cpn 60 Gene Based Multiplex-PCR Assay for Simultaneous Identification of Streptococcal Species

Dmitr iev A. , M. Bhide, I . Mikula: cpn60 Gene Based Multiplex-PCR Assay for Simultaneous Identification of Streptococcal Species. Acta Vet. Brno 2006, 75: 235-240. The cpn60 genes of Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis were sequenced and a certain polymorphism of cpn60 genes was revealed. Presumable species-specific pairs of primers were designed and their specificity was confirmed by PCR. Based on these data, the cpn60 gene-based multiplex-PCR assay was developed. It was found to be effective for simultaneous identification of S. agalactiae, S. dysgalactiae and S. uberis strains. Streptococci, cpn60 gene, multiplex-PCR Bovine mastitis is a major cause of economic loss in dairy industry (Allmann et al. 1995; Keefe 1997). In the United States, for example, the economic loss is estimated to be approximately $1,8 billion annually. Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis are the major streptococcal species among causative agents of bovine mastitis (Keefe et al. 1997). Obviously, the rapid and specific identification of streptococci is desirable for prevention of infectious diseases and monitoring mastitis. At present, ribosomal operon-based PCR is used for identification of many bacterial species, and numerous approaches have been applied for detection of mastitis-causative bacteria in milk products (Mart inez et al. 2001; Phuektes et al. 2001; Riffon et al. 2001). However, due to high homology of the ribosomal operon genes among different species, the ribosomal operon based PCR can result in a false-positive diagnosis (Backman et al. 1999; Hassan et al. 2000; MeiriBendek et al. 2002). These observations reduce the value of ribosomal operon-based PCR approach for identification of causative bacterial species. Obviously, some other genes have to be tested as potential targets for speciesspecific diagnosis. Recently it was demonstrated that phylogenetic cpn60 gene sequence analysis of streptococcal strains could be used for precise identification of several streptococcal species (Goh et al. 1998; Brousseau et al. 2001; Hung et al. 2005) that correlated with the ribosomal operon sequence analysis (Teng et al. 2002). However, S. agalactiae, S. dysgalactiae and S. uberis as important pathogens for animals and humans, were not tested using cpn60 gene as a potential discriminative marker. The goal of the present study was to develop a cpn60 gene based multiplex-PCR assay for simultaneous identification of these streptococcal species. Materials and Methods Bacter ia l s t ra ins A total of 68 streptococcal strains were used in this study. Forty two S. agalactiae strains, nine S. dysgalactiae strains and seven S. uberis strains were isolated from dairy cows in different regions of the Czech Republic and Slovakia, and ten S. agalactiae strains were isolated from pregnant women in Russia. Bacteria were grown in ToddHewitt broth or on 1.5% horse blood agar at 37 °C overnight. Other animal pathogens, i.e., Escherichia coli, Staphylococcus aureus and Salmonella typhimurium were also used in this study. ACTA VET. BRNO 2006, 75: 235–240 Address for correspondence: Alexander Dmitriev, Ph.D., Dr.Sc. Institute of Experimental Medicine acad. Pavlov str., 12, 197376, Saint-Petersburg, Russia Phone: 007-812-234-05-42 Fax: 007-812-234-94-77 E-mail: admitriev10@yandex.ru http://www.vfu.cz/acta-vet/actavet.htm General DNA techniques Most of the molecular genetic procedures were conducted according to previously published protocols (Mania t i s et al. 1982). In order to prepare DNA samples for PCR, a single bacterial colony was resuspended in 100 μl of distilled water and incubated at 97 °C for 5 minutes. The samples were centrifuged, and 5 μl of supernatant was used in PCR reaction. PCR was carried out in the final volume of 25 μl using the primers listed in Table 1. A pre-PCR step at 94 °C for 2 min was applied. A total of 30 PCR cycles were used under the following conditions: denaturation at 94 °C for 30 s, annealing at 52 °C for 1 min and extension at 72 °C for 30 s. After the final cycle, the additional incubation at 72 °C for 10 min was done to complete the reaction. For a multiplex-PCR assay, all the primers were added in the same reaction mixture. Sequencing of PCR products was performed using ABI PrismTM 377 Perkin-Elmer Sequencer and Big Dye Terminator Kit (Applied Biosystems, USA). Computer analysis of DNA Nucleotide sequences of cpn60 genes were accessed through the GenBank database (http://www.ncbi.nlm.nih.gov/entrez). Computer analysis of DNA was accomplished using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) and MULTALIN. Sequences of the primers were designed using the computer program “OLIGO”. The 100 bp ladder (New England Biolabs) was used as a DNA molecular weight standard. The sequences data reported in this study were deposited in the GenBank Nucleotide Sequence Database under accession numbers AY487423, AY487424, AY496926 and AY508648.

Bovine mastitis is a major cause of economic loss in dairy industry (Allmann et al. 1995;Keefe 1997).In the United States, for example, the economic loss is estimated to be approximately $1,8 billion annually.Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis are the major streptococcal species among causative agents of bovine mastitis (Keefe et al. 1997).Obviously, the rapid and specific identification of streptococci is desirable for prevention of infectious diseases and monitoring mastitis.
At present, ribosomal operon-based PCR is used for identification of many bacterial species, and numerous approaches have been applied for detection of mastitis-causative bacteria in milk products (Martinez et al. 2001;Phuektes et al. 2001;Riffon et al. 2001).However, due to high homology of the ribosomal operon genes among different species, the ribosomal operon based PCR can result in a false-positive diagnosis (Backman et al. 1999;Hassan et al. 2000;Meiri-Bendek et al. 2002).These observations reduce the value of ribosomal operon-based PCR approach for identification of causative bacterial species.Obviously, some other genes have to be tested as potential targets for speciesspecific diagnosis.Recently it was demonstrated that phylogenetic cpn60 gene sequence analysis of streptococcal strains could be used for precise identification of several streptococcal species (Goh et al. 1998;Brousseau et al. 2001;Hung et al. 2005) that correlated with the ribosomal operon sequence analysis (Teng et al. 2002).However, S. agalactiae, S. dysgalactiae and S. uberis as important pathogens for animals and humans, were not tested using cpn60 gene as a potential discriminative marker.The goal of the present study was to develop a cpn60 gene based multiplex-PCR assay for simultaneous identification of these streptococcal species.

Bacterial strains
A total of 68 streptococcal strains were used in this study.Forty two S. agalactiae strains, nine S. dysgalactiae strains and seven S. uberis strains were isolated from dairy cows in different regions of the Czech Republic and Slovakia, and ten S. agalactiae strains were isolated from pregnant women in Russia.Bacteria were grown in Todd-Hewitt broth or on 1.5% horse blood agar at 37 °C overnight.Other animal pathogens, i.e., Escherichia coli, Staphylococcus aureus and Salmonella typhimurium were also used in this study.

General DNA techniques
Most of the molecular genetic procedures were conducted according to previously published protocols ( M a n i a t i s et al. 1982).In order to prepare DNA samples for PCR, a single bacterial colony was resuspended in 100 µl of distilled water and incubated at 97 °C for 5 minutes.The samples were centrifuged, and 5 µl of supernatant was used in PCR reaction.PCR was carried out in the final volume of 25 µl using the primers listed in Table 1.A pre-PCR step at 94 °C for 2 min was applied.A total of 30 PCR cycles were used under the following conditions: denaturation at 94 °C for 30 s, annealing at 52 °C for 1 min and extension at 72 °C for 30 s.After the final cycle, the additional incubation at 72 °C for 10 min was done to complete the reaction.For a multiplex-PCR assay, all the primers were added in the same reaction mixture.Sequencing of PCR products was performed using ABI Prism TM 377 Perkin-Elmer Sequencer and Big Dye Terminator Kit (Applied Biosystems, USA).

Computer analysis of DNA
Nucleotide sequences of cpn60 genes were accessed through the GenBank database (http://www.ncbi.nlm.nih.gov/entrez).Computer analysis of DNA was accomplished using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) and MULTALIN.Sequences of the primers were designed using the computer program "OLIGO".The 100 bp ladder (New England Biolabs) was used as a DNA molecular weight standard.The sequences data reported in this study were deposited in the GenBank Nucleotide Sequence Database under accession numbers AY487423, AY487424, AY496926 and AY508648.

Results
Previously, the polymorphism of cpn60 gene sequences was used as a marker for identification of Streptococcus iniae (Goh et al. 1998), for differentiation of mutans streptococci and Streptococcus suis strains (Brousseau et al. 2001;Hung et al. 2005), and for determination of evolutional relationship of 10 streptococcal species belonging to viridans group streptococci (Teng et al. 2002).The cpn60 gene phylogenetic tree of streptococcal species was similar to the tree constructed based on 16S rRNA sequences (Brousseau et al. 2001;Teng et al. 2002) .
In this study we hypothesized that polymorphism of cpn60 genes could be used for differentiation of S. agalactiae, S. dysgalactiae and S. uberis strains.
In order to analyze this hypothesis we compared the complete S. agalactiae cpn60 gene with cpn60 gene fragments of S. dysgalactiae and S. uberis available in GenBank database.After the comparison, a certain polymorphism of cpn60 genes was revealed (Fig. 1).Based on these data, the presumed species-specific primers were designed and used in PCR (Table 1).
All 52 S. agalactiae strains tested produced the expected 310 bp amplification products when analyzed by PCR with primers AGAFor and AGARev.It is important that both human and bovine S. agalactiae strains of different serotypes produced amplification products of the same size (Fig. 2).PCR analysis of S. dysgalactiae and S. uberis strains with pairs of the primers DYSFor, DYSRev and UBEFor, UBERev, respectively, revealed PCR products of the expected sizes 192 bp and 400 bp, respectively (Fig. 3).Specificity of each pair of primers was confirmed when other streptococcal species did not produce any PCR products (Fig. 3).Furthermore, specificity of primers was demonstrated by PCR analysis of other veterinary pathogens, i.e., E. coli, S. aureus and S. typhimurium (Fig. 2).
Three PCR products of different sizes (192 bp, 310 bp and 400 bp) were isolated from agarose gel and sequenced.As expected, their sequences corresponded to the previously published streptococcal cpn60 gene sequences available in the cpnDB Database (http://cpndb.cbr.nrc.ca).
In order to evaluate the possibility of simultaneous identification of streptococcal species, the multiplex-PCR with three pairs of primers AGAFor, AGARev, DYSFor, DYSRev, UBEFor and UBERev was employed (Fig. 3).Results of this analysis demonstrated species-specificity of the selected primers and indicated the value of cpn60 gene polymorphism for differentiation of S. agalactiae, S. dysgalactiae and S. uberis strains.

Discussion
Bovine mastitis is a major cause of economic loss in dairy industry due to reduced milk yield and quality, discarded milk, expensive treatment etc. (Allmann et al. 1995;Keefe 1997).Streptococci can widely spread within the herd due to specific life style and the machine milking technology.Additionally, humans can be infected by streptococci due to the close contact of farm staff with the infected animals and raw milk tanks.Thus, early identification of streptococci in the milk product is necessary for effective animal treatment and for monitoring streptococcal diseases.
The ribosomal operon-based PCR is often used for identification of streptococcal species in milk products (Martinez et al. 2001;Phuektes et al. 2001;Riffon et al. 2001).However, due to the high homology of ribosomal operons, the false-positive ribosomal operon-based PCR results can occur that reduce the specificity of this approach to 0.87-0.96.For example, the false-positive results were observed when S. agalactiae and S. pneumoniae as well as S. agalactiae and S. difficile strains were analyzed (Backman et al. 1999;Hassan et al. 2000;Meiri-Bendek et al. 2002).When S. agalactiae and S. dysgalactiae were analyzed, no false-positive results were observed (Riffon et al. 2001), however, the 6 bp difference between species-specific PCR products resulted in difficulties during comparison (Phuektes et al. 2001).
Obviously, a larger number of species simultaneously analyzed by ribosomal operonbased PCR can lead to a higher possibility of an incorrect diagnosis.Therefore, the search of another potential discriminative marker is important for veterinary and dairy industry.
The Cpn60 protein, also known as GroEL and HSP60, is a 60 kDa heat-shock protein that assists in the correct folding of most bacterial proteins under both normal and stress conditions (Ranford and Henderson 2002).The Cpn60 proteins showed extensive sequence similarity in bacterial species; typically around 70% (Ranford and Henderson 2002).It is significantly smaller than the sequence similarity of 16S rRNA genes (91% -93%) as it was determined for different streptococcal species (Bentley et al. 1991).
The In contrast to scpB gene-based detection, the use of cpn60 gene as a target in PCR can be effectively used for both human and bovine S. agalactiae strains (Fig. 2).The cpn60 gene-based multiplex-PCR approach demonstrated the possibility of simultaneous identification of S. agalactiae, S. dysgalactiae and S. uberis in the same sample (Fig. 3).An additional advantage of this approach is significantly reduced cost of analysis in comparison with three independent PCR assays, which can be attractive for dairy industry.
Based on the results of this study, we also hypothesize that cpn60 gene polymorphism can be used for identification of any other bacterial pathogens.Clearly, additional study is necessary to analyze this hypothesis.
In conclusion, the data of the present study demonstrate that cpn60 gene-based multiplex-PCR assay can be effectively used for simultaneous identification of S. agalactiae, S. dysgalactiae and S. uberis strains.This novel approach can be employed for analysis of the milk products and for monitoring mastitis.

Fig. 1 .
Fig.1.Comparative analysis of cpn60 gene fragments of S. agalactiae, S. dysgalactiae and S. uberis strains.Species-specific primers used in this study correspond to underlined regions.Numbers correspond to nucleotide numbers in complete S. agalactiae cpn60 gene.
cpn60 gene-based PCR assay developed in this study avoided the disadvantages of ribosomal operon-based PCR.It demonstrated high specificity and simplicity in interpreting the results.It is important to note that the previously described scpB genebased PCR detection of S. agalactiae in human sera and blood ( D m i t r i e v et al. 2004) cannot be used for the analysis of bovine S. agalactiae because the scpB gene was present only in 20% of bovine strains ( D m i t r i e v et al. 1999; F r a n k e n et al. 2001).

Table 1 .
Primers used in the present study