Malaya and the China Medical Board. REFERENCES
1)
Altwegg, M., Hickman -Brenner, F.W., and Farmer, J. J. 1989. Ribosomal RNA gene restriction patterns provide increased sensitivity for typing Salmonella typhi strains. J. Infect. Dis. 160: 145-149. 2) Cowan, S.T., and Steel, J. 1974. In Cowan & Steel's Manual for the identification of medical bacteria, 2nd ed, Cambridge University Press, Cambridge. 3) Edelman, R., and Levine, M.M. 1986. Summary of an international workshop on typhoid fever. Rev. Infect. Dis. 8: 329-349. 4) Eisenstein, B.I. 1990. New molecular techniques for microbial epidemiology and diagnosis of infectious diseases. J. Infect. Dis. ,161: 595-602. 5) Heinene, W., Matar, G., Reeves, M., and Swaminathan, B. 1991. Molecular characterization of Salmonellatyphi isolates from patients with severe and mild typhoid fever. Eur. J. Epidemiol. 7: 192-193. 6) Iglewski, B.H., and Clark, V.L. (eds), 1990. Molecular basis of bacterial pathogenesis, 473 pp., Academic Press, London. 7) Jimenez- Lucho, V., and Foulds, J. 1990. Heterogeneity of lipopolysaccharide phenotype among Salmonellatyphi strains. J. Infect. Dis. 162: 763-764. 8) Koblavi, S., Grimont, F., and Grimont, P.A.D. 1990. Clonal diversity of Vibrio cholerae O1 evidenced by rRNA gene restriction patterns. Res. Microbiol. 141: 645-657. 9) Martinetti, G., and Altwegg, M. 1990. rRNA gene restriction patterns and plasmid analysis as a tool for typing Salmonella enteritidis. Res. Microbiol. 141: 1151-1162. 10) Murphy, J.R., Baqar, S., Losonsky, G., Tacket, C., Lindberg, A.A., and Levine, M.M. 1987. Human immunogenicity of orally administered typhoid vaccines, Ty21 a and 541Ty- 543Ty. Microbiol. Ther. 17: 107-116.
NOTES
543
11) Reeves, M.W., Evins, G.M., Heiba, A.A., Plikaytis, B.D ., and Farmer III, J. J. 1989. Clonal nature of Salmonellatyphi and its genetic relatedness to other Salmonellae as shown by multilocus enzyme electrophoresis and proposal of Salmonella bongori comb . nov. J. Clin. Microbiol. 27: 313-320. (Received for publication, December 20, 1991)
Genetic
Variation
as Detected
among
by Ribosomal
Tikki
PANG, 1, * Martin Chong
Lek
KoH,3
Malaysian
Isolates
of Salmonella typhi
RNA Gene Restriction ALTWEGG,2 and
Savithri
Gladys
Patterns
MARTINETTI,2
PUTHUCHEARY4
1Institute
for AdvancedStudies, Universityof Malaya, Kuala Lumpur, Malaysia, of Medical Microbiology,Universityof Zurich, Zurich, Switzerland, 3Departmentof Geneticsand Cellular Biology, and 4Departmentof Medical Microbiology,Universityof Malaya, Kuala Lumpur, Malaysia
2Department
(Accepted for publication, February 1, 1992) Abstract Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.
Typhoid fever remains an important public health problem in many parts of the tropical world (3). Endemic regions often experience periodic outbreaks which emphasize the need to have better markers to identify and monitor isolates of epidemiologic importance (4). In addition, efforts to effectively control this disease have been hampered by the unavailability of a universally effective vaccine. One currently used formulation appears to be showing variable effectiveness in different parts of the world (10), with lowest efficacy in highly endemic areas with high attack rates. This observed variability could be due to many reasons, including the possibility of genetic variation in strains prevalent in various geographical regions. Studies to determine genetic variation among S. typhi by multilocus enzyme electrophoresis indicated limited diversity in that all strains analysed had identical isoenzyme patterns suggesting that they belong to a single clone (11). However, analysis of ribosomal RNA (rRNA) gene restriction patterns indicated considerable variation at the DNA level (1). Heterogeneity has also been noted with lipopolysaccharide (LPS) phenotypes among S. typhi strains (7). It would seem to be of interest to apply molecular genetic analysis to S. typhi strains from different parts of the world, especially in endemic areas which periodically experience 'epidemics' of typhoid fever. We report here the results of a study to determine rRNA gene (rDNA) restriction patterns among S. typhi strains isolated in Malaysia. Isolates of S. typhi from either blood or stool cultures were used in this study. These strains were isolated using standard methods (2) from patients admitted to 539
540
T. PANG
ET AL
the University Hospital, Kuala Lumpur. Strains 2-11 were isolated between January- September, 1987, and strains 103-113 between January- September, 1990, a period of increased disease activity. Susceptibility testing was performed on Mueller-Hinton agar (BBL Co., U.S.A.) by the disc-diffusion method. All strains were sensitive to ampicillin, chloramphenicol, and co-trimoxazole. Phage typing of the strains was performed according to standard procedures by the Salmonella Reference Centre at the Institute for Medical Research, Kuala Lumpur. Anlysis of rRNA gene restriction patterns was performed according to previously published procedures (9). Briefly, chromosomal DNA digested by restriction endonucleases Smal or Pstl was separated by agarose gel electrophoresis, transferred to a nylon membrane and then probed with biotinylated plasmid pKK3535 DNA. pKK3535
Table
1.
Phage
type and rDNA gene restriction
patterns
of S. typhi isolates
NOTES
541
is a pBR322-derived plasmid containing one copy each of the genes coding for 5S RNA, 16S RNA, 23S RNA and tRNAgiu from E. coli. Of the 20 strains analyzed, 18 strains belonged to 7 different phage types and two were untypeable (Table 1). Considerable variability among strains was detected when rRNA gene restriction patterns (Fig. 1) were analyzed with eight different pattern combinations present (Table 1). The variability of the patterns also depended upon the restriction endonuclease used; Pstl produced more different patterns than did Smal (Table 1). Interestingly, pattern combinations 6, 7, and 8 were found only in strains 103 to 113 while pattern combination 2 was found only in strains 2 to 11 (Table 1). Our results clearly show that during both periods, typhoid fever was not carried by a single strain but rather by a multitude of different clones. The suspected clusters were, therefore, not epidemics in the strictest sense and the increase in observed cases, seen during the time of isolation of strains 103 to 113, was not due to single strains of increased virulence. However, the factor(s) responsible for the higher incidence of this disease during certain periods remain to he elucidated. In light of continuing disease activity in many parts of the developing world,
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Fig. 1. rDNA gene restriction patterns of S. typhi (St) isolates following digestion with PstI and hybridization with plasmid pKK3535. Lane 1 =Serratia fonticola, 2=St111, 3=St109, 4=St105, 5=St104, 6=St11, 7=St110, 8=St7, 9=St10, 10=St5, 11=St3, 12=St113, 13=St112, 14=St108, 15=St107, 16=St103, 17=St9, 18=St8,19=St6,20=St4, 21 = St2, 22 =lambda HindII I markers.
542
T. PANG
ET AL
documentation of the extent of genetic variation among strains of S. typhi is of utmost importance. In addition to the obviously important implications for the design of more effective vaccines, there has also been recent interest in correlating bacterial population genetics with bacterial pathogenesis (6). With regards to typhoid fever it might be possible that disease severity is associated with particular genotypes although such a correlation has not been documented so far (5). The findings in the present study, with strains isolated in an endemic area, confirmed those published previously which indicated considerable genetic variation among S. typhi strains (1) and suggest that rRNA gene restriction patterns are of value in differentiating individual strains and may complement phage typing for epidemiological purposes. These two approaches may help to determine if epidemics are due to a single clone or to the simultaneous activity of several different clones. Clonal diversity based on rRNA gene restriction patterns has also been observed with Vibrio cholerae(8). However, it is not clear at this stage what these genetic variations mean at the phenotype level and whether the variations are in any way related to virulence of individual strains. The collection of information pertaining to genetic variation among S. typhi strains globally may ultimately provide an invaluable database for comparison, epidemiological surveillance, and effective implementation of vaccination strategies. The above research project was funded by grants from the Working Group on Biotechnology Ministry of Science, Technology & Environment, Malaysia, the University of Malaya and the China Medical Board. REFERENCES
1)
Altwegg, M., Hickman -Brenner, F.W., and Farmer, J. J. 1989. Ribosomal RNA gene restriction patterns provide increased sensitivity for typing Salmonella typhi strains. J. Infect. Dis. 160: 145-149. 2) Cowan, S.T., and Steel, J. 1974. In Cowan & Steel's Manual for the identification of medical bacteria, 2nd ed, Cambridge University Press, Cambridge. 3) Edelman, R., and Levine, M.M. 1986. Summary of an international workshop on typhoid fever. Rev. Infect. Dis. 8: 329-349. 4) Eisenstein, B.I. 1990. New molecular techniques for microbial epidemiology and diagnosis of infectious diseases. J. Infect. Dis. ,161: 595-602. 5) Heinene, W., Matar, G., Reeves, M., and Swaminathan, B. 1991. Molecular characterization of Salmonellatyphi isolates from patients with severe and mild typhoid fever. Eur. J. Epidemiol. 7: 192-193. 6) Iglewski, B.H., and Clark, V.L. (eds), 1990. Molecular basis of bacterial pathogenesis, 473 pp., Academic Press, London. 7) Jimenez- Lucho, V., and Foulds, J. 1990. Heterogeneity of lipopolysaccharide phenotype among Salmonellatyphi strains. J. Infect. Dis. 162: 763-764. 8) Koblavi, S., Grimont, F., and Grimont, P.A.D. 1990. Clonal diversity of Vibrio cholerae O1 evidenced by rRNA gene restriction patterns. Res. Microbiol. 141: 645-657. 9) Martinetti, G., and Altwegg, M. 1990. rRNA gene restriction patterns and plasmid analysis as a tool for typing Salmonella enteritidis. Res. Microbiol. 141: 1151-1162. 10) Murphy, J.R., Baqar, S., Losonsky, G., Tacket, C., Lindberg, A.A., and Levine, M.M. 1987. Human immunogenicity of orally administered typhoid vaccines, Ty21 a and 541Ty- 543Ty. Microbiol. Ther. 17: 107-116.
NOTES
543
11) Reeves, M.W., Evins, G.M., Heiba, A.A., Plikaytis, B.D ., and Farmer III, J. J. 1989. Clonal nature of Salmonellatyphi and its genetic relatedness to other Salmonellae as shown by multilocus enzyme electrophoresis and proposal of Salmonella bongori comb . nov. J. Clin. Microbiol. 27: 313-320. (Received for publication, December 20, 1991)
