Identification and genotypic analysis of Streptococcus spp. isolated from Giant Pandas in China by PCR-based methods

1 College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, P. R. China. 2 Entry-exit Inspection and Quarantine of Sichuan province, Cheng Du 610041, P. R. China China. 3 Conservation and Research Center for Giant Panda, Ya’an 625000, P. R. China. 4 Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, P. R. China. 5 Key laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya’an 625014, P. R. China.


INTRODUCTION
The Giant panda, Ailuropoda melanoleuca, is one of the most rare species in the world and are unique to China, they are the First-grade state protected animal in China, who are praised as "Living Fossil" and "National Treasure" of our country.Streptococcus species are widely distributed in nature, such as water, dust, alimentary tract, respiratory and genitourinary tracts, milk, etc.They are major pathogens for animals and human beings.The species of Streptococcus spp.are diversified *Corresponding author.E-mail: yanqigui@126.com.
including non-pathogenic normal bacteria in human or animal, and those which cause human or animal diseases.The cases of Giant Panda infected by βhemolytic Streptococcus have been reported (Zheng et al., 1999).
Identification of streptococcal species is currently based on observation of the cultural and morphological characteristics, determination of the biochemical pattern or antigenic characteristics.There are different typing methods for Streptococcus spp.The traditional classification is based on hemolytic or antigenic characteristics.However, these methods have their own limits.With the development of molecular biology, methods based on PCR technique had been recognized as having the great discriminative ability to differentiate isolated strains by producing DNA fingerprints that are specific for individual strains.REP-PCR and ERIC-PCR methods were widely used for analyzing Streptococcus spp.(Alam et al., 1999;Coffey et al., 1995;Harrington et al., 2007).Analysis of 16SrRNA squence of Streptococcus spp.by restriction fragment length polymorphism PCR (RFLP-PCR) can be a reliable method to distinguish different species of Streptococcus (McDonald et al., 2005).A new gene glucose kinase (gki) -PCR-denaturing gradient gel electrophoresis (DGGE) technique used to discriminate different Streptococcus mitis group strains had been reported van Vliet et al. (2009).These methods were proved to be fast, sensitive and reliable for differentiation of microorganisms.
The aim of this study was to isolate Streptococcus from Giant pandas in China, identify them by amplifying 16S ribosomal RNA sequences, and trying to analyze the genetic diversity among Streptococcus spp.isolated in Giant pandas by REP-PCR and ERIC-PCR techniques.

Bacterial isolation
Streptococcus strains were isolated from samples collected from different locations of different Giant pandas in Giant Panda Conservation and Research Center in Ya'an Bi Feng Xia Base of China Suspected streptococcus strains were purified and initially identified by biochemical tests and kept at -20°C in 30% glycerol sera medium to be further identified.

DNA preparation
Genomic DNA of Streptococcus spp. was extracted by the CTAB/NaCI mini-prep protocol (Barney et al., 2001).All streptococcus isolates were grown in 5mL of Luria-Bertani (LB) broth (Oxoid) containing 10% sera with 160 rpm shaking for 18 h at 37°C.Then, 1.5 ml LB broth culture was centrifuged at 10,000 g for 2 min.The cell pellet was re-suspended in 567 μl TE and mixed with 30 μl 10% SDS and 3 μl 20 mg/ml protase K; then the mixture was kept for 1 h at 37°C; 100 μl CTAB and 100 μl 5 mol/L NaCl were added in the mixture, 65°C for 10 min, DNA templates were extracted by phenol/chloroform method, and DNA were stored at -20°C for amplification of 16S ribosomal DNA sequence.
The purified PCR product was cloned into pMD18 Simple vector; the recombinant plasmid was transformed into competent cell DH5α, the positive plasmid screened were sent to Invitrogen in Shanghai to sequence.Wan et al. 1381 Serotyping Determinations of the serotypes of Streptococcus strains identified by16S rRNA was performed according to the procedures of specification of Strep Latex Slide Agglutination Test Kit (purchase from MICROGEN).This Kit was used for only separating A, B, C, D, F and G serotype of Streptococcus spp.
The reaction products were stored at 4°C until they were electrophoresed on a 2%(w/v) agarose gel containing 1×TAE and 0.5 μg mL -1 ethidium bromide along with the DNA markers IV (TIANGEN) in 2 to 3 h.

ERIC-PCR and REP-PCR fingerprints analyses
ERIC-PCR and REP-PCR fingerprints of amplified DNA fragments obtained from the agarose gel electrophoreses were recorded.REP-PCR and ERIC-PCR fingerprints were converted to a twodimensional binary matrix and analyzed by using the biostatistical analysis program NTSYS-pc (Version 2.10).From bottom to top of the gel, if a band was present, it was assigned a value of 1, if it was absent at same horizontal place, it was assigned a value of 0. According to previous rules, the DNA sequence data was recorded in Ntedit and saved with format of nts.Dendrograms were generated based on Dice's similarity coefficient (SD) and the unweighted pair group method using arithmetic averages (UPGMA).

Serotyping
The serotype results were listed in Table 1. S. dysgalactiae was serotype C, S. alactolyticus and S. pluranimalium were serotype D, 2 other species of Streptococcus spp.were serotype B, the serotype of S. suis was not determined because of the limitation of the Kit.8 Streptococcus strains were divided into 4 groups according to the serotype of each strain.
strain gave almost a different REP-PCR fingerprinting although a common band about 700 bp was observed in 7 strains.Dendrogram was constructed by analysing the similarity among the different profiles with the cluster analysis software (Figure 3).According to dendrogram analysis, 2 S. dysgalactiae with 0.88 similarity formed cluster as one type, S. suis and one of S. alactolyticus shared 0.84 similarity were clustered as a type with another S. alactolyticus at 0.81 similarity.The remaining strains were distinct with similarity of lower than 0.80.

ERIC-PCR
With primer ERIC1and ERIC2, ERIC-PCR finger-printing profiles were obtained (Figure 4).Analysis of these strains with ERIC-PCR yielded six to eleven bands ranging in size from 50bp to 2500bp.ERIC-PCR fingerprinting profiles of individual strain was different.The profiles were repeated three times, however, some of minor amplification bands were inconsistent making the analysis more difficult.Dendrogram was constructed by the same method (Figure 5).Similar to REP-PCR, only few strains formed clusters as most strains have unique REP-PCR fingerprinting profile.The first cluster sharing 0.9 similarities consisted of 2 S. dysgalactiae isolated from feet and teeth of Giant panda 2, respectively.The second cluster with 0.85 similarities was composed of 2 S. alactolyticus isolated from anus and nasal of Giant panda4.The remaining strains were distinct with similarity of lower than 0.80.

DISCUSSION
Giant pandas as the First-grade state protection animal in China are important for biological diversity.In order to protect Giant pandas, we have conducted experiments to isolate and identifying microorganisms in Giant panda.Streptococcus spp.widely existing in nature may cause suppurative inflammation.In our study eight strains of Streptococcus spp.were isolated from different locations of Giant pandas some of which belonged to the same species such as S. dysgalactiae or S. alactolyticus.(isolated from anua of panda 3); 2. S. dysgalactiae (isolated from sole of panda 2); 3. Unknown (isolated from uterus of panda 1); 4. Unknown (isolated from vaginal of panda 1); 5. S. dysgalactiae (isolated from teeth of panda 2); 6. S. suis (isolated from throat of panda 5); 7. S. alactolyticus (isolated from nasal of panda 4); 8. S. alactolyticus (isolated from anus of panda 4).
However, their ERIC-PCR and REP-PCR finger-printing profiles were inconsistent.Streptococci isolated from different Giant pandas were different.Edwards et al had reported the entire gene of 16S rDNA about 1.5kb of Streptococcus which had been used for identification of Streptococcus because of its conservation and specificity for Streptococcus species.Identification of Streptococci by analysis of 16S rRNA had been reported (Hassan et al., 2001).
S. dysgalactiae is an important pathogenic bacterium which is reason of acute or chronic mastitisthe for cattle, goats and sheep (Chenier et al., 2008) even for fish (Nomoto et al., 2006).S. pluranimalium was isolated from panda 3 when it was Alvei profluvium.Seimiya et al. (2007) has reported S. pluranimalium was isolated from the brain and cerebrospinal fluid of a neonatal calf (Seimiya et al., 2007), so the isolated S. pluranimalium may cause Giant panda Alvei profluvium. 2 S. alactolyticus was isolated from nasal cavity and anus of panda4 who is a wild Giant panda.It is not confirmed that S. alactolyticus is pathopoiesis to Giant pandas.S. suis is another important pathogenic bacteria that can cause both animal and human arthrophlogosis and meningitis, it was outbreak that human was infected in Sichuan, China in 2006 (journal.shouxi.net),follow by human infected S. suis was reported in Vietnam ( 2006 Only Zhang et al has reported that panda infected βhemolytic streptococci, however, Streptococcus isolated in this study has not been reported so for.Accordingly, this study has considerable contribution to Streptococcicosis of Giant panda. PCR-based methods for genotypic analysis of Streptococcus spp.are widely applied.Valdes et al had used different molecular typing methods including REP-PCR and ERIC-PCR to evaluate the genetic diversity of S. phocae (Valdes et al., 2009).In this study, REP-PCR and ERIC-PCR were used for genotypic analysis of 8 Streptococcus REP-PCR fingerprinting profiles of each Streptococcus were inconsistent.It demonstrated genetic diversity of Streptococcus of Giant pandas.According to REP-PCR dendrogram analysis results, 2 S. dysgalactiae belonged to serotype C were divided into 2 subgenotypes at 0.88 similarity, 2 S. alactolyticus and 1 S. pluranimalium belonged to serotype D were also separated into 3 sub-genotypes, the species of S. pluranimalium is not the same as S. alactolyticus, so they were separated from each other with lower similarity.Unfortunately, S. alactolyticus and S. suis sharing relatively high similarity with 0.84 forming one cluster.Although the 2 unknown Streptococcus spp.belonged to identical serotype D, they did not form into the same cluster due to genetic variability.
Compared with REP-PCR, ERIC-PCR has a higher discriminative ability. 2 S. alactolyticus and 2 S. dysgalactiae were divided into 2 sub-genotypes with similarity 0.90 and 0.85, respectively.The remaining strains were distinct with similarity of lower than 0.80.
REP-PCR can be used for molecular epidemiologic analysis of Streptococcus (Al-Ghamdi et al., 2000;Matsumoto et al., 2001).REP-PCR and ERIC-PCR fingerprinting profiles suggested that there are genetic differences among to the identical Streptococcus species isolated from the same Giant pandas.It was demonstrated that REP-PCR and ERIC-PCR analysis can rapidly type 8 Streptococcus, and the results indicated that the genetic diversity of Streptococcus spp. in Giant pandas is high.
As a result, The REP-PCR and ERIC-PCR fingerprinting analysis results were correlated with serotype results.REP-PCR and ERIC-PCR are rapid methods for typing as well as investigating of the molecule epidemiology of Streptococcus spp.

Table 1 .
The 8 Streptococcus strains identified by 16SrRNA in this study.