Abstract
The use of rrs (16S rRNA) gene is widely regarded as the “gold standard” for identifying bacteria and determining their phylogenetic relationships. Nevertheless, multiple copies of this gene in a genome is likely to give an overestimation of the bacterial diversity. In each of the 50 Streptococcus genomes (16 species, 50 strains), 4–7 copies of rrs are present. The nucleotide sequences of these rrs genes show high similarity within and among genomes, which did not allow unambiguous identification. A genome-wide search revealed the presence of 27 gene sequences common to all the Streptococcus species. Digestion of these 27 gene sequences with 10 type II restriction endonucleases (REs) showed that unique RE digestion in purH gene is sufficient for clear cut identification of 30 genomes belonging to 16 species. Additional gene-RE combinations allowed identification of another 15 strains belonging to S. pneumoniae, S. pyogenes, and S. suis. For the rest 5 strains, a combination of 2 genes was required for identifying them. The proposed strategy is likely to prove helpful in proper detection of pathogens like Streptococcus.
Electronic supplementary material
The online version of this article (doi:10.1007/s12088-015-0561-5) contains supplementary material, which is available to authorized users.
Keywords: Biomarkers, Diagnosis, Genome, In silico, Restriction endonuclease, Streptococcus
Introduction
Streptococcus species cause severe diseases such as bacteremia, pneumonia, meningitis, and otitis media. Since these are responsible for high morbidity and mortality rates, clinicians and microbiologists have been constantly struggling to develop assays to identify them [1, 2]. The diseases caused by these pathogens may assume an epidemic dimension, if their growth is unchecked. The process of identification has progressed dramatically from conventional biochemical assays to molecular techniques [3]. Yet, in spite of a large number of identification strategies being developed in the past few decades, there seems to be no universal procedure and characteristic, which can be used to target all the bacteria. Evaluation of pathogens belonging to the genus Streptococcus has been highly problematic and hampered due to: (1) the close phylogenetic relationship among its species, and (2) sharing of phenotypic traits through horizontal gene transfer [4]. These events blur the boundaries and lead to difficulties in unequivocal identification of bacterial isolates [5]. There is a lack of sensitivity and specificity of the identification assays. Efforts to improve the quality of these assays have been made using diverse characteristics. The tube bile solubility test is widely employed for the identification of Streptococcus pneumoniae [6]. The major limitations are time consuming and difficulties in interpreting the results. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is another tool, which has also been proving quite effective in bacterial identification [3, 7–9]. This procedure has been reported to be not very effective in closely related Streptococcus species.
The reliability and reproducibility of molecular techniques, especially amplification of genes has taken precedence over culture based methods. The gene most widely used for identifying bacteria is the house keeping gene (HKG) −16S rRNA (rrs), which is highly conserved among all prokaryotes. The full length sequence of rrs of Streptococcus spp.: S. pneumoniae, S. pseudopneumoniae, S. mitis, and S. oralis show more than 99 % similarity leading to unsuccessful or misleading results [10]. Recent studies have elucidated certain latent but unique characteristics in rrs: (1) 30–50 nucleotides (nts) long signatures, and (2) restriction endonuclease (RE) digestion patterns [12–16]. These features enable easy identification of organisms up to the species level. However, rrs gene does not prove helpful in the following scenarios: (1) in phylogenetically very closely related strains, which differ in less than 1 % nts along their length, and (2) in strains with multiple copies of this gene in each genome [17–20]. An obvious solution to circumvent this problem is to use other HKGs [13, 21]. Streptococcus poses a big challenge as most pathogenic strains are genetically very close to each other and have multiple copies of rrs per genome. A few genes which are frequently used for the identification of Streptococcus include genes: cpsA, gdh, groESL, lytA, ply, psaA, pspA, recA, recN, rpoA, rpoB, sodA, tuf, wzg, 16S-23S ribosomal DNA spacer region, and the DNA fragment Spn9802 [3, 4, 11, 22–31]. Optochin susceptibility testing (CO2 atmosphere) in combination with amplification of Spn9802 fragment and the autolysin genes: lytA, rpoB, and tuf, have proved effective in identifying S. pneumoniae [32]. It must be realized that the most idyllic rrs gene is offering no escape as human beings are struggling to reach a consensus, while pathogenic bacteria are threatening our long-term survival. Although, multilocus sequence analysis (MLSA) has been relatively quite successful in the identification of a wide range of bacterial species [33], however, using sequences of 7 HKGs: guaA, map, pfl, ppaC, pyk, rpoB, and sodA, also did not prove suitable for rapid identification of Streptococcus species [21]. MLSA using gdh, ddl along with guaA, map, pfl, ppaC, pyk, rnpB, rpoB, sodA, and tuf have been useful in identifying Streptococcus species [32, 34, 35].
Although many molecular techniques are available, however, they display variable specificity and reliability, and thus continue to be inconvenient for routine diagnostic assays. There is an urgent necessity to look for a procedure, which can discriminate Streptococcus species with higher precision than the existing ones. It must be based on certain genes with unique and easily identifiable characteristics. It should have the strength to be implemented in routine diagnostic procedures; otherwise the rapid escalation in expenditures on health may lead towards an otherwise avoidable economic collapse.
We have selected genes common to all the species of Streptococcus and systematically identified unique RE digestion patterns. These gene-RE combinations have the potential for being used in diagnosis of Streptococcus even among a large population of distantly or closely related organisms.
Materials and Methods
Sequence Data and Comparative Genome Analysis
Completely sequenced genomes of the 50 strains of 16 species belonging to genus Streptococcus were retrieved (http://www.ncbi.nlm.nih.gov/): S. agalactiae, S. dysgalactiae, S. gordonii, S. intermedius, S. macedonicus, S. mitis, S. mutans (2 strains), S. oralis, S. parasanguinis, S. pasteurianus, S. pneumoniae (20 strains), S. pyogenes (7 strains), S. salivarius, S. sanguinis, S. suis (9 strains), and S. uberis (Table S1). Characteristics of the Streptococcus genomes such as Accession number, GC percentage, size, and number of genes has been presented (Table S1). Based on GenBank (Full) data of Streptococcus genomes, we could trace 27 common genes, varying in size from 471 to 2514 nucleotides (nts; Tables S1 and S2). Gene, rrs was also taken into consideration. Sequence analysis and their orientation were done using BioEdit [36].
Restriction Endonuclease Analysis of Common Genes
A total of 10 Type II REs were considered for digestion on the basis of our previous works [18–20]. The following REs were used: (1) 4 base cutters AluI (AG’CT), BfaI (C’TA_G), BfuCI (_GATC’), CviAII (C_AT’G), HpyCH4V (TG’CA), RsaI (GT’AC), TaqI (T_CG’A), Tru9I (T_TA’A), and (2) 6 base cutters HaeI (WGG’CCW), Hin1I (GR_CG’YC). Cleaver (http://cleaver.sourceforge.net/) was used to get RE digestion patterns of the 27 common gene sequences (Table S2). Data matrices of REs producing 5–15 fragments were taken into consideration for further analysis [18–20].
Results
In Silico RE Digestion Analysis of rrs Gene
The genomes of Streptococcus strains have 4–7 copies of rrs gene. The different copies of rrs within a genome are exactly similar among themselves in 44 out of 50 sequenced genomes. Multiple sequence alignments of 217 copies of rrs belonging to 50 Streptococcus genomes resulted in segregating them into 40 different groups. The number of rrs copies in each group varied from 1 to 20 copies such that 150 copies cannot be distinguished from each other. On the other hand, in silico digestion of rrs gene sequences with 10 REs, allowed us to identify certain unique RE digestion patterns. The RE digestion patterns of all the rrs copies were unique to a strain (but exactly similar to each other), in the cases of: (1) S. agalactiae 2603V/R, (2) S. dysgalactiae subsp. equisimilis RE378, (3) S. intermedius JTH08, (4) S. pasteurianus ATCC 43144, (5) S. pneumoniae R6, (6) S. pneumoniae TCH8431/19A, (7) S. pneumoniae 670-6B, (8) S. pyogenes MGAS15252, (9) S. salivarius 57.I, (10) S. suis TL13, (11) S. suis TL15, and (12) S. uberis 0140J. This analysis revealed that 12 genomes belonging to 9 Streptococcus species can be easily distinguished because of the unique digestion pattern of their rrs gene copies with REs—AluI, BfaI, BfuCI, CviAII, HpyCH4V, RsaI, TaqI, and Tru9I (Table 1). In the cases of S. gordonii str. Challis substr. CH1, S. parasanguinis FW213, S. pneumoniae CGSP14, S. pneumoniae SPN034156, S. pyogenes MGAS9429, S. pyogenes NZ131, S. sanguinis SK36, and S. suis D12, the 4–6 copies of rrs could be segregated into two groups. However, only one of the two groups in the latter set of genomes had unique RE digestion patterns (Table 1). It is concluded that rrs may not prove as a very good candidate gene for identifying Streptococcus species in an unambiguous manner. It indicates that there is a need to look for other genes, which may be highly conserved and should have certain latent features like unique RE digestion patterns and prove helpful in deriving useful information.
Table 1.
In silico restriction endonuclease (RE) digestion patterns (5′–3′) of rrs genes of Streptococcus strains
| Streptococcus spp. | GenBank ID | Copies of rrs | Unique RE digestion pattern |
|---|---|---|---|
| RE-AluI | |||
| S. gordonii str. Challis substr. CH1 | CP000725.1 | 4 | 74•90•86•186•429•207•207•231 |
| S. pyogenes MGAS9429 | CP000259.1 | 6 | 122•429•207•207•266 |
| S. pyogenes MGAS15252 | CP003116.1 | 5 | 149•86•186•429•207•207•328•5•232 |
| S. pyogenes NZ131 | CP000829.1 | 6 | 63•86•186•429•207•207•157 |
| S. salivarius 57.I | CP002888.1 | 6 | 74•86•86•186•56•373•207•207•261 |
| S. uberis 0140 J | AM946015.1 | 4 | 160•86•186•429•161•46•207•269 |
| RE-BfaI | |||
| S. pasteurianus ATCC 43144 | AP012054.1 | 5 | 79•52•89•27•578•186•137•195•194 |
| S. pneumoniae CGSP14 | CP001033.1 | 4 | 206•577•186•137•195•111 |
| S. pyogenes MGAS9429 | CP000259.1 | 2/6 | 515•186•137•195•198 |
| 4/6a | 236•578•186•137•195•314•67•116 | ||
| RE-BfuCI | |||
| S. parasanguinis FW213 | CP003122.1 | 4 | 213•119•931•166 |
| S. pneumoniae 670-6B | CP002176.1 | 4 | 246•119•932•174•8 |
| S. pneumoniae R6 | AE007317.1 | 4 | 4•294•119•932•165 |
| S. pneumoniae TCH8431/19A | CP001993.1 | 4 | 7•294•119•932•174•8 |
| S. pyogenes MGAS9429 | CP000259.1 | 2/6 | 112•932•175•12 |
| 4/6a | 292•119•932•175•120•191 | ||
| S. salivarius 57.I | CP002888.1 | 6 | 7•296•119•412•520•174•8 |
| RE-CviAII | |||
| S. dysgalactiae subsp. equisimilis RE378 | AP011114.1 | 5 | 53•140•289•203•269•106•148•125•217 |
| S. parasanguinis FW213 | CP003122.1 | 4 | 97•492•268•106•148•125•193 |
| S. pneumoniae 670-6B | CP002176.1 | 4 | 130•492•269•106•148•125•209 |
| S. pneumoniae SPN034156 | FQ312045.1 | 1/4 | 29•138•492•269•106•150•33•90•127 |
| 3/4a | 29•138•492•269•106•148•125•127 | ||
| S. pyogenes MGAS9429 | CP000259.1 | 6 | 369•269•106•148•125•214 |
| S. pyogenes MGAS15252 | CP003116.1 | 5 | 36•140•492•269•106•148•125•436•77 |
| S. suisTL15 | CP006246.1 | 4 | 47•142•492•269•106•148•125•210 |
| S. uberis 0140 J | AM946015.1 | 4 | 47•123•17•492•269•106•148•125•217 |
| RE-HpyCH4 V | |||
| S. dysgalactiae subsp. equisimilis RE378 | AP011114.1 | 5 | 56•28•130•6•443•396•262•229 |
| S. gordonii str. Challis substr. CH1 | CP000725.1 | 1/4 | 46•144•28•443•396•262•191 |
| 3/4a | 46•144•22•6•443•396•262•191 | ||
| S. pneumoniae CGSP14 | CP001033.1 | 1/4 | 11•134•22•6•443•88•308•262•139 |
| 3/4a | 11•134•22•6•443•395•262•139 | ||
| S. pyogenes MGAS9429 | CP000259.1 | 2/6 | 347•396•262•226 |
| 4/6a | 39•28•579•396•262•313•125•87 | ||
| S. pyogenes NZ131 | CP000829.1 | 2/6 | 560•396•262•117 |
| 4/6a | 51•28•579•396•262•117 | ||
| S. suis D12 | CP002644.1 | 1/4 | 78•579•396•262•222 |
| 3/4a | 50•28•579•396•262•222 | ||
| S. suis TL13 | CP003993.1 | 4 | 47•28•108•471•396•262•230 |
| RE-RasI | |||
| S. intermedius JTH08 | AP010969.1 | 4 | 389•405•143•212•146•40 |
| S. parasanguinis FW213 | CP003122.1 | 4 | 539•262•355•146•128 |
| S. pyogenes MGAS9429 | CP000259.1 | 2/6 | 319•262•143•212•146•149 |
| 4/6a | 618•262•143•212•146•448 | ||
| S. pyogenes NZ131 | CP000829.1 | 2/6 | 532•262•143•212•146•40 |
| 4/6a | 631•262•143•212•146•40 | ||
| S. uberis 0140 J | AM946015.1 | 4 | 183•8•700•143•212•146•152 |
| RE-TaqI | |||
| S. parasanguinis FW213 | CP003122.1 | 1/4 | 672•199•559 |
| 3/4 | 88•584•198•559 | ||
| S. pneumoniae 670-6B | CP002176.1 | 4 | 705•199•575 |
| S. pyogenes MGAS9429 | CP000259.1 | 2/6 | 452•199•580 |
| 4/6a | 751•199•687•192 | ||
| S. pyogenes NZ131 | CP000829.1 | 1/6 | 8•658•199•471 |
| 5/6a | 764•199•583 | ||
| S. suis D12 | CP002644.1 | 1/4 | 82•196•484•199•576 |
| 3/4a | 278•484•199•576 | ||
| RE-Tru9I | |||
| S. agalactiae 2603 V/R | AE009948.1 | 7 | 181•18•394•12•14•251•86•134•44•150•223 |
| S. dysgalactiae subsp. equisimilis RE378 | AP011114.1 | 5 | 1•186•10•8•394•12•265•86•134•44•150•260 |
| S. intermedius JTH08 | AP010969.1 | 4 | 102•242•48•116•41•224•86•134•342 |
| S. parasanguinis FW213 | CP003122.1 | 4 | 515•14•27•223•86•134•194•236 |
| S. pasteurianus ATCC 43144 | AP012054.1 | 5 | 181•409•15•14•26•225•86•134•194•253 |
| S. salivarius 57.I | CP002888.1 | 6 | 605•265•86•134•194•252 |
| S. sanguinis SK36 | CP000387.1 | 1/4 | 2•200•410•14•27•224•86•134•454 |
| 3/4 | 2•610•14•27•224•86•134•454 | ||
| S. uberis 0140 J | AM946015.1 | 4 | 199•406•14•251•86•134•44•150•260 |
Symbol (•) indicates RE site in the gene sequences
aThis pattern is not unique. It has been presented to indicate the RE digestion pattern of the rest of the rrs copies
In Silico RE Digestion Analysis of Common Genes
In this analysis, we have identified 27 genes (in addition to rrs), common to all the 50 sequenced genomes of Streptococcus. These 27 genes varied from 471 to 2514 nts, in size (Tables S2). In silico digestion of 27 genes with all the REs revealed unique features, on the basis of which the Streptococcus genomes can be easily distinguished. Although all the 27 genes proved to have certain unique features, however, only 7 genes, namely purH, dnaA, dnaK, fabG, mraY, purK and pyrH can be recommended for usage in identification process.
purH
In silico digestion of purH gene (1548 nts) was observed with 8 REs: AluI, BfaI, BfuCI, CviAII, HpyCH4V, RsaI, TaqI, and Tru9I. REs—AluI, BfuCI, CviAII, TaqI and Tru9I were effective in providing unique digestion patterns in purH gene present in 18–21 genomes. Together these gene-RE combinations encompassed 30 genomes, which represented all the 16 species used in this study. The unique feature of this gene is that the number of RE sites varied from 2 to 6 and exceptionally it varied up to 13. The fragment sizes ranged from 18 to 500 nucleotides (nts) in most of the cases and exceptionally up to 1100 nts (Table 2a, b).
Table 2.
Unique in silico restriction endonuclease digestion patterns (5′–3′) of purH gene of Streptococcus genomes
| Streptococcus spp. | Restriction endonucleases | |||
|---|---|---|---|---|
| AluI | BfaI | BfuCI | CviAII | |
| (a) | ||||
| S. agalactiae 2603 V/R | 400•42•75•57•138•8•148•21•63•66•60•18•452 | 416•63•536•360•173 | 891•384•158•81•34 | – |
| S. dysgalactiae subsp. equisimilis RE378 | 223•33•20•352•30•291•426•170 | 1012•360•173 | 282•150•360•96•579•78 | 409•421•112•556•47 |
| S. gordonii str. Challis | 403•72•99•29•28•89•130•18•84•46•20•144•72•255•59 | –a | 83•151•1119•195 | 412•869•231•36 |
| S. intermedius JTH08 | 403•138•141•186•84•66•63•345•15•107 | – | 719•183•531•115 | 211•36•21•144•413•8•448•207•60 |
| S. macedonicus ACA-DC 198 | – | – | 285•150•906•207 | 211•201•413•8•448•231•36 |
| S. mitis B6 | 259•20•295•146•232•46•215•21•114•141•59 | – | 1275•184•89 | 412•221•192•8•131•302•282 |
| S. mutans LJ23 | 406•138•179•148•84•66•63•345•15•107 | – | 905•451•195 | 214•36•21•144•413•8•448•267 |
| S. mutans UA159 | 418•72•66•179•148•84•66•63•153•114•78•15•107 | – | 917•451•195 | 15•211•36•21•144•413•8•448•267 |
| S. oralis Uo5 | 541•33•294•84•46•164•386 | – | 285•974•289 | 633•192•8•131•317•231•36 |
| S. parasanguinis FW213 | 406•279•168•102•46•61•289•200 | 253•166•666•466 | 557•19•45•158•63•75•528•72•34 | 828•8•131•317•231•36 |
| S. pasteurianus ATCC 43144 | – | – | 285•150•1035•78 | 211•201•413•456•231•36 |
| S. pneumoniae CGSP14 | – | – | 456•456•134•328•195 | 433•413•8•448•231•36 |
| S. pneumoniae G54 | 279•295•146•232•46•20•144•386 | – | – | 412•221•192•8•448•267 |
| S. pneumoniae Hungary19A-6 | – | – | 435•590•328•17•178 | – |
| S. pneumoniae INV200 | – | – | 435•456•134•328•195 | – |
| S. pneumoniae TCH8431/19A | – | – | 456•590•328•195 | 268•119•438•390•273•60 |
| S. pyogenes A20 | 279•238•203•169•129•60•18•452 | 158•258•599•360•173 | – | – |
| S. pyogenes MGAS5005 | 309•238•203•169•129•60•18•422 | 7•181•258•599•360•143 | – | 855•8•448•220•11•6 |
| S. salivarius 57.I | 403•135•36•138•8•130•18•21•63•46•164•51•21•114•141•59 | 416•666•466 | 1459•55•34 | 211•614•8•448•220•47 |
| S. sanguinis SK36 | 78•7•372•75•24•62•18•99•148•84•46•20•63•81•267•119 | 130•900•533 | 32•418•138•222•96•462•161•34 | 15•412•221•879•36 |
| S. suis D12 | 259•450•26•263•20•351•179 | – | 435•699•414 | 268•119•828•273•24•36 |
| S. suis ST1 | 72•184•235•50•168•26•154•129•195•213•122 | – | 435•360•339•219•195 | 268•557•687•36 |
| S. suis T15 | 491•50•168•180•129•530 | – | 117•318•438•261•414 | 268•119•438•52•338•273•60 |
| S. suis TL13 | 491•50•168•26•133•150•408•122 | – | – | – |
| S. uberis 0140 J | 355•48•105•9•24•120•207•84•46•20•60•81•54•108•27•81•12 | 289•127•44•516•39•178•167•19•169 | 435•918•195 | 211•201•413•676•47 |
| Streptococcus spp. | Restriction endonucleases | |||
|---|---|---|---|---|
| HpyCH4 V | RsaI | TaqI | Tru9I | |
| (b) | ||||
| S. agalactiae 2603 V/R | –a | – | 146•222•972•208 | 16•172•306•129•303•321•298•3 |
| S. dysgalactiae subsp. equisimilis RE378 | 10•108•131•148•159•201•203•585 | – | 750•185•610 | 61•124•126•408•21•48•27•429•189•109•3 |
| S. gordonii str. Challis | 198•361•147•257•168•417 | 1243•130•175 | 91•25•123•36•93•609•507•64 | 743•183•622 |
| S. intermedius JTH08 | 943•20•93•75•417 | – | – | – |
| S. macedonicus ACA-DC 198 | 13•246•300•210•194•28•411•24•122 | – | 91•1057•400 | 299•254•160•30•75•108•619•3 |
| S. mitis B6 | 349•6•204•105•42•257•28•140•417 | – | 91•55•138•84•320•172•117•363•21•123•64 | 16•49•123•126•296•112•204•312•307•3 |
| S. oralis Uo5 | – | 1243•85•220 | 91•55•213•9•492•195•429•64 | 16•727•802•3 |
| S. parasanguinis FW213 | 667•299•585 | – | 149•93•36•93•492•117•507•64 | 332•819•288•109•3 |
| S. pasteurianus ATCC 43144 | 13•246•300•210•194•28•438•119 | – | 91•1057•336•64 | 299•254•190•75•108•619•3 |
| S. pneumoniae 70585 | – | – | – | 16•244•54•15•414•324•369•109•3 |
| S. pneumoniae D39 | – | – | 91•55•714•117•507•64 | 16•172•534•204•141•369•109•3 |
| S. pneumoniae G54 | 355•204•404•28•140•417 | – | 146•222•609•507•64 | 16•298•15•738•369•109•3 |
| S. pneumoniaeI NV104 | – | – | 91•55•222•492•117•507•64 | – |
| S. pneumoniae R6 | – | – | 112•55•714•117•507•64 | 5•32•172•534•204•141•369•109•3 |
| S. pyogenes MGAS5005 | – | – | 176•129•93•972•178 | 25•21•172•416•19•303•321•271 |
| S. pyogenes NZ131 | 55•711•177•20•168•169•248 | – | – | – |
| S. salivarius 57.I | 963•28•411•146 | – | 146•93•36•9•84•492•624•64 | 722•426•397•3 |
| S. sanguinis SK36 | 267•711•28•140•417 | 117•1427•19 | 798•77•78•210•264•136 | 329•477•754•3 |
| S. suis D12 | 252•307•147•257•168•417 | 102•1141•258•47 | 368•533•439•208 | 16•283•422•97•730 |
| S. suis ST1 | 252•220•24•21•189•257•118•50•298•119 | – | 91•707•103•439•208 | 16•172•416•117•827 |
| S. suis T15 | 198•54•220•24•21•189•257•109•59•417 | – | – | 16•588•117•70•27•618•112 |
| S. suis TL13 | – | – | – | 16•283•305•117•70•757 |
| S. uberis 0140 J | 13•950•28•432•66•21•38 | – | 368•66•1114 | 299•15•155•84•190•75•727•3 |
Symbol (•) indicates RE site in the gene sequences
aNo unique pattern observed
dnaA, dnaK, fabG, mraY, purK and pyrH
Gene—purH did not provide information on unique RE patterns in the rest of the 20 genomes: S. pneumoniae (11/20 strains), S. pyogenes (4/7 strains), and S. suis (5/9 strains). We looked for unique RE digestion patterns in other genes that can be used for identification. This analysis revealed that the following combinations can be used: (a) dnaA:HpyCH4V for S. pneumoniae SPNA45; (b) dnaK: (1) Tru9I for S. pneumoniae 670-6B, (2) TaqI for S. pneumoniae SPN034156, and (3) Tru9I for S. suis ST3; (c) fabG: Tru9I for S. pneumoniae ATCC700669; (d) mraY: (1) AluI for S. pneumoniae SPN994038, and (2) AluI for S. pneumoniae Taiwan 19F-14; (e) purK: (1) BfaI for S. pneumoniae OXC141, (2) AluI for S. pneumoniae P1031; (3) AluI for S. pyogenes MGAS315 (4) AluI for S. pyogenes MGAS9429; (5) Tru9I for S. suis BM407; (f) pyrH: (1) HpyCH4V for S. pneumoniae JJA, (2) Tru9I for S. pyogenes MGAS1882, (3) AluI for S. suis D9 (Table 3). This strategy allowed us to distinguish 15 out of 20 genomes, which could not be segregated using purH gene.
Table 3.
Unique in silico restriction endonuclease digestion patterns (5′–3′) of common genes (other than purH) of Streptococcus genomes
| Streptococcus spp. | Gene | RE | RE digestion pattern |
|---|---|---|---|
| S. pneumoniae SPNA45 | dnaA | HpyCH4 V | 622•348•329 |
| S. pneumoniae SPN034156 | dnaK | TaqI | 557•555•33•198•487 |
| S. pneumoniae 670-6B | Tru9I | 428•380•160•399•258•84•115 | |
| S. suis ST3 | Tru9I | 968•36•555•265 | |
| S. pneumoniae ATCC 700669 | fabG | Tru9I | 100•193•57•57•186•128•11 |
| S. pneumoniae SPN994038 | mraY | AluI | 139•207•8•166•189•155•117 |
| S. pneumoniae Taiwan19F-14 | AluI | 139•215•166•189•91•64•117 | |
| S. pneumoniae P1031 | purK | AluI | 5•509•174•69•335 |
| S. pneumoniae OXC141 | BfaI | 125•249•5•120•180•422 | |
| S. pyogenes MGAS315 | AluI | 202•8•238•9•141•68•354 | |
| S. pyogenes MGAS9429 | AluI | 78•142•72•8•247•141•68•354 | |
| S. suis BM407 | Tru9I | 418•496•163•3 | |
| S. pneumoniae JJA | pyrH | HpyCH4 V | 103•84•89•45•127•290 |
| S. pyogenes MGAS1882 | Tru9I | 25•4•443•64•14•79•100 | |
| S. suis D9 | AluI | 279•115•66•272 |
Symbol (•) indicates RE site in the gene sequences
As no single gene-RE combination worked for identifying the 5 genomes of S. pneumoniae SPN034183, S. pneumoniae SPN994039, S. suis P1/7, S. suis SC84, and S. pyogenes MGAS15252, combinations of 2–3 gene-RE patterns were used. Two strains viz., S. suis P1/7, S. suis SC84, were found to have identical RE patterns with argR and dnaA genes of S. suis B407, and S. suis D12, respectively, whereas S. pyogenes MGAS15252 and S. pyogenes MGAS1882 shared their RE patterns for the following genes: argR, argS, cysS, dnaK, glyA, gyrB, parE, purH, purK, and purR. These three groups of two strains each could be distinguished by using additional gene-RE combinations: purK-Tru9I, purH-BfuCI, and pyrH-Tru9I (Table 4). Using this strategy, we could not distinguish S. pneumoniae SPN034183 and S. pneumoniae SPN994039, from each other.
Table 4.
Identification of Streptococcus strains using in silico restriction endonuclease digestion patterns (5′–3′) of two common genes
| Streptococcus spp. | RE digestion patterns | |
|---|---|---|
| argR-Tru9I | purK-Tru9I | |
| S. suis P1/7 | 25•397•19 | 251•167•496•163•3 |
| S. suis B407a | 25•397•19 | 418•496•163•3 |
| argR-BfuCI | purH-BfuCI | |
|---|---|---|
| S. suis SC84 | 29•445 | 435•699•219•195 |
| S. suis D12a | 29•445 | 436•699•414 |
| argR-Tru9I | pyrH-Tru9I | |
|---|---|---|
| S. pyogenes MGAS15252 | 25•51•30•189•27•75•74 | 25•4•443•64•14•179 |
| S. pyogenes MGAS1882b | 25•51•30•189•27•75•74 | 25•4•443•64•14•79•100 |
The strategy of screening all the 50 genomes for searching genes which were common to all of them and subjecting each one of them to 10 different Res, allowed us to find unique RE digestion patterns in a few genes. purH alone proved effective in segregating 30 out of 50 genomes. Identification of an additional 18 genomes, was possible by employing other gene-RE combinations. No unique gene—RE combinations could be deduced for two strains of S. pneumoniae.
Discussion
Bacterial identification methods have graduated from those based on morphological and metabolic characteristics to molecular methods. Among the various genes based identification methods, the most widely employed has been the usage of rrs gene [12–16]. It has proved instrumental in bacterial identification; however, the major difficulty encountered is in the cases where the organism has multiple copies of rrs within the genome [13, 14, 18–20]. The issue becomes more complicated when rrs genes from different species show high sequence similarity among themselves. In order to circumvent these issues, the information is complemented by employing other highly conserved genes. However, it involves more inputs and selection of other genes out of a few thousand genes within the genome is not an easy task. In spite of the fact that around 23 genes have been used frequently in many studies carried out for identifying Streptococcus, no consensus gene has been identified [3, 4, 11, 21–35]. It has also not been realized that except recA, the rest of the 22 genes are not present in all the species of Streptococcus. Although, recA is one of those genes which are used widely for identification of Streptococcus [27], however, our analysis revealed that it is not among the best candidates, which can be exploited for detection of Streptococcus infections especially in the cases of S. mutans (2 strains), S. pneumoniae (20 strains), S. pyogenes (3/7 strains), and S. suis (5/9 strains) (Table 5a, b). In the similar manner, analysis of gyrB gene, commonly used as biomarker for identification in general [13], was also not very effective as the unique RE patterns could not be deduced in the following cases: S. pneumoniae (18/20 strains), S. pyogenes (4/7 strains), and S. suis (6/9 strains) (Table 6a, b).
Table 5.
Unique in silico Restriction Endonuclease digestion patterns (5′–3′) of recA gene of Streptococcus genomes
| Streptococcus spp. | Restriction endonucleases | |||
|---|---|---|---|---|
| AluI | BfaI | BfuCI | CviAII | |
| (a) | ||||
| S. agalactiae 2603 V/R | 136•144•261•282•245•22•50 | 706•178•256 | 753•342•45 | 271•646•223 |
| S. dysgalactiae subsp. equisimilis RE378 | 61•111•57•138•89•7•228•65•267 | –a | 113•109•645•27•84•45 | 157•57•198•311•300 |
| S. gordonii str. Challis | 64•97•217•103•60•264•347 | 22•336•765•29 | 166•170•645•66•92•13 | 156•115•57•233•276•315 |
| S. intermedius JTH08 | 136•34•140•171•389•276 | – | 336•443•109•93•27•138 | 156•115•57•198•116•195•309 |
| S. macedonicus ACA-DC 198 | 28•84•58•116•57•35•163•36•293•285 | – | – | – |
| S. mitis B6 | 161•182•227•346•138•27•50•24 | – | 336•117•702 | 117•154•57•233•81•195•318 |
| S. oralis Uo5 | 161•380•36•293•279 | – | 86•693•109•64•29•99•69 | 117•211•233•276•80•232 |
| S. parasanguinis FW213 | 161•416•228•282•59 | – | 86•23•109•118•98•345•173•85•109 | 117•39•115•57•497•12•309 |
| S. pasteurianus ATCC 43144 | 28•84•58•116•57•35•163•36•246•47•285 | – | – | – |
| S. pyogenes MGAS5005 | 138•21•90•57•138•96•102•191•304 | – | – | 234•57•198•35•276•337 |
| S. pyogenes MGAS9429 | – | – | 336•117•639•45 | – |
| S. pyogenes NZ131 | – | – | 336•645•111•45 | – |
| S. salivarius 57.I | 175•203•54•49•89•300•40•230 | 172•186•526•227•29 | 952•29•27•132 | 271•57•198•35•579 |
| S. sanguinis SK36 | 175•257•189•184•65•40•69•170 | – | 336•443•202•168 | 156•172•198•311•312 |
| S. suis D12 | 164•84•35•75•77•172•219•326 | – | – | 120•39•172•198•116•195•312 |
| S. suis ST1 | 164•84•35•75•77•172•219•255•71 | – | 339•98•319•32•331•33 | 159•172•198•20•15•81•195•312 |
| S. suis T15 | 173•75•35•75•77•172•219•255•68 | – | – | 120•39•172•198•20•15•81•195•309 |
| S. suis TL13 | 164•84•35•75•77•391•326 | 574•543•35 | – | 120•39•115•371•195•312 |
| S. uberis 0140 J | 265•87•788 | 67•57•54•714•248 | 51•693•237•159 | 156•115•57•198•391•223 |
| Streptococcus spp. | Restriction endonucleases | |||
|---|---|---|---|---|
| HpyCH4 V | RsaI | TaqI | Tru9I | |
| (b) | ||||
| S. agalactiae 2603 V/R | 424•54•59•379•110•114 | – | 57•167•243•363•310 | 365•219•38•169•80•4•265 |
| S. dysgalactiae subsp. equisimilis RE378 | –a | – | 203•150•670 | 251•219•30•117•60•24•210•109•3 |
| S. gordonii str. Challis | 307•15•102•54•59•346•269 | 488•213•140•311 | 467•36•504•135•10 | 731•51•33•337 |
| S. intermedius JTH08 | 76•111•9•111•171•99•27•542 | 488•250•408 | 467•669•10 | 80•285•249•69•132•87•244 |
| S. macedonicus ACA-DC 198 | – | – | – | 85•280•318•108•24•60•280 |
| S. mitis B6 | 150•124•57•93•113•618 | 488•353•81•233 | 224•93•18•132•672•16 | 80•735•340 |
| S. oralis Uo5 | 307•45•96•48•41•346•266 | 488•235•118•81•227 | 89•135•93•150•426•240•16 | 80•735•331•3 |
| S. parasanguinis FW213 | 136•138•33•42•188•346•263 | 149•339•353•20•61•103•121 | 89•135•93•54•66•30•36•504•33•106 | 614•69•132•60•268•3 |
| S. pasteurianus ATCC 43144 | 196•78•150•113•618 | – | – | 85•280•219•99•108•24•60•280 |
| S. pyogenes A20 | – | – | – | 13•352•219•30•117•60•24•198•12•109•3 |
| S. pyogenes MGAS5005 | – | – | – | 328•219•30•117•60•24•198•12•109•24•13•3 |
| S. pyogenes MGAS9429 | – | 841•20•158•118 | – | – |
| S. salivarius 57.I | 112•24•60•126•126•30•18•198•446 | 488•213•12•306•6•115 | 317•99•724 | 365•249•168•9•24•325 |
| S. sanguinis SK36 | 478•59•142•470 | 488•353•81•51•176 | 317•186•646 | 80•285•249•177•84•274 |
| S. suis D12 | 352•75•54•18•41•157•455 | – | 182•138•186•646 | 818•331•3 |
| S. suis T15 | – | – | – | 617•201•328•3 |
| S. suis TL13 | 277•75•75•72•41•40•117•319•136 | – | – | – |
| S. uberis 0140 J | 196•111•42•6•69•180•201•111•224 | – | 89•378•76•159•438 | 79•286•318•48•51•9•24•60•262•3 |
Symbol (•) indicates RE site in the gene sequences
aNo unique pattern observed
Table 6.
Unique in silico restriction endonuclease digestion patterns (5′–3′) of gyrB gene of Streptococcus genomes
| Streptococcus spp. | Restriction endonucleases | |||
|---|---|---|---|---|
| AluI | BfaI | BfuCI | CviAII | |
| (a) | ||||
| S. agalactiae 2603 V/R | 82•102•111•315•489•36•264•51•90•123•45•213•32 | 1711•54•185•3 | 483•1323•81•66 | 90•12•220•51•486•1094 |
| S. dysgalactiae subsp. equisimilis RE378 | 121•156•195•423•291•9•132•165•461 | 812•363•778 | 112•371•838•334•124•27•147 | 322•51•425•22•11•1044•6•72 |
| S. gordonii str. Challis | 793•378•9•132•12•123•90•141•27•245 | 191•221•286•474•540•238 | 109•55•327•13•89•1087•96•27•59•88 | 817•39•15•530•111•288•150 |
| S. intermedius JTH08 | 76•622•92•198•180•12•141•179•192•255 | 44•131•426•94•474•332•446 | 501•765•507•108•66 | 367•458•43•405•596•78 |
| S. macedonicus ACA-DC 198 | 184•306•214•191•279•225•51•56•202•80•165 | 812•350•238•553 | 382•1397•27•147 | 514•284•22•39•545•177•372 |
| S. mitis B6 | 698•611•105•180•252•101 | 136•559•551•165•177•235•124 | 106•1209•458•27•81•66 | 418•48•326•22•11•28•15•1079 |
| S. mutans LJ23 | 82•96•117•879•141•84•309•245 | –a | 24•516•732•140•271•96•108•66 | 322•51•425•22•11•28•15•530•177•222•150 |
| S. mutans UA159 | 82•96•117•311•568•141•84•309•80•165 | – | 24•516•732•411•96•108•66 | 322•476•22•11•28•15•530•177•222•150 |
| S. oralis Uo5 | 76•102•520•290•189•68•64•42•596 | 136•39•13•221•192•94•551•701 | 106•371•57•781•334•28•123•81•66 | 418•48•326•33•28•15•242•288•48•351•150 |
| S. parasanguinis FW213 | 793•99•41•238•528•251 | 73•118•413•94•716•536 | 109•395•188•213•413•58•132•57•60•151•108•66 | 817•11•28•15•641•438 |
| S. pasteurianus ATCC 43144 | 82•102•306•214•32•159•204•75•81•144•51•56•202•245 | 812•588•553 | 1341•438•27•147 | 373•141•284•22•39•545•111•66•372 |
| S. pneumoniae TCH8431/19A | 395•659•69•647•174 | 55•75•262•578•69•12•240•294•105•231•23 | 100•83•1241•75•445 | 235•72•584•543•75•66•25•185•78•81 |
| S. pneumoniae Hungary19A-6 | 1309•285•108•80•165 | – | – | – |
| S. pyogenes MGAS5005 | – | – | 514•57•533•610•123•81•35 | – |
| S. pyogenes MGAS9429 | – | – | – | 90•283•249•176•33•28•15•641•438 |
| S. pyogenes NZ131 | – | – | – | 90•283•458•28•15•641•438 |
| S. salivarius 57.I | 606•444•49•258•93•21•482 | 607•793•17•525•11 | 112•1571•270 | 514•306•39•545•399•150 |
| S. sanguinis SK36 | 411•171•119•92•40•100•114•85•60•132•273•188•70•95 | 1048•124•778 | 537•1347•66 | 319•150•359•43•530•111•360•78 |
| S. suis D12 | – | – | 1157•622•174 | – |
| S. suis TL13 | – | – | 540•1205•34•174 | – |
| S. uberis 0140 J | 295•501•40•59•270•9•9•12•120•12•30•63•533 | 50•131•721•375•676 | 483•57•1192•47•27•81•66 | 322•351•147•39•15•641•438 |
| Streptococcus spp. | Restriction endonucleases | |||
|---|---|---|---|---|
| HpyCH4 V | RsaI | TaqI | Tru9I | |
| (b) | ||||
| S. agalactiae 2603 V/R | 193•207•401•40•342•144•144•378•104 | 891•857•205 | 115•6•986•764•19•63 | 203•192•84•137•295•50•27•19•63•491•97•295 |
| S. dysgalactiae subsp. equisimilis RE378 | 135•649•17•382•288•378•104 | 527•1411•15 | 158•57•51•140•482•321•92•70•63•98•384•37 | 461•167•90•193•30•47•82•314•315•251•3 |
| S. gordoniistr. Challis | 132•666•193•135•57•663•104 | 516•196•176•660•387•15 | 163•4•467•106•160•75•256•137•161•384•37 | 419•585•63•570•313 |
| S. intermedius JTH08 | 24•163•102•546•288•54•267•168•83•148•104 | 100•493•292•1047•15 | 223•13•268•1406•37 | 934•130•314•256•182•128•3 |
| S. macedonicus ACA-DC 198 | 135•237•429•394•81•425•252 | 106•354•410•21•660•115•272•15 | 87•71•12•59•13•1059•615•37 | 395•516•50•46•63•491•79•18•164•128•3 |
| S. mitis B6 | 24•771•334•144•674 | 799•86•660•387•15 | 109•114•37•22•500•513•231•358•26•37 | 389•191•975•97•36•259 |
| S. mutans LJ23 | –a | 43•63•785•829•218•15 | 288•500•295•870 | – |
| S. mutans UA159 | – | 43•63•785•829•218•15 | 788•295•870 | – |
| S. oralis Uo5b | 366•429•670•210•272 | 799•65•21•1047•15 | 109•506•9•32•639•247•342•26•37 | 580•1054•54•259 |
| S. parasanguinis FW213b | 798•526•144•234•248 | 888•1047•15 | 163•76•174•46•248•268•105•218•231•358•26•27•10 | 583•354•48•19•63•588•295 |
| S. pasteurianus ATCC 43144 | 193•128•480•394•81•425•252 | 106•764•21•660•115•272•15 | 87•71•71•1072•615•37 | 395•516•50•46•63•491•97•164•128•3 |
| S. pneumoniae TCH8431/19A | 154•107•643•107•933 | 489•79•115•104•1016•141 | 103•55•33•1398•355 | 23•357•210•41•1310•3 |
| S. pyogenes MGAS9429 | – | – | 115•184•1002•652 | – |
| S. salivarius 57.I | 135•160•77•429•817•63•27•245 | 86•231•202•351•21•660•197•190•15 | 158•12•72•24•396•605•34•70•161•339•45•37 | 13•382•27•476•13•96•63•570•18•295 |
| S. sanguinis SK36 | 369•429•514•210•324•104 | 510•6•80•1339•15 | 112•100•922•779•27•10 | – |
| S. suis ST1 | – | – | 87•71•12•402•766•140•54•373•11•37 | – |
| S. uberis 0140 J | 135•666•724•176•226•26 | 35•71•211•187•15•939•480•15 | 23•64•79•63•13•46•455•433•21•104•570•24•58 | 395•66•50•117•90•193•30•129•314•177•97•295 |
Symbol (•) indicates RE site in the gene sequences
aNo unique pattern observed
bWith RE-Hin1I, unique digestion patterns were recorded for S. oralis Uo5: 282•1658•7, and S. parasanguinis FW213: 44•1050•856
The present study has shown that (1) purH bears unique RE digestion characteristics for identifying 60 % of the strains representing all the 16 species, (2) a few other genes can be used for identifying another 36 % of the strains. For identifying the clinical isolates, the following protocol may be adopted. DNA extracted from the infected sample can be used to amplify the biomarker gene(s) with specific primer sets using standard molecular techniques. The amplified gene product can be either subjected to RE digestion and run on the gel or the gene can be sequenced and subjected to in silico RE digestion, and compare the patterns to identify the strain. This approach of using genes which are common to all the species enhances the chances of identifying the potential organism, as has been proposed for Clostridium, Yersinia, and Vibrio. Although, purH is also present in Staphylococcus, the RE digestion patterns did not match with that of Streptococcus (data not shown). These biomarkers have the potential for being applied and used in diagnostic kits for Streptococcus, a deadly pathogen for which drug targets are being search furiously [37, 38].
Electronic supplementary material
Acknowledgments
We are thankful to the Director of CSIR-Institute of Genomics and Integrative Biology (IGIB), and CSIR projects—GENESIS (BSC0121) and INDEPTH (BSC0111) for providing the necessary funds, facilities and moral support. Authors are also thankful to the Academy of Scientific & Innovative Research (AcSIR), New Delhi.
References
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