The rising antibiotic resistance of bacteria imposes a severe threat on human health. Inhibition of bacterial virulence is an alternative approach to develop new antimicrobials. Molecules targeting antibiotic resistant enzymes have been used in combination with cognate antibiotics. It might be ideal that a molecule can simultaneously suppress virulence factors and antibiotic resistance. Here we combined genetic and computer-aided inhibitor screening to search for such molecules against the bacterial pathogen Pseudomonas aeruginosa. To identify target proteins that control both virulence and antibiotic resistance, we screened for mutants with defective cytotoxicity and biofilm formation from 93 transposon insertion mutants previously reported with increased antibiotic susceptibility. A pyrD mutant displayed defects in cytotoxicity, biofilm formation, quorum sensing and virulence in an acute mouse pneumonia model. Next, we employed a computer-aided screening to identify potential inhibitors of the PyrD protein, a dihydroorotate dehydrogenase (DHODase) involved in pyrimidine biosynthesis. One of the predicted inhibitors was able to suppress the enzymatic activity of PyrD as well as bacterial cytotoxicity, biofilm formation and antibiotic resistance. A single administration of the compound reduced the bacterial colonization in the acute mouse pneumonia model. Therefore, we have developed a strategy to identify novel treatment targets and antimicrobial molecules.

Tryptophan (Trp) plays an important role in piglet growth. However, the effect of dietary Trp on microbial flora is still poorly understood. A total of 40 28-d weaned piglets were allocated to four groups with 10 barrows per group and one pig per replicate. Piglets were fed a corn and soybean meal-based diet with 0.14%, 0.21%, 0.28%, or 0.35% Trp for four weeks. Five piglets from each diet group were euthanized, and blood and tissue samples were collected. The average daily body weight gain, average daily feed intake, feed conversion ratio, spleen index, pancreas index, longissimus dorsi muscle index, plasma insulin, 5-hydroxytryptamine, kynurenine, and Trp concentrations of weaned piglets increased in a dose-dependent manner (p < 0.05). Compared with the 0.14% Trp diet, the adequate-Trp diets (0.21%, 0.28%, or 0.35%) down-regulated the relative abundances of 12 genera including Turicibacter, Prevotella, Mitsuokella, Anaerovibrio, Megasphaera, Succinivibrio, Sutterella, Desulfovibrio, and Methanobrevibacter (p < 0.05); up-regulated the abundances of Ruminococcaceae, Lactobacillus, and Muribaculaceae in the colon (p < 0.05); and augmented the mRNA level and concentration of porcine β-defensin 2 in the small intestinal mucosa (p < 0.05). Moreover, Trp-adequate diets increased the abundances of Trp hydroxylase, indoleamine 2,3-dioxygenase, porcine β-defensin 2, phosphorylated mammalian target of rapamycin, and phosphorylated protein kinase B in the small intestinal mucosa (p < 0.05). We noted that a corn and soybean meal-based diet with 0.35% Trp may be a nutritional strategy to improve growth performance, intestinal mucosal barrier integrity, and intestinal microbial ecology in weaned piglets.