Streptococcus suiscaracterización fenotípica y molecular de aislados y desarrollo de una nueva vacuna recombinante

Resum

Respiratory diseases are currently one of the most critical health issues in the porcine industry. They are often the result of a combination of primary and opportunistic infectious agents. In pig farming, the main threat comes from the Porcine Respiratory Disease Complex (PRDC), a term coined for describing the viral and bacterial etiology pneumonia processes developed in pigs between 16 and 20 weeks of age. Some of the most relevant agents in this group include the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Glaesserella parasuis, Pasteurella multocida and Streptococcus suis. Along with these microorganisms, non-infectious factors (such as animal handling and controlling environmental factors) contribute decisively to respiratory disease, increasing the dissemination of pathogens and creating unfavorable conditions that lead to stress for the animals. In these terms, S. suis is one of the main post-weaning pathogens in the porcine industry. This Gram-Positive bacterium resides asymptomatically in the upper respiratory tract, intestine, and genitalia of pigs as part of their microbiota. Colonization by S. suis typically occurs through close contact with the mother and between the piglets themselves during and after farrowing. However, it can become pathogenic when it penetrates the mucosal barrier. Once it passes those barriers, the bacterium enters the bloodstream. At this point, the CPS of S. suis allows protection against phagocytosis and monocyte and macrophage-mediated killing. Hence, the bacteria can survive in blood and spread throughout the central nervous system and other host organs, commonly causing meningitis, septicemia, arthritis, endocarditis, septicemia, or sudden death in diseased pigs. Currently, the most common classification method of the bacteria is based on the antigens on its cell surface, which allows the detection of 29 serotypes. The most prevalent serotype worldwide is serotype 2, while in Europe, serotypes 2 and 9 are the most prevalent and tend to be the most virulent. However, the recent advances in multilocus sequence typing (MLST) have allowed S. suis strain classification according to their sequence type, detecting up to 616 sequence types, 61 % of which belong to specific serotypes. This Doctoral Thesis addressed the analysis of the serotype distribution of 909 S. suis isolates from different regions of Spain. The most common serotypes among the isolates were, in descending order, 9, 1-14, 2-1/2, 7, 3, and 8. Furthermore, the PCR detection of virulence determinants was studied, as epf, mrp, sly, gapdh and luxS genes can provide valuable data about the virulence profile of the strains. In this Doctoral Thesis, these five genes were studied in 909 isolates. In decreasing order, the most detected virulence factors among the isolates were luxS, mrp, gapdh, sly and mrp. The most observed virulence pattern was the epf + /mrp + /sly + /luxS + /gapdh + pattern. In addition, it was determined whether there was an association between isolates belonging to the different serotypes and the expression of specific virulence markers. The statistical analyses gave rise to three statistically significant associations. The strongest association was the correlation between isolate belonging to one of the most significant serotypes, with its virulence profile including the epf + / mrp + / sly + pattern, with a p-value less than 0.0001. On the other hand, concerning the high variability between S. suis strains, different sequence types and serotypes are the main difficulties in developing an effective vaccine formulation which can provide broad and long-lasting protection. Furthermore, the bacterium can evade the host's immune system and survive within the host. This ability makes the task of developing a universal vaccine even more difficult. Although many studies have tried to develop vaccines against S. suis, an utterly effective solution has not been found. An effective vaccine should be able to stimulate an immune response that can recognize and neutralize all variants of the bacteria. For this reason, in this Doctoral Thesis, two highly conserved proteins were chosen as vaccine candidates to achieve complete and heterologous protection against S. suis. Two recombinant proteins were designed from the sequences of these proteins, which were obtained through protein expression and purification protocols. After that, three vaccine formulations were prepared with the corresponding adjuvant and an immunization test was carried out in mice to assess the titer of antibodies developed by the vaccine. Both B and A proteins proved to be proteins capable of causing seroconversion in BALB/c mice after intraperitoneal administration. However, they could not protect mice against the challenge of a virulent strain of S. suis serotype 2. Likewise, the A protein was more immunogenic than the B protein and the combination of both. However, the experimental test conditions in mice and the recombinant protein expression protocols should be optimized to allow better extrapolation of the obtained results, achieve greater efficiency in the performed procedures and eventually transfer the experimental test to pigs. Nevertheless, in addition to the threat posed by the bacterium, the resistance it is developing to antibiotics, like other pathogens, is becoming an emerging problem. In fact, according to a 2019 World Health Organization (WHO) report, around 2.4 million people could die in developed countries between 2015 and 2050 without a strategic plan to control antibiotic resistance. Specifically, we evaluated the prevalence in our isolates of six antibiotic-resistance genes (ARG) and the susceptibility to eighteen antibiotics to determine the most effective treatment of streptococcal diseases in pigs. In addition, we determined if there was an association between the presence of ARGs against a specific family of antibiotics and the resistance observed at the phenotypic level when the isolate was exposed to that family of antimicrobials. The most detected resistance genes among the 303 isolates studied were, in decreasing order, tet(O), erm(B), lsa(E) and lnu(B), tet(M) and mef(A/E). Likewise, the most detected resistance pattern was the pattern erm(B) + / mef(A/E) − / tet(M) − / tet(O) + / lsa(E)/lnu(B) −. On the other hand, regarding antimicrobial susceptibility, the most effective antibiotics to inhibit the growth of S. suis were, in decreasing order, ampicillin, gentamicin and ceftiofur. Likewise, the least effective antibiotics to inhibit bacterial growth were sulfadimethoxine, chlortetracycline and oxytetracycline. Finally, the study of the association between the presence of specific antibiotic resistance genes and the resistance observed at the phenotypic level allowed up to seven statistically significant associations to be detected. The strongest association was the one relating the presence of the tet(O) gene with the resistance observed at the phenotypic level for chlortetracycline and oxytetracycline, with a p-value less than 0.0001. Therefore, it has been demonstrated that S. suis isolates used in this study exhibited worrisome AMR patterns, as antibiotic susceptibility testing showed overall high resistance levels. Besides, the two vaccinal candidates effectively induced seroconversion in BALB/c mice. However, optimization protocols will be required to enhance the obtained IgG titers.