PL226609B1 - The use of polynucleotide, a set of primers and the method of determining the degree of relationship of Staphylococcus strains - Google Patents

Abstract

The new method for species determination and intra-species diversity analysis of Staphylococcus strains was revealed. An exemplary way to achieve this is the use of the polymerase chain reaction (PCR) to obtain the product on the conserved locus template, which is subsequently digested with restriction enzymes (method known as restriction fragments length polymorphism, RFLP). This allows to obtain restriction patterns allowing for species determination. Intra-species diversity analysis for staphylococci is also possible when the RFLP is carried out for properly selected enzymes.

Description

The invention relates to a method for determining the degree of relatedness of strains of the genus Staphylococcus based on the analysis of sequence polymorphism of a selected gene.

In recent years, many methods of identifying and typing isolates of bacteria of the genus Staphylococcus (staphylococcus) have been developed and developed. Two groups can be distinguished among these methods: phenotypic and genotypic methods. Identification of staphylococcal species by methods of the first group is based on the morphological characteristics, physiological properties and the chemical composition of the cell wall. The results obtained using commercial tests based on the profiles of biochemical and immunological reactions are burdened with errors due to the variable expression of some phenotypic features and the ambiguity in the interpretation of the results. The overall accuracy of conventional tests is low, ranging from 50 to 70%.

Among the genotypic taxonomic methods, comparative DNA-DNA hybridization and sequence analysis of the gene encoding 16S rRNA have defined species within the genus Staphylococcus and are recommended for confirming the results of new methods that are introduced into widespread use (Takahashi et al. 1999). Other methods of genetic identification include: genetic probes, dot-blot hybridization, ribotyping by Southern blot hybridization, fluorescent in situ hybridization (FISH), amplification by PCR and species-specific (selective) PCR, PCR-RFLP - restriction fragment length polymorphism analysis (Bentley et al. 1993; Kempf et al. 2000; Krimmer et al. 1999; Yugueros et al. 2000; Yugueros et al. 2001; Zakrzewska-Czerwinska et al. . 1992). Recently, for phylogenetic and taxonomic analysis of prokaryotes, a comparison of the DNA sequences of genes encoding highly conserved proteins has been used, such as: heat shock protein HSP60 encoded by the cpn60 gene, an enzyme catalyzing the successive incorporation of the second and third glycine into the transverse bridge in the muropeptide encoded by the femA gene, β subunit of bacterial RNA polymerase encoded by the rpoB gene, Tu elongation factor necessary in the formation of a peptide chain encoded by the tuf manganese-dependent superoxide oxidase (Mn-SOD) gene encoded by the sodA gene (Blaiotta et al. 2005; Blaiotta et al. 2004; Ghebremedhin et al. 2008; Goh et al. 1996; Goh et al. 1997; Sivadon et al. 2005; Voytenko et al. 2006).

The aim of the invention is to provide a new, more sensitive and at the same time universal method for determining the degree of relationship of Staphylococcus strains and the means that can be used for its implementation.

The subject of the invention is the use of a polynucleotide having the sequence of the hyptf gene for determining the degree of relatedness of strains of the genus Staphylococcus, the hyptf gene encoding a protein having the amino acid sequence shown as Seq. Id. No. 2.

Preferably, determining the degree of relatedness comprises restriction polymorphism analysis. Preferably, the hyptf gene has the nucleotide sequence shown as Seq. Id. No. 1.

Another subject of the invention is a method for determining the degree of relatedness of Staphylococcus strains, characterized by isolating the hyptf gene sequence from the genome of the typed bacterial strain, comparing the isolated sequence with the corresponding sequence of the hyptf gene from a known strain of Staphylococcus genus and determining the degree of similarity of these sequences. , wherein the predetermined degree of sequence similarity of the hyptf gene is a measure of the relatedness between a typed bacterial strain with a known strain of the genus Staphylococcus.

Preferably, the hyptf gene encodes a protein having the amino acid sequence shown as Seq. Id. No. 2. Preferably, determining the degree of relatedness comprises restriction polymorphism analysis. Preferably, the hyptf gene has the nucleotide sequence shown as Seq. Id. No. 1. Preferably, the sequence of the hyptf gene is isolated by PCR using the oligonucleotides indicated in Table 1 as primers. Preferably, the method of the invention comprises digesting the isolated sequence of the hyptf gene or a fragment thereof with a restriction enzyme selected from the group consisting of the following restriction enzymes: TaiI, Tsp509I, AluI and MseI.

Another object of the invention is a set of primers for determining the degree of relatedness of Staphylococcus strains by the RFLP technique, which consists of the consensus primers indicated in Table 1 of the bacteria specific for the hyptf gene of the Staphylococcus genus.

The invention discloses a new use of the genomic hyptf locus in microbiological diagnostics, in particular in determining species affiliation and diversity analysis.

Intraspecific strains of bacteria within the genus Staphylococcus. For example, it can be based on the use of PCR (polymerase chain reaction) to obtain a product on the template of a conserved genomic locus, digest this product with restriction enzymes and analyze the resulting image (RFLP) in order to determine species affiliation. RFLP analysis with appropriately selected restriction enzymes also allows for the analysis of intraspecific variability within the genus Staphylococcus.

Figure 1 shows a set of RFLP images obtained for 25 species of the genus Staphylococcus using restriction polymorphism analysis for the hyptf gene. Sequence of lanes for individual restriction enzymes: TaiI, Tsp509I, AluI, MseI.

P r z k ł a d 1.

Determining the degree of relatedness of Staphylococcus strains through RFLP analysis of the hyptf gene

Materials and methods

Bacterial strains

The bacterial strains of the genus Staphylococcus used came partly from the own collection of the Department of Microbiology (Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University in Kraków, Poland) and partly from foreign collections (obtained through the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wrocław, Poland).

Determination of DNA concentration

The concentration of the obtained aqueous DNA solutions was determined by measuring the absorbance at 260 and 280 nm wavelengths using the ND-1000 spectrophotometer (Thermo Scientific, Wilmington, USA).

Genomic DNA isolation

Bacteria were grown in 20 ml of liquid LB medium (Sigma Aldrich, Poznań, Poland) at 37 ° C for 14 h. 2 ml were taken from the culture and centrifuged 2 min at 5000 g. The medium was decanted and the bacterial pellet was frozen at -20 ° C. After thawing, the pellet was resuspended in 200 µl of EC buffer (6 mM Tris; 1 M NaCl; 100 mM EDTA; 0.5% Brij 58; 0.5% Sarkozy; pH 7.6) to which 1 µl RNase A (Thermo Scientific , Schwerte, Germany) and 1 µl of Lysostaphine (Thermo Scientific). The sample prepared in this way was incubated at 37 ° C for 14 h. In the next stages of purification the Genomic Mini kit (A&A Biotechnology, Gdańsk, Poland) was used and the protocol attached by the manufacturer was followed. In the last step, the DNA was eluted with 50 µl of distilled and filtered water (ddH2O) and its concentration was determined.

Primers used for the PCR reaction

The primers were synthesized and supplied by Genomed (Warsaw, Poland).

Table 1. Consensus primers specific for the hyptf gene of Staphylococcus bacteria

Starter name

1. GEN0MIC-SigEndDeg2RevB: 2. GEN0MIC-SigEndDeg2RevA:

3. GENOMIC-SiglntDeglForA:

4. GENOMIC-SiglntDeglForB:

5. GENOMIC-SigExtraForA:

6. GENOMIC ~ SigExtraRevB:

7. GENOMlC-SigFinalRev:

Sequence

ΆθΆ 7 \ * X *

RAATAJRNCKNKS GAAGAAACC

ATGYTAACNAAAGAATTYGC

ATGYTAACNAAAGAATTYGCNCARCG

AT G T T AAC T AAAGAAT T T G CAC

TGCGAAGAAACCTTTTTTCT

AATAAACGTGCAAAGAANCCYTT

Primers were designed based on the sequences of the hyptf locus gene and homologous loci in various species of bacteria of the genus Staphylococcus whose genomic sequences are available in the NCBI Nucleotide database, http://www.ncbi.nlm.nih.gov/nuccore (species name and strain designation; accession number of the genomic sequence for a given species in the NCBI Nucleotide database; accession number of the sequence of the protein encoded by the hyptf gene or its homologue in the NCBI Protein database; Locus tag of the hyptf gene or its homologue):

PL 226 609 B1

1. S. arlettae CVD059; NZ_ALWK01000016; ZP_10998849; SARL_04191

2. S. aureus ED98; NC_013450; YP_003281300; SAAV_0351

3. S. capitis SK14; NZ_ACFR01000004; ZP_03613546; STACA0001_1451

4. S. caprae C87; NZ_GL545278; ZP_07842400; HMPREF0786_01751

5. S. carnosus TM300; NC_012121; YP_002633143; Sca_0043

6. S. epidermidis VCU071; NZ_AGUB01000006; ZP_12936352; SEVCU071_1788

7. S. equorum Mu2; NZ_CAJL01000015; ZP_10336487; SEQMU2_13725

8. S. haemolyticus JCSC1435; NC_007168; YP_254503; SH2588

9. S. hominis SK119; NZ_ACLP01000001; ZP_04058912; STAHO0001_2121

10. S. ludginensis VCU139; NZ_AHLK01000025; ZP_13198142; SEVCU139_2215

11. S. massiliensis S46; NZ_AMSQ01000007; ZP_18856164; C273_05867

12. S. pettenkoferi VCU012; NZ_AGUA01000079; ZP_12935319; SEVCU012_0617

13. S. pseudintermedius HKU10-03; NC_014925; YP_004148220; SPSINT_0055

14. S. saprophyticus ATCC15305; NC_007350; YP_302418; SSP2328

15. S. simiae CCM 7213 NZ_AEUN01000034; ZP_09145692; SS7213T_01868

16. S. simulans ACS-120-V-Sch1; NZ_KB373326; ZP_11416468; HMPREF9310_00842

17. S. warnerii L37603; NZ_ACPZ01000041; ZP_04678206; STAWA0001_2413 The prototype of this locus is the SAAV_0351 locus of Staphylococcus aureus ED98 (NCBI

Nucleotide DB: NC_013450) with the following gene sequence:

Seq. Id. No. 1 :

AT GT TAACTAAAGAAT T TGCACAACGCG TAGAAC TAAGT GAGAAGCAAGTCCG TAAAAT T

GT TCAACAT T TAGAACGTGGT TACC AACT AAGCAAGAC TGAATATC GTGGT CGTGAA

G CAAC T G AT T T CAAAGAAGAAGATAT C GAG C T T T T CAAAGACAT T G C C GAC AAAG T AAAA ^ jz ^ 2 \ A.C2rL? K2 \ .Tj? KGT TJ? \ .T G.?lTC Tj ^ \. GCGT T T G.A.T T TCCTGCAA.

CTCjArTGTC / AAAACGACCACAAAAACTTACCIACTAATCAAAATCTCCCTCAACTAGTA

GAAGATTTACGCCTAGAAATCCAGAAAATGCGCGAAGAACGTCACCTACTTGGTCAAATG _ATG ^ _TC ^ _GTACATCAGC ^ _C ^ _C ^ _G ^ _{TTAAAAGAACTTCAAAATCAACTTACATCT}

AAAATCGATTCAAATAGCGAATCCTTAAAAGCCATCCAAACATCACAAGAGGCTATCCAA

GAAGCACAAGCATCTCAAGCTAAAGTATTGGCTGAATCAACAAATAAAGTTGAAAAGAAT

GCTGTTACTGAAGATAAAGCTGACAGTAAAGATTCGAAGGTTGCTGGAGTGAATACATCA

ACAGACGCTAAGACGGATACTAAAGCAGAAAATGCAGGCGATGGCACTGCTACTAAAGTA

GATAAAGAGGATCAAATTTCTGCAACTGAAGCAATTGAAAAAGCCAGCGTTGAGCAGTCT

AAAAATGAAAATGCAGCTGAGACTTCAAATAAAGAAGCTACAGTAGATGCAGACGCGCAA

CATGATGCTGAACAACAAGTAGCAGAAGCCCATGCAGAAGCTTCTAAACAAGCTACTTCG

AAC GAT T CAT T GGAAGC GAAAG C AGAAAAT GAC AG C ACAGC T T C ACAG T C AGAAAT G T CA

GAAC C AAAAC C T CAAGAAGAGAAAAAAG GTTTCTTCG CAC G T T T AT T CAAC C T G T AA and the coded protein sequence used to look for homologues of this gene in other species:

PL 226 609 B1

Seq. Id. No. 2:

MLTKEFAQRVELSEKQVRKIVQHLEERGYQLSKTEYRGREATDFKEEDIELFKDIADKVK

QTNSYDLAFDELEKEKDFLQVIVKNDDKNLPTNQNVAQLVEDLRLEIQKMREERHLLGQM

MNQVHQQQQELKELQNQLTSKIDSNSESLKAIQTSQEAIQEAQASQAKVLAESTNKVEKN

AVTEDKADSKDSKVAGVNTSTDAKTDTKAENAGDGTATKVDKEDQISATEAIEKASVEQS

KNENAAET3NKEATVEADAQHDAEQQVAEAHAEASKQATSND3LEAKAENDSTA3QSEMS

EPKPQEEKKGFFARLFNL

Polymerase chain reaction (PCR)

The reaction was performed in a volume of 10 µL (for analytical purposes) or five replicates of 20 µL (for preparative purposes for restriction enzyme digestion). Each 10 μΐ reaction was prepared with 6.50 μΐ ddH ₂ O, 1.0 μl 10 × concentrated buffer for RUN polymerase with magnesium ions (A&A Biotechnology), 1.0 μl 2 mM dNTP (A&A Biotechnology), 0.5 μl 10 μM mixture of primers (see Primers used for PCR reactions) and 0.5 μl of Run DNA polymerase (A&A Biotechnology) and 0.5 μl of isolated genomic DNA solution (50-100 ng of DNA). The PCR reaction was performed according to the following program:

1.94 ° C, 2 min.

2.94 ° C, 30 sec.

3.50 ° C, 30 sec.

4.74 ° C, 1 min 30 sec.

5.30 repetitions from step 2.

6. 74 ° C, 10 min.

7.4 ° C, pause.

Purification of the PCR reaction product

The PCR product to be digested with restriction enzymes was purified using the Clean Up kit (A&A Biotechnology) according to the protocol provided by the manufacturer. In the last step, the DNA was eluted with 50 μl of ddH ₂ O and its concentration was determined.

Digestion with restriction enzymes

500 ng of the PCR product was independently digested with the four restriction enzymes FastDigest® TaiI, FastDigest® Tsp509I, FastDigest® AluI, FastDigest® MseI (Thermo Scientific) according to the manufacturer's instructions. The digested DNA fragments were subjected to agarose gel electrophoretic separation along with the GeneRuler® 50 bp DNA Ladder DNA fragment standard (Thermo Scientific) containing DNA fragments ranging from 50 to 1000 base pairs in length.

Agarose gel electrophoresis

The electrophoretic separation was carried out on a 3% solution of purified agarose (Bioshop, Burlington, Canada) in TAE buffer (40 mM Tris; 20 mM acetic acid; 1 mM EDTA; pH 8.0) with the addition of ethidium bromide (Sigma Aldrich) at a concentration of 0, 5 µg / ml at a voltage of 5 V / cm for 1 h. The effect of the separation was visualized by highlighting the gel with DNA fragments with UV light and taking a picture with a camera.

Results

Among the methods of typing species membership within the genus Staphylococcus, the method of restriction fragment length polymorphism (RFLP) analysis of the gap gene (encoding glyceraldehyde-3-phosphate 2000 dehydrogenase) using the enzyme AluI (Yugueros et al. ; Yugueros et al. 2001). A novelty compared to this method is the use of a unique pattern of hyptf gene variability, a variability significantly greater than that of the gap gene and other genes used in typing methods for bacteria of the genus Staphylococcus. This allows for obtaining restriction images for four different enzymes (not one), which are characterized by high unambiguity and repeatability, and the possibility of easy interpretation even in the case of the appearance of intraspecific polymorphisms. The obtained PCR products for the hyptf gene and its homologues were 600-1000 base pairs long. Digestion of these products independently with four different restriction enzymes and electrophoretic separation of the obtained DNA fragments on an agarose gel allowed to obtain highly differentiating images (Fig. 1). Thanks to them, it was possible to differentiate even very closely

From related species such as S. delphinii, S. intermedius and S. pseudintermedius, which is not possible with other RLFP based methods including the gap method.

Bibliography

Bentley, R. W., Harland, N. M., Leigh, J. A. and Collins, M. D. 1993. A Staphylococcus aureus-specific oligonucleotide probe derived from 16S rRNA gene sequences. Lett Appl Microbiol 16, 203-6.

Blaiotta, G., Casaburi, A. and Villani, F. 2005. Identification and differentiation of Staphylococcus carnosus and Staphylococcus simulans by species-specific PCR assays of sodA genes. Syst Appl Microbiol 28, 519-26.

Blaiotta, G., Ercolini, D., Mauriello, G., Salzano, G. and Villani, F. 2004. Rapid and reliable identification of Staphylococcus equorum by a species-specific PCR assay targeting the sodA gene. Syst Appl Microbiol 27, 696-702.

Ghebremedhin, B., Layer, F., Konig, W. and Konig, B. 2008. Genetic classification and distinguishing of Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene sequences. J Clin Microbiol 46, 1019-25.

Goh, S. H, Potter, S., Wood, J. O., Hemmingsen, S. M., Reynolds, R. P. and Chow, A. W. 1996. HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. J Clin Microbiol 34, 818-23.

Goh, S. H, Santucci, Z., Kloos, WE, Faltyn, M., George, CG, Driedger, D. and Hemmingsen, SM 1997. Identification of Staphylococcus species and subspecies by the chaperonin 60 gene identification method and reverse checkerboard hybridization. J Clin Microbiol 35, 3116-21.

Kempf, V. A., Trebesius, K. and Autenrieth, I. B. 2000. Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol 38, 830-8.

Krimmer, V., Merkert, H., von Eiff, C., Frosch, M., Eulert, J., Lohr, J. F., Hacker, J. and Ziebuhr, W.

1999. Detection of Staphylococcus aureus and Staphylococcus epidermidis in clinical samples by 16S rRNA-directed in situ hybridization. J Clin Microbiol 37, 2667-73.

Sivadon, V., Rottman, M., Chaverot, S., Quincampoix, JC, Avettand, V., de Mazancourt, P., Bernard, L., Trieu-Cuot, P., Feron, JM, Lortat-Jacob, A., Piriou, P., Judet, T. and Gaillard, JL 2005. Use of genotypic identification by sodA sequencing in a prospective study to examine the distribution of coagulase-negative Staphylococcus species among strains recovered during septic orthopedic surgery and evaluate their significance . J Clin Microbiol 43, 2952-4.

Takahashi, T., Satoh, I. and Kikuchi, N. 1999. Phylogenetic relationships of 38 taxa of the genus Staphylococcus based on 16S rRNA gene sequence analysis. Int J Syst Bacteriol 49 Pt 2, 725-8.

Voytenko, AV, Kanbar, T., Alber, J., Lammler, C., Weiss, R., Prenger-Berninghoff, E., Zschock, M., Akineden, O., Hassan, AA and Dmitrenko, OA 2006. Identification of Staphylococcus hyicus by polymerase chain reaction mediated amplification of species specific sequences of superoxide dismutase A encoding gene sodA. Vet Microbiol 116, 211-6.

Yugueros, J., Temprano, A., Berzal, B., Sanchez, M., Hemanz, C., Luengo, J. M. and Naharro, G.

2000. Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp. J Clin Microbiol 38, 4351-5.

Yugueros, J., Temprano, A., Sanchez, M., Luengo, J. M. and Naharro, G. 2001. Identification of Staphylococcus spp. By PCR-restriction fragment length polymorphism of gap gene. J Clin Microbiol 39, 3693-5.

Zakrzewska-Czerwinska, J., Gaszewska-Mastalarz, A., Pulverer, G. and Mordarski, M. 1992. Identification of Staphylococcus epidermidis using a 16S rRNA-directed oligonucleotide probe. FEMS Microbiol Lett 79, 51-8.

Claims (10)

CLAIMS 1. The use of a polynucleotide having the sequence of the hyptf gene for determining the degree of relatedness of strains of the genus Staphylococcus, wherein the hyptf gene encodes a protein having the amino acid sequence shown as SEQ. Id. No. 2. 2. The use according to claim 1 The method of claim 1, wherein the determination of the degree of relatedness comprises restriction polymorphism analysis. 3. Use according to claim 1, characterized in that the hyptf gene has the nucleotide sequence shown as Seq. Id. No. 1. PL 226 609 B1 4. The method of determining the degree of relatedness of the Staphylococcus genus strains, characterized by isolating the hyptf gene sequence from the genome of the typified bacterial strain, comparing the isolated sequence with the corresponding sequence of the hyptf gene from a known strain of Staphylococcus genus and determining the degree of similarity of these sequences, while the established degree of sequence similarity of the hyptf gene is a measure of the relationship between a typed bacterial strain with a known strain of the genus Staphylococcus. 5. The method according to p. The method of claim 4, wherein the hyptf gene encodes a protein having the amino acid sequence shown as SEQ. Id. No. 2. 6. The method according to p. The method of claim 4, wherein the determination of the degree of relatedness comprises restriction polymorphism analysis. 7. The method according to p. The method of claim 4, wherein the hyptf gene has the nucleotide sequence shown as Seq. Id. No. 1. 8. The method according to p. 4. The method of claim 4, wherein the sequence of the hyptf gene is isolated by PCR using the oligonucleotides indicated in Table 1 as primers. 9. The method according to p. The method of claim 4 or 6, characterized in that it comprises digesting the isolated sequence of the hyptf gene or a fragment thereof with a restriction enzyme selected from the group consisting of the following restriction enzymes: TaiI. Tsp509I, AluI and MseI. 10. A set of primers for determining the degree of relationship of Staphylococcus strains by the RFLP technique, characterized in that it consists of the consensus primers indicated in Table 1 of Staphylococcus species specific for the hyptf gene.