AUTHOR=Yi Li , Jin Manyu , Gao Mengxia , Wang Haikun , Fan Qingying , Grenier Daniel , Sun Liyun , Wang Shaohui , Wang Yang 

TITLE=Specific quantitative detection of Streptococcus suis and Actinobacillus pleuropneumoniae in co-infection and mixed biofilms

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2022.898412

DOI=10.3389/fcimb.2022.898412

ISSN=2235-2988

ABSTRACT=<p>Respiratory infections seriously affect the swine industry worldwide. Co-infections of two vital pathogenic bacteria <italic>Streptococcus suis</italic> (<italic>S. suis</italic>) and <italic>Actinobacillus pleuropneumoniae</italic> (<italic>A. pleuropneumoniae</italic>), colonizing the respiratory tract often occurs in veterinary clinical practice. Moreover, our previous research found that <italic>S. suis</italic> and <italic>A. pleuropneumoniae</italic> can form biofilm <italic>in vitro</italic>. The formation of a mixed biofilm not only causes persistent infections, but also increases the multiple drug resistance of bacteria, which brings difficulties to disease prevention and control. However, the methods for detecting <italic>S. suis</italic> and <italic>A. pleuropneumoniae</italic> in co-infection and biofilm are immature. Therefore, in this study, primers and probes were designed based on the conservative sequence of <italic>S. suis gdh</italic> gene and <italic>A. pleuropneumoniae apx</italic>IVA gene. Then, a TaqMan duplex real-time PCR method for simultaneous detection of <italic>S. suis</italic> and <italic>A. pleuropneumoniae</italic> was successfully established <italic>via</italic> optimizing the reaction system and conditions. The specificity analysis results showed that this TaqMan real-time PCR method had strong specificity and high reliability. The sensitivity test results showed that the minimum detection concentration of <italic>S. suis</italic> and <italic>A. pleuropneumoniae</italic> recombinant plasmid was 10 copies/μL, which is 100 times more sensitive than conventional PCR methods. The amplification efficiencies of <italic>S. suis</italic> and <italic>A. pleuropneumoniae</italic> were 95.9% and 104.4% with R<sup>2</sup> value greater than 0.995, respectively. The slopes of the calibration curves of absolute cell abundance of <italic>S. suis</italic> and <italic>A. pleuropneumoniae</italic> were 1.02 and 1.09, respectively. The assays were applied to cultivated mixed biofilms and approximately 10<sup>8</sup> CFUs per biofilm were quantified when 10<sup>8</sup> CFUs planktonic bacteria of either <italic>S. suis</italic> or <italic>A. pleuropneumoniae</italic> were added to biofilms. In summary, this study developed a TaqMan real-time PCR assay for specific, accurate quantification of <italic>S. suis</italic> or <italic>A. pleuropneumoniae</italic> in mixed biofilms, which may help for the detection, prevention and control of diseases caused by a bacterial mixed infection involving <italic>S. suis</italic> and <italic>A. pleuropneumoniae</italic>.</p>