METHODOLOGY: Here, we further confirmed and characterized this bacterial species using PCR, histological staining, whole-genome sequencing, and bioinformatics approaches. PCR assays with in-house designed primer sets and 16S universal primers showed clear positive bands in the cerebrum, cerebellum, lung, and blood of UM3 suggesting that UM3 might have developed septicaemia. Histological staining showed the presence of Gram-negative rod-shaped bacteria in the pangolin brain and lungs, indicating the colonization of the bacteria in these two organs. In addition, PCR screening of UM3's fetal tissues revealed the presence of P. fungorum in the gastrocnemius muscle, but not in other tissues that we examined. We also sequenced and reconstructed the genome of pangolin P. fungorum, which has a genome size of 7.7 Mbps.
CONCLUSION: Our study is the first to present detailed evidence of the presence of P. fungorum in a pangolin and her fetus (although preliminary results were presented in our previous article). Here, we raise the concern that P. fungorum may potentially infect humans, especially YOPI (young, old, pregnant, and immunocompromised) people. Therefore, caution should be exercised when using this bacterial species as biodegradation or bioremediation agents in agriculture.
METHODS: To determine the antibacterial effectiveness of the MAC against Pseudomonas aeruginosa, we conducted minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques were employed to observe bacterial morphology and biofilm formation. We further performed a biofilm inhibition assay to assess the effect of the MAC on biofilm formation. Whole-transcriptome sequencing and bioinformatics analysis were employed to elucidate the antibacterial mechanism of the MAC. Additionally, the expression levels of differentially expressed genes were validated using the real-time PCR approach.
RESULTS: Our findings demonstrated the antibacterial activity of the MAC against P. aeruginosa. SEM analysis revealed that the MAC can induce morphological changes in bacterial cells. The biofilm assay showed that the MAC could reduce biofilm formation. Whole-transcriptome analysis revealed 1093 differentially expressed genes consisting of 659 upregulated genes and 434 downregulated genes, in response to the MAC treatment. Mechanistically, the MAC inhibited P. aeruginosa growth by targeting metabolic processes, secretion system, signal transduction, and cell membrane functions, thereby potentially compromising the survival of this human pathogen. This study provides valuable insights into the antibacterial and antibiofilm activities of the MAC, a synergistic and cost-effective malic acid combination, which holds promise as a potential therapeutic drug cocktail for treating human infectious diseases in the future.