The Potential Applications Of Phage SMP To Control Streptococcus Suis Infection And The Kinetics Proliferation

Streptococcus suis infection has been a major diseases troubled pig industry,especially with the overuse of antibiotics. It showed serious resistance and multi-drug resistance, and has brought new difficulties to the prevention and control of the disease. The development of novel antibacterial agents is very important, and bacteriophage as an alternative antibacterial agents show a great potential for applications.In this article, the genome sequence of Streptococcus suis serotype 2 and its phage SMP was analyzed, and the general PCR methods had been developed to the detecte the phage and Streptococcus suis simultaneously. We also developed the real-time PCR for detecting Streptococcus suis type 2 or its phage, respectively. After challenge CD1 mice with Streptococcus suis, the injection of phage SMP showed a good potential to control the bacterial infection. The preliminary study about the kinetics between Streptococcus suis and its phage was carried out. The results implied that the phage can be used to control the Streptococcus suis infection.It was found that there is a big difficulty in the isolation of Streptococcus suis phage, and there are many limitations when the traditional method,such as double-layer agar culture was used to pick plaque for phage isolation. In order to solve this problem, Streptococcus suis serotype 2 was used as a template, which isolated from nasal swab samples of healthy Bama pigs, and PCR methods were developed to detect the phage and Streptococcus suis at the same time. Compared the genome of phage SMP or the other 99 isolates of Streptococcus suis, many of the conservative fragments in the SMP were found, but they are very short. Among them single- stand binding gene appears to be the most conservative, so primers were designed based on the single-stand binding gene sequence, and PCR methods were developed. The results showed that the target gene can be found in many Streptococcus suis and phage SMP. The high specificity of the PCR was evaluated by other bacteria or phage, such as E. coli and its bacteriophage. PCR was optimized and the detection limit was up to 103cfu/mL. This method can be simultaneously used to screen a bacteriophage or related host bacteria, which can increase the probability to isolate the phage. In order to separate the phage from the bacteria, the 200nm membrane filtration can be used. The further confirmation of the presence of phage could be carried out by the fluorescence quantitative PCR described below. So the combination of these methods can accelerate the isolation of phage. And this is the first report try to detect the prophage of Streptococcus suis.To assess the antibacterial effect of potent phage, the real-time PCR were developed to determine the accurate growth and decline of Streptococcus suis or the phage, respectively. In this work, the primers target the mrp gene(muramidase-released protein, MRP)were designed to detect Streptococcus suis type 2. The results show that the real-time PCR can identified Streptococcus suis type 2 within 30 minutes with the detection limit less than 30 templates per ml. The specificity test showed that it can distinguish Streptococcus suis type 2 from E. coli and its phage, Bacillus subtilis, Staphylococcus aureus and its own phage SMP, etc.To detect the phage by real-time PCR, the target gene lysin was selected. The phage SMP could be determined within 30 minutes with sensitive limit less than 30 templates per ml. The specificity test showed that it can distinguish phage SMP from E. coli and its phage, Bacillus subtilis, Staphylococcus aureus and Streptococcus suis, etc.To find the kinetics of phage replication on Streptococcus suis in the view of qualitative analysis, the co-culture of phage and Streptococcus suis was carried out. For further quantitative analysis, the co-cultures of Streptococcus suis and phage were sampled at certain intervals and quantified by real-time PCR. To detect and explain the relationship between phage and its host bacteria, a mathematical model of predators has been developed after statistically analyzing the experimental data. The results showed that phage SMP had a relatively good germicidal and antibacterial capacity. It's the first time that using ecological mathematical models to simulate relationship between the micro-organisms, which provides a theoretical basis for the phage treatment.To confirm the role of phage SMP in the treatment of Streptococcus suis infection, the protection test in mice was carried out. The in vivo antibacterial activity of phage SMP was tested by using CD1 mice as infection model of S. suis serotype 2. The comparasion of bacterial counts between phage treated groups and S. suis challenging group were caaried out, The results revealed the good bactericidal effect of phage in vivo. In detail, the mice were divided into four groups, namely, PBS negative control group, phage injection group, bacterial challenge group, and phage treated bacterial challenge group. Injections were done through tail-vein, and orbital blood was sampled at regular intervals. The number of bacteria and phages were counted. The results showed that the phage have bactericidal effect on Streptococcus suis, which can accelerates the reduction of bacteria in mice, while the peak of bacteria reduced, so that the phage has an obvious protective effect in mice. This suggests that SMP can be used as a highly efficient anti-infective agent to prevent and control the disease caused by Streptococcus suis.