World Journal of Microbiology & Biotechnology 12, 405-407
Short Communication: Restriction fragment length polymorphism (RFLP) of Salmonella organisms
S.I. Koh-Luar,* S.T. Chew, E. Lau and S.B. Chua Genetic relatedness of 20 Salmonella isolates comprising 16 serotypes was analysed by restriction endonuclease digestion of the total DNA with six endonucleases individually. The rDNA fingerprints generated by EcoRI were more polymorphic, each serotype showed a unique fingerprint sharing several core (monomorphic) bands with several polymorphic bands. Eight characteristic Ncfl rDNA fingerprints were found. Similar rDNA RFLP patterns were observed in strains of Salmonella from different serotypes. Key words: Food pathogen, Salmonella serotypes, RFLP, 16S rDNA.
The genus Salmonella contains about 2000 different serotypes that can be transmitted between animals and humans, and all are pot:entially pathogenic to humans. With the exception of disease caused by Salmonella typhi, paratyphi A and paratyphi C, Salmonella infections are zoonoses. Salmonella typhi, paratyphi A and paratyphi C, which cause typhoid fever or enteric fever, are specific to humans and do not have a reservoir in animals. Advances in molecular biological techniques have provided yet another avenue for epidemiology and identification studies of many pathogens such as Mycobacterium, Vibrio and Candida. Unlike serotyping of specific surface antigens, nucleic acid-based techniques such as restriction fragment length polymorphism (RFLP) followed by hybridization with gene-specific probes (Knight et at. I990) and the polymerase chain reaction (PCR) (Stone et al. 1994) depend on the genotypes of the organisms concerned and not on their phenotypic expression. In this paper, we describe some preliminary observations on RFLP analysis of Salmonella isolates. We sought to find genetic relationships between strains of different serotypes which may be of value in monitoring trends in the prevalent Salmonella infections.
S.I. Koh-Luar and E. Lau are with the Department of Chemical Process & Biotechnology, Singapore Polytechnic, 500 Dover Road, Singapore 139651. Republic of Singapore; fax: (065) 7721976. S.T. Chew is with the Veterinary Public Health Laboratory (VPHL), Primary Production Department (PPD), 51 Jalan Buroh, Singapore 619415. Republic of Singapore. S.B. Chua is with the Veterinary Public Health Division, Primary Production Department, 5 Maxwell Road, No. 03-00, Tower Block, MND Complex, Singapore 069110. Republic of Singapore. * Corresponding author.
Materials and Methods Microorganisms and rDNA Probe Salmonella organisms used for DNA analyses were isolated at the Veterinary Public Health Laboratory, Primary Production Department according to the standard methods (US Food & Drug Administration 1992). I6S rDNA gene (as described by Edwards et al. I989) of Salmonella wandsworth synthesized by polymerase chain reaction was radioactively labelled with [~-32P]dCTP (Amersham) by the megaprime labelling technique (Amersham).
Genomic DNA Isolation All Salmonella strains were grown in LB medium on a rotary shaker at 37°C until late exponential phase. Cells were harvested by centrifugation and DNA was extracted by the phenol-chloroform method (Sambrook et al. 1989).
Restriction Enzyme Digestion and Gel Electrophoresis Three /lg of genomic DNA were digested separately with the restriction enzymes (2 units//~g) according to the supplier's specifications. Digested DNAs were separated by electrophoresis in a 0.8% agarose gel in 1 x TBE buffer.
Southern Hybridisation and Detection Gels were treated sequentially with 0.25 M HCL 0.5 m NaOH/ 1.5 M NaCl and 0.5 M Tris/HCl/1.5 M NaCl as described by Sambrook et al. (1989). The fractionated restriction fragments were quantitatively transferred to Hybond-N membranes (Amersham) by capillary transfer method and fixed by baking at 80°C for 2 h (Sambrook et aI. 1989). Salmonella wandsworth 16S rDNA was used as a probe in this work because the serotype appears to be the predominant serotype isolated. Southern hybridization and autoradiography were carried out according to Sambrook et al. (1989).
Q 1996 Rapid Science Publishers World Journal of M~crobiolagy ~ Biotechnology, Vol 12, 1996
405
5.I. Koh-Luar, 5.T. Chew, E. Lau and 5.B. Chua (A)
(B)
Figure 1. A representative photograph (A) and diagram (B) of the agarose gel (0.8%) of EcoRI rDNA fingerprints of Salmonellae. The arrows indicate the two constant bands which were found in all Salmonellae analysed in this study. Sizes of markers in kb are as indicated. S3--S. wandsworth; S4--S. hadar; $9---S. mbandaka; S10~S. indiana; S 1 2 ~ . anatam.
Figure 2. (A) An example of a typical Ncil rDNA fingerprints of: $29~S. agona; $ 3 2 ~ . amsterdam; $33~S. dublin; $36~S. havana and $ 3 7 ~ . weltevreden. (B) A schematic diagram of summarised Ncil rDNA RFLPs. Sizes of markers in kb are indicated. The arrows indicate the two constant bands found in all Salmonellae analysed in this study. Profile I ~ . indiana; Profile I 1 ~ . hadar, S. chester, S. senftenburg, S. lexington; Profile II1--~. dublin, S. wandsworth, S. anatam, S. weltevreden; Profile I V Y . blockley, S. mbandaka, S. agona; Profile V ~ . havana; Profile Vl--S. amsterdam; STM--S. typhimurium; S T Y . typhi.
Results and Discussion Genomic DNA of the Salmonella isolates was digested separately with six restriction enzymes (Bglll, PvulI, PstI, XhoI, EcoRI and NciI) that recognized different penta- and hexanucleotide sequences. Two enzymes, EcoRI and NciI, produced clearly well-resolved rDNA fingerprinting patterns within the range of the standard size markers. Agreement between independent isolates of the same serotype was excellent. Duplicate samples of the 20 strains gave reproducible fingerprints when probed.
406
World Journal of A/h'crobiology& Biotechnology, VoI 12, I996
Digestion with EcoRI generated an average of seven to eight fragments per strain ranging in size from 1.0 to 21.1 kb. Most strains displayed a common set of fragments at 1.8 (doublet) and 1.4 kb (Figure i). However, considerable variations between different strains were evident. The polymorphism appeared to be more marked for fragments of molecular weight from 6.0 to 21.1 kb. An average of five or six bands in the size range 1.0 to 2.4 kb were observed in NciI digested I6S rDNA (Figure 2). Two monomorphic bands of 1.5 and 2.2 kb, were seen for all Salmonella organisms in this study including S. typhimurium and S. typhi. However, two additional bands, of 0.9 and 1.4 kb, were also present in all non-typhi
RFLP of Salmonella Salmonella. The smallest 0.9 kb fragment was found in S. typhi but was absent from the other three isolates of S. typhimurium analysed. Conversely, the 1.4 kb fragment found in S. typhimurium and all other serovars, was absent from the genome of S. typhi. Further investigation will be carried out to identify the nature of this difference. Generally, EcoRI fragments are much larger than the Ncil fragments. Therefore, it is not surprising that the EcoRI fragments are more polymorphic particularly in the higher molecular weight range. This is due to the fact that large DNAs are more likely to undergo mutation than smaller ones. The additional or missing 16S rDNA bands in some strains suggest that mutation events such as gain or loss of restriction sites, insertion or deletion of bases may have taken place. The use of EcoRI restriction enzyme may be potentially applicable to the generation of 16S rDNA RFLPs for specific typing of Salmonella isolates since none of the 16 serotypes shared similar EeoRI RFLP fingerprints. Our 20 isolates comprising 16 serotypes gave eight different NciI RFLP profiles. All the non-typhi Salmonellae were closely related to S. typhimurium and S. typhi ( > 55%). At this stage, it may be premature to use this technique to characterise the Salmonella organisms unless a more comprehensive study of ribotyping or other fingerprinting techniques such as pulsed-field gel electrophoresis (PFGE) on all available Salmonella serotypes was carried out. More characterization of other non-typhi Sahnonella strains not listed here is needed before any possible taxonomic distinction between groups or subgroups could become evident. Nevertheless, we observed that S. hadar, S. chester, S. senftenburg and S, lexington share a common NciI profile (denoted profile II); S. dublin, S. wandsworth, S. anatam and S. weltevreden, exhibit profile III and S. blockley, S. mbandaka and S. agona, illustrate profile IV (see Figure 2B). Our studies clearly show that different serotypes of Salmonella can give similar 16S rDNA fingerprints which reflect the close genetic relationship of the serotypes. Tompkins et al. (i987) reported a close genetic relationship between some strains of Legionella pneumophila from different serogroups based on alloenzyme analysis. Likewise, Ketel (1988) observed similar DNA restriction endonuclease profiles in strains of L. pneumophila from different serogroups. Hence, it is not at all surprising to observe similar RFLP
patterns generated from different serotypes of Salmonella in the present study. These findings suggest that genotypes and phenotypes of organisms in environmental sources may not be as stable as we usually presume. The genetic similarities illustrated indicate that the organisms may have originated from the same clone. Strains originating from the same clone may acquire mutations in genes other than the rDNA genes, thus giving rise to common rDNA fingerprints even though they belong to different serotypes. Further investigations are necessary to confirm our hypothesis.
Acknowledgements This work was supported by the Singapore Polytechnic and the Primary Production Department, Singapore. We thank B.L. Koh for his critical review.
References Edwards, U., Rogall, T., Blocker, H., Emde, M. & B6ttger, E.C. 1989 Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. NucleicAcids Research 17, 7843-7853. Ketel, R.J. 1988 Similar DNA restriction endonuclease profiles in strains of Legionella pneumophila from different serogroups. Journal of ClinicalMicrobiology 26, 1838-1841. Knight, I.T., Shults, S., Kaspar, C.W. & Colwell, R.R. 1990 Direct detection of salmonella spp. in estuaries by using a DNA probe. Applied and Environmental]vlicrobiology56, 1059-1066. Sambrook, J., Maniatis, T. & Fritsch, E.F. 1989 Molecular Cloning:a Laboratory Manual, 2nd edition, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Stone, G.G., Oberst, R.D., Hays, M.P., McVey, S. & Chengappa, M.M. 1994 Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure. Journal of ClinicalMicrobiology 32, 1742-I749. Tompkins, L.S., Troup, N.J., Woods, T., Bibb, W.F. & McKinney, R.M. 1987 Molecular epidermiology of Legionella species by restriction endonuclease and alloenzyme analysis. Journal of ClinicalMicrobiology 25, I875-1880. US Food & Drug Administration. 1992 Bacteriologicalanalytical manual, 7th edition. Washington, DC: Association of Official Analytical Chemists.
(Received in revised form II March I996; accepted II March I996~
World Journal of Microbiology & Biotechnology, I/oi I2, 1996
40 7
Short Communication: Restriction fragment length polymorphism (RFLP) of Salmonella organisms
S.I. Koh-Luar,* S.T. Chew, E. Lau and S.B. Chua Genetic relatedness of 20 Salmonella isolates comprising 16 serotypes was analysed by restriction endonuclease digestion of the total DNA with six endonucleases individually. The rDNA fingerprints generated by EcoRI were more polymorphic, each serotype showed a unique fingerprint sharing several core (monomorphic) bands with several polymorphic bands. Eight characteristic Ncfl rDNA fingerprints were found. Similar rDNA RFLP patterns were observed in strains of Salmonella from different serotypes. Key words: Food pathogen, Salmonella serotypes, RFLP, 16S rDNA.
The genus Salmonella contains about 2000 different serotypes that can be transmitted between animals and humans, and all are pot:entially pathogenic to humans. With the exception of disease caused by Salmonella typhi, paratyphi A and paratyphi C, Salmonella infections are zoonoses. Salmonella typhi, paratyphi A and paratyphi C, which cause typhoid fever or enteric fever, are specific to humans and do not have a reservoir in animals. Advances in molecular biological techniques have provided yet another avenue for epidemiology and identification studies of many pathogens such as Mycobacterium, Vibrio and Candida. Unlike serotyping of specific surface antigens, nucleic acid-based techniques such as restriction fragment length polymorphism (RFLP) followed by hybridization with gene-specific probes (Knight et at. I990) and the polymerase chain reaction (PCR) (Stone et al. 1994) depend on the genotypes of the organisms concerned and not on their phenotypic expression. In this paper, we describe some preliminary observations on RFLP analysis of Salmonella isolates. We sought to find genetic relationships between strains of different serotypes which may be of value in monitoring trends in the prevalent Salmonella infections.
S.I. Koh-Luar and E. Lau are with the Department of Chemical Process & Biotechnology, Singapore Polytechnic, 500 Dover Road, Singapore 139651. Republic of Singapore; fax: (065) 7721976. S.T. Chew is with the Veterinary Public Health Laboratory (VPHL), Primary Production Department (PPD), 51 Jalan Buroh, Singapore 619415. Republic of Singapore. S.B. Chua is with the Veterinary Public Health Division, Primary Production Department, 5 Maxwell Road, No. 03-00, Tower Block, MND Complex, Singapore 069110. Republic of Singapore. * Corresponding author.
Materials and Methods Microorganisms and rDNA Probe Salmonella organisms used for DNA analyses were isolated at the Veterinary Public Health Laboratory, Primary Production Department according to the standard methods (US Food & Drug Administration 1992). I6S rDNA gene (as described by Edwards et al. I989) of Salmonella wandsworth synthesized by polymerase chain reaction was radioactively labelled with [~-32P]dCTP (Amersham) by the megaprime labelling technique (Amersham).
Genomic DNA Isolation All Salmonella strains were grown in LB medium on a rotary shaker at 37°C until late exponential phase. Cells were harvested by centrifugation and DNA was extracted by the phenol-chloroform method (Sambrook et al. 1989).
Restriction Enzyme Digestion and Gel Electrophoresis Three /lg of genomic DNA were digested separately with the restriction enzymes (2 units//~g) according to the supplier's specifications. Digested DNAs were separated by electrophoresis in a 0.8% agarose gel in 1 x TBE buffer.
Southern Hybridisation and Detection Gels were treated sequentially with 0.25 M HCL 0.5 m NaOH/ 1.5 M NaCl and 0.5 M Tris/HCl/1.5 M NaCl as described by Sambrook et al. (1989). The fractionated restriction fragments were quantitatively transferred to Hybond-N membranes (Amersham) by capillary transfer method and fixed by baking at 80°C for 2 h (Sambrook et aI. 1989). Salmonella wandsworth 16S rDNA was used as a probe in this work because the serotype appears to be the predominant serotype isolated. Southern hybridization and autoradiography were carried out according to Sambrook et al. (1989).
Q 1996 Rapid Science Publishers World Journal of M~crobiolagy ~ Biotechnology, Vol 12, 1996
405
5.I. Koh-Luar, 5.T. Chew, E. Lau and 5.B. Chua (A)
(B)
Figure 1. A representative photograph (A) and diagram (B) of the agarose gel (0.8%) of EcoRI rDNA fingerprints of Salmonellae. The arrows indicate the two constant bands which were found in all Salmonellae analysed in this study. Sizes of markers in kb are as indicated. S3--S. wandsworth; S4--S. hadar; $9---S. mbandaka; S10~S. indiana; S 1 2 ~ . anatam.
Figure 2. (A) An example of a typical Ncil rDNA fingerprints of: $29~S. agona; $ 3 2 ~ . amsterdam; $33~S. dublin; $36~S. havana and $ 3 7 ~ . weltevreden. (B) A schematic diagram of summarised Ncil rDNA RFLPs. Sizes of markers in kb are indicated. The arrows indicate the two constant bands found in all Salmonellae analysed in this study. Profile I ~ . indiana; Profile I 1 ~ . hadar, S. chester, S. senftenburg, S. lexington; Profile II1--~. dublin, S. wandsworth, S. anatam, S. weltevreden; Profile I V Y . blockley, S. mbandaka, S. agona; Profile V ~ . havana; Profile Vl--S. amsterdam; STM--S. typhimurium; S T Y . typhi.
Results and Discussion Genomic DNA of the Salmonella isolates was digested separately with six restriction enzymes (Bglll, PvulI, PstI, XhoI, EcoRI and NciI) that recognized different penta- and hexanucleotide sequences. Two enzymes, EcoRI and NciI, produced clearly well-resolved rDNA fingerprinting patterns within the range of the standard size markers. Agreement between independent isolates of the same serotype was excellent. Duplicate samples of the 20 strains gave reproducible fingerprints when probed.
406
World Journal of A/h'crobiology& Biotechnology, VoI 12, I996
Digestion with EcoRI generated an average of seven to eight fragments per strain ranging in size from 1.0 to 21.1 kb. Most strains displayed a common set of fragments at 1.8 (doublet) and 1.4 kb (Figure i). However, considerable variations between different strains were evident. The polymorphism appeared to be more marked for fragments of molecular weight from 6.0 to 21.1 kb. An average of five or six bands in the size range 1.0 to 2.4 kb were observed in NciI digested I6S rDNA (Figure 2). Two monomorphic bands of 1.5 and 2.2 kb, were seen for all Salmonella organisms in this study including S. typhimurium and S. typhi. However, two additional bands, of 0.9 and 1.4 kb, were also present in all non-typhi
RFLP of Salmonella Salmonella. The smallest 0.9 kb fragment was found in S. typhi but was absent from the other three isolates of S. typhimurium analysed. Conversely, the 1.4 kb fragment found in S. typhimurium and all other serovars, was absent from the genome of S. typhi. Further investigation will be carried out to identify the nature of this difference. Generally, EcoRI fragments are much larger than the Ncil fragments. Therefore, it is not surprising that the EcoRI fragments are more polymorphic particularly in the higher molecular weight range. This is due to the fact that large DNAs are more likely to undergo mutation than smaller ones. The additional or missing 16S rDNA bands in some strains suggest that mutation events such as gain or loss of restriction sites, insertion or deletion of bases may have taken place. The use of EcoRI restriction enzyme may be potentially applicable to the generation of 16S rDNA RFLPs for specific typing of Salmonella isolates since none of the 16 serotypes shared similar EeoRI RFLP fingerprints. Our 20 isolates comprising 16 serotypes gave eight different NciI RFLP profiles. All the non-typhi Salmonellae were closely related to S. typhimurium and S. typhi ( > 55%). At this stage, it may be premature to use this technique to characterise the Salmonella organisms unless a more comprehensive study of ribotyping or other fingerprinting techniques such as pulsed-field gel electrophoresis (PFGE) on all available Salmonella serotypes was carried out. More characterization of other non-typhi Sahnonella strains not listed here is needed before any possible taxonomic distinction between groups or subgroups could become evident. Nevertheless, we observed that S. hadar, S. chester, S. senftenburg and S, lexington share a common NciI profile (denoted profile II); S. dublin, S. wandsworth, S. anatam and S. weltevreden, exhibit profile III and S. blockley, S. mbandaka and S. agona, illustrate profile IV (see Figure 2B). Our studies clearly show that different serotypes of Salmonella can give similar 16S rDNA fingerprints which reflect the close genetic relationship of the serotypes. Tompkins et al. (i987) reported a close genetic relationship between some strains of Legionella pneumophila from different serogroups based on alloenzyme analysis. Likewise, Ketel (1988) observed similar DNA restriction endonuclease profiles in strains of L. pneumophila from different serogroups. Hence, it is not at all surprising to observe similar RFLP
patterns generated from different serotypes of Salmonella in the present study. These findings suggest that genotypes and phenotypes of organisms in environmental sources may not be as stable as we usually presume. The genetic similarities illustrated indicate that the organisms may have originated from the same clone. Strains originating from the same clone may acquire mutations in genes other than the rDNA genes, thus giving rise to common rDNA fingerprints even though they belong to different serotypes. Further investigations are necessary to confirm our hypothesis.
Acknowledgements This work was supported by the Singapore Polytechnic and the Primary Production Department, Singapore. We thank B.L. Koh for his critical review.
References Edwards, U., Rogall, T., Blocker, H., Emde, M. & B6ttger, E.C. 1989 Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. NucleicAcids Research 17, 7843-7853. Ketel, R.J. 1988 Similar DNA restriction endonuclease profiles in strains of Legionella pneumophila from different serogroups. Journal of ClinicalMicrobiology 26, 1838-1841. Knight, I.T., Shults, S., Kaspar, C.W. & Colwell, R.R. 1990 Direct detection of salmonella spp. in estuaries by using a DNA probe. Applied and Environmental]vlicrobiology56, 1059-1066. Sambrook, J., Maniatis, T. & Fritsch, E.F. 1989 Molecular Cloning:a Laboratory Manual, 2nd edition, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Stone, G.G., Oberst, R.D., Hays, M.P., McVey, S. & Chengappa, M.M. 1994 Detection of Salmonella serovars from clinical samples by enrichment broth cultivation-PCR procedure. Journal of ClinicalMicrobiology 32, 1742-I749. Tompkins, L.S., Troup, N.J., Woods, T., Bibb, W.F. & McKinney, R.M. 1987 Molecular epidermiology of Legionella species by restriction endonuclease and alloenzyme analysis. Journal of ClinicalMicrobiology 25, I875-1880. US Food & Drug Administration. 1992 Bacteriologicalanalytical manual, 7th edition. Washington, DC: Association of Official Analytical Chemists.
(Received in revised form II March I996; accepted II March I996~
World Journal of Microbiology & Biotechnology, I/oi I2, 1996
40 7
