Indian Journal of Biotechnology Vol 8, July 2009, pp 276-279
Differentiation of Staphylococcus aureus strains based on 16S-23S ribosomal RNA intergenic space polymorphism Ashok Dubey, S K Ghorui1 and S K Kashyap* College of Veterinary and Animal Science, Bikaner 334 001, India National Research Centre on Camel, Jorbeer, Bikaner 334 001, India
1
Received 29 January 2008; revised 22 December 2008; accepted 10 March 2009 Discrimination of Staphylococcus aureus strains, isolated from camel abscesses and mastitic milk of camel, cattle and goats, on the basis of 16S-23S ribosomal RNA intergenic space polymorphism was carried out. Two sets of primers were used for amplification of DNA of intergenic space; the one having a highly conserved sequence in eubacterial 23S rRNA transcript, while the other having less conserved sequence of 16S rRNA, reported earlier by other workers. Of the two sets of primers used, amplification could be achieved with one set of primers. Of 60 strains of S. aureus tested, amplification could be achieved in only 18 strains. In these strains the most frequent bands of DNA were of 350, 500, 750 and 1500 base pairs. Polymorphism was noted in the number of the rRNA transcripts and size of the 16S-23S rRNA intergenic space, as evident by variable band pattern in different strains of S. aureus. Keywords: Ribosomal RNA, 16S-23S rRNA intergenic space, Staphylococcus aureus
Introduction: Staphylococcus aureus is an important pathogen associated with several pyogenic conditions in man and animals. It is the main pathogen causing mastitis in all milch animals leading to great economic losses. The pathogenic potential of different strains of S. aureus varies greatly owing to colonization to different tissues and host species or adaptation to varying microenvironment. The pathogenic strains are known to be capable of producing more than 30 extracellular enzymes and toxins. Ability to produce coagulase has been considered as the primary criterion to differentiate pathogenic and relatively non pathogenic strains1. Biotyping, serotyping and bacteriophage typing methods have been used for discrimination of different strains or isolates of S. aureus. Of late, molecular typing methods, such as, genomic hybridization, plasmid characterization, detection of ribosomal RNA homology, etc., have been employed, and were found to be of great value in discrimination of otherwise homogeneous strains. Strain discrimination of bacteria has been found useful in epidemiological studies mainly in determining clonal relationship of the bacterial isolates and tracing the origin and course of spread of _____________ *Author for correspondence: Tel: 91-151-2544158; Fax: 151-2549348 E-mail:
[email protected]
the organism in a population2. The present study was envisaged to discriminate coagulase producer (CPS) and coagulase non-producer (CNS) strains of S. aureus isolated from abscess in camel and mastitic milk of goats, cattle and camel on the basis of their intergenic space polymorphism between 16S and 23S ribosomal RNA transcripts amplified using polymerase chain reaction. In this technique, primers are designed such that they bind to certain conserved sequence in the 16S rRNA and 23S rRNA coding regions. The PCR products formed on amplification, thus, consist of the intergenic spacer region. The length of the spacer region can vary in different copies of the rRNA operon within the same chromosome. The internal spacer region is believed to be under least selection pressure and, thus, may have variation in length and sequence within the organisms of the same genus and the same species3. Hence, after electrophoresis, more than one band may be obtained but in a particular strain it will be constant. The variation in the number and length of the spacer DNA among different isolates can be exploited to differentiate bacterial strains of the same genus or the species4. Materials and Methods Isolation and Identification of S. aureus
A total of 60 S. aureus isolates were cultured and identified on the basis of established morphological
DUBEY et al.: RIBOSOMAL RNA INTERGENIC SPACE POLYMORPHISM IN S. AUREUS
and biochemical procedures5, and grouped based on the source of isolation, i.e., camel abscess, camel mastitic, goat mastitic, and cattle mastitic milk samples, 15 in each group. Ability to produce coagulase was determined by tube test employing human plasma. In each group, 10 isolates were chosen as coagulase producer (CPS) and 5 as coagulase nonproducer (CNS). Determination of 16S-23S rRNA Intergenic Space Polymorphism
Primers—The primers used for amplification of 16S-23S ribosomal RNA intergenic space were having the following pairs with sequence as described earlier3: Primer set 1
Primer set 2
16S F- 5’ GGATACCAGAAGTAGGGT 3’ 23S R- 5’ CCAAGGGCATCCACCGT 3’ 23S R- 5’ CCAAGGGCATCCACCGT 3’ 16S F- 5’ TCGGTTTTGTGAGATTGGC 3’
(18 bases)
P1
(17 bases)
P2
(17 bases)
P2
(19 bases)
P3
Of these, primer P2 (common in both sets) was selected from highly conserved region of 23S rRNA of E. coli and is known to be conserved among eubacteria. P1 and P3 primers were chosen from relatively less conserved regions of 16S rRNA of Neisseria gonorrhoeae. Isolation of DNA—Bacterial cultures were grown in Luria Bartani broth and DNA was separated by following the established method6. The quality of DNA was checked by electrophoresis. Amplification Conditions—Varying conditions of annealing temperature, Mg++ ion and primer concentrations were employed for amplification. Annealing temperatures varied from 45o to 55.4oC in gradient manner, Mg++ ion concentrations 1.5, 2.0, 2.5, 3.0 and 3.5 μM, and primer concentrations varied from 0.1, 0.2, 0.3, 0.4 and 0.5 μM. dNTPs 2 mM and Taq polymerase 1.5 units were used in the reaction. Results Isolation and Identification of Bacteria
S. aureus strains were identified on the basis of primary and secondary tests based on morphological and biochemical properties standard for it5. 16S-23S rRNA Intergenic Space Polymorphism
Good quality DNA was obtained. Amplification was optimized at annealing temperature 55oC, MgCl2
277
concentration 1.5-3.5 μM and primer concentration 0.3-0.4 μM. Amplification of DNA was achieved in conditions of PCR optimized as specified above with primer set 2. All attempts to amplify the intergenic space with primers pair set 1 failed at all annealing temperatures and concentrations of MgCl2. With primer pair set 2, the DNA was amplified only from 18 samples, out of total 60 samples. In remaining isolates no DNA band was observed despite similar conditions of DNA isolation and amplification. The amplified DNA bands from the intergenic space of strains show that these strains vary in terms of the number and pattern of the bands (Table 1; Figs 1a & b). DNA band of 350 bp in 6 isolates, bands of 500 and 1500 bp in 4 isolates, band of 650 bp in 3 isolates, and bands of 700 and 1000 bp in 2 isolates were amplified, while DNA bands of 320, 550 and 850 bp were amplified only in one isolate. Maximum number of bands, i.e, 5 appeared in one isolate, whereas 3 bands appeared in one isolate, 2 bands in two isolates and only one band in 14 isolates. It was also observed that isolates 3, 10, 28 and 33 showed similar band pattern (single band of 350 bp) and, thus, may belong to one clone. Isolates 32, 37, 50 and 51 also showed similar band pattern (single band at 500 bp) and may be clonally related. Similarly, isolates 17 and 34 (single band of 650 bp) and isolates 2 and 36 (single band of 600 bp) might belong to other clones. Discussion A number of procedures for comparison of bacterial isolates are being recognized in the investigations of strain origin, clonal relatedness among strains and epidemiology7. In the present investigation, 16S-23S rRNA intergenic space polymorphism allowed discrimination of S. aureus strains isolated from camel, cattle and goat appearing similar in many phenotypic characteristics. The primer set 1, which was used for the amplification of eubacterial strains (M.M.), did not result in any amplification in any of the isolates. It could have been due to non availability of the sequence in S. aureus as one of the primers in set 1 consisted of less conserved region of N. gonorrhoeae. With primer set 2 also, the amplification was achieved in only 18 samples, out of total 60 samples attempted from the same DNA samples in empirically determined optimum condition of temperature and Mg++ ions (Figs 1a & b) indicating that this sequence was shared by only few S. aureus isolates.
INDIAN J BIOTECHNOL, JULY 2009
278
In total, 26 DNA bands of varying size appeared at different positions in 18 isolates of S. aureus after PCR amplification (Table 1). Frequency distribution of different bands shows that majority of bands that appeared had base pairs about 350, 500, 600, 650, 700 and 1500 bp designated as band I, H, F, E, and A, respectively. These results indicate that 4 clones of S.
Fig. 1 (a-b)—PCR amplified 16S-23S rRNA intergenic space band pattern of S. aureus: a. Bovine and caprine isolates; b. Camel wound and milk isolates.
aureus are in circulation in the camel, cattle and caprine population of this area. Clone I includes isolate 3, 10, 33 and 28, which were associated with camel wound (3 & 10), camel milk (33) and bovine milk (28). Clone II consists of isolates 32, 37, 50 and 51, which were associated with camel milk (32 & 37) and bovine milk (50 & 51). Clone III consists of the isolates 2 and 36 that were associated with camel wound (2) and camel milk (36); and clone IV has isolates 17 and 34 associated with caprine milk and camel milk, respectively. Remaining isolates of S. aureus displayed different patterns of ribosomal transcript intergenic space and, thus, may not be genetically related. However, some of these strains do show one or two bands at similar positions. The isolate 8 showed 5 bands indicating many copies of ribosomal transcripts with some bands at positions also shared by isolate 26. On comparing CNS isolates from CPS isolates, it was observed that each clone consists of CPS as well as CNS isolates indicating no clear band pattern difference and correlation between coagulase production and length of ribosomal rRNA intergenic space. It is also obvious from above results that the instances of similar band patterns in the S. aureus strains of different host origin are very few considering all the strain put together, which is indicative of modifications in the process of adaptation into different conditions of a host species. The present findings are in accordance with the earlier reports in which polymorphism in the spacer region between 16S and 23S rRNA gene was used in the typing S. aureus and this method was found to provide a good discrimination between unrelated isolates8. In an earlier study of ribotyping-PCR, 5 to 8 bands in the range of approximately 650-1100 bp were generated in S. aureus strains and 11 isolates could be classified into seven ribotypes (R1-R7)9.
Table 1—Schematic representation of PCR amplified 16S-23S rRNA intergenic space band pattern of S. aureus isolates (Not to scale) Amplified band in bp 1500-A 1100-B 850-C 700-D 650-E 600-F 550-G 500-H 350-I 320-J
2 cps
3 cns
4 cps
8 cns
▬
▬ ▬ ▬ ▬
10 cps
32 cps
33 cns
34 cps
35 cps
36 cps
37 cps
59 cns
51 cps
50 cps
28 cns
▬
26 cns ▬ ▬
▬
▬
▬
17 cps
16 cps ▬
▬
▬ ▬ ▬ ▬
▬
▬ ▬
▬ ▬
▬
▬ ▬
▬
DUBEY et al.: RIBOSOMAL RNA INTERGENIC SPACE POLYMORPHISM IN S. AUREUS
Similarly, in another study significant variation in the sequences of 16S-23S rRNA spacer region was recorded for nine bacterial species, which cause mastitis with the lengths of the spacers varying from 240 to 461 bp. Between genera the spacers shared only short conserved regions (8-9 bp) and within genera the sequence identities varied from 53 to 85%10. In investigations to determine whether 16S23S rDNA spacer length polymorphism was suitable for identification of Staphylococcus strains, it was observed that each species represented a specific 16S23S pattern made of 1-8 fragments ranging from 104 to 771 bp and that S. aureus was the most heterogeneous species (five different pattern for eight strains) 11. Host specific band pattern could not be recorded in the present investigation as was observed in an earlier study in which twelve 16S-23S ribosomal DNA internic/intergenic band types were found among 57 S. intermedius isolates and amongst them six ITS-PCR types were host specific, while most human and canine strains belonged to same types. Pigeon, horse and mink strains, however, appeared more heterogeneous12. Polymorphism has also been observed in the sequence of the 16S-23S rRNA spacer region of S. aureus strains along with polymorphism in the number of bands with range of length of bands 160 to 1100 bp, predominant being from 460 to 50013. It can, thus, be concluded that 16S-23S rDNA intergenic space analysis is a useful tool for identification when there are so many isolates to deal with and appears to be a rapid and reliable method as has been found earlier in case of classifying bovine staphylococcal isolates at species and subspecies levels14. Acknowledgement The facilities provided by Director, NRC on Camel, Bikaner during the course of study is acknowledged with thanks.
2
3
4
5
6
7
8
9
10
11
12
13
References 1
Thakker M, Park J S, Carey V & Lee J C, Staphylococcus aureus serotype 5 capsular polysaccharide is anti-phagocytic and enhances bacterial virulence in a marine bacteremia model, Infect Immun, 66 (1998) 5183-5189.
14
279
Sleander R K, Korhonen T K, Vaisanen Rhem V, William P H, Pattison P E et al, Genetic relationship and clonal structure of strains of Escherichia coli in neonatal septicemia and meningitis, Infect Immun, 52 (1986) 213-222. McLaughlin G L, Howe D K, Biggs D R, Smith A R, Ludwinski P et al, Amplification of rDNA loci to detect and type Neisseria meningitidis and other eubacteria, Mol Cell Probes, 7 (1993) 7-17. Kostman J R, Edlind T D, Lipuma J J & Stull T L, Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reaction: Ribotyping, J Clin Microbiol, 30 (1992) 2084-2087 Quinn P J, Carter M E, Markey B K & Carter G R, Clinical veterinary microbiology (Wolfe Publishing, London) 1994, 118-126. Sambrook J, Fristch E F & Maniatis T, Molecular cloning: A laboratory manual (Cold Spring Habour Laboratory Press, USA) 1990, 121-152. Van Belkum A, Kluytmans J, van Leeuwen W, Bax R, Quint W et al, Multicenter evaluation of arbitary primed PCR for typing of Staphylococcus aureus strains, J Clin Microbiol, 33 (1995) 1537-1547. Dolzani L, Tonil E, Lagatolla C & Monty-Bargadin C, Typing of Staphylococcus aureus by amplification of the 16S-23S rRNA intergenic spacer sequences, FEMS Microbiol Lett, 119 (1994) 167-173. Pererira M S V, Leal N C, Leal T C A, Sobreira M, De Almedia A M P et al, Typing of human and bovine Staphylococcus aureus by RAPD-PCR and ribotyping-PCR, Lett Appl Microbiol, 35 (2002) 32-37. Forsman P, Tilsala-Timisiarvi A & Alafossava T, Identification of staphylococcal and streptococcal causes of bovine mastitis using 16S-23S rRNA spacer reason, Microbiology, 143 (1997) 3491-3500. Mendoza M, Meugnier H, Bes M, Etienne J & Freney J, Identification of Staphylococcus speices by 16S-23S rDNA entergenic spacer PCR analysis, Int J Syst Bacteriol, 48 (1998) 1049-1055. Bes M, Slimsaidi L, Decharnia F, Meugnier H, Vandenesch F et al, Population diversity of Staphylococcus intermedius isolates from various host species: Typing by 16S-23S intergenic ribosomal DNA spacer polymorphism analysis, J Clin Microbiol, 40 (2002) 2275-2277. Kim K, Yoon Y, Kim K & Yoon Y, Identification of Staphylococcus aureus on length and sequence polymorphism of 16S-23S rRNA spacer region and the comparison of sequence alignment, J Anim Sci Technol, 43 (2001) 545-552. Bes A M, Gwrin-Fabulee V, Meugnier H, Etienne J & Freney J, Improvement of the identification of staphylococci isolated from bovine mammary infection using molecular methods, Vet Microbiol, 71 (2000) 287-294
