Discovery of isatin-ß-methyldithiocarbazate derivatives as New Delhi metallo- ß-lactamase-1 (NDM-1) inhibitors against NDM-1 producing clinical isolates.

New Delhi metallo-ß-lactamase-1 (NDM-1) poses a threat to public health due to its capability to hydrolyze nearly all ß-lactam antibiotics, leaving limited treatment options for NDM-1 positive pathogens. Regrettably, there are presently no effective NDM-1 inhibitors in clinical use. This compels us to seek new compounds to combat multi-drug resistant bacterial infections (MDR). In our study, Zndm19 was identified as a new NDM-1 inhibitor through virtual screening and an NDM-1 enzyme activity inhibition assay. Subsequently, we employed the checkerboard method, time-killing assay, and combined disk test to investigate the synergistic bactericidal efficacy of Zndm19 in combination with meropenem (MEM). Meanwhile, molecular docking and site-directed mutagenesis were conducted to uncover the crucial amino acid residues engaged in Zndm19 binding. Finally, we established a mice peritonitis infection model to assess the synergistic effect of Zndm19 and MEM in vivo. Our findings demonstrated that 16 µg/mL of Zndm19 inhibited NDM-1 activity without affecting NDM-1 expression, restoring the bactericidal activity of MEM against NDM-1-positive Escherichia coli in vitro. Furthermore, MET-67, ASP-124, HIS-189, and HIS-250 amino acid residues constituted the active site of Zndm19 in NDM-1. Importantly, this combination therapy exhibited synergistic anti-infection activity in the mice peritonitis infection model, leading to an approximate 60% increase in survival rates and reduction of tissue bacterial load, effectively combating bacterial infection in vivo. In summary, our research validates that the synthetic novel NDM-1 inhibitor Zndm19 holds promise as a drug to treat drug-resistant bacterial infections, especially those harboring NDM-1.

Shionone-Targeted Pneumolysin to Ameliorate Acute Lung Injury Induced by Streptococcus pneumoniae In Vivo and In Vitro.

Streptococcus pneumoniae (S. pneumoniae), as a Gram-positive bacterium, can cause severe bacterial pneumonia, and result in high morbidity and mortality in infected people. Meanwhile, isolated drug-resistant S. pneumoniae is growing, which raises concerns about strategies for combatting S. pneumoniae infection. To disturb S. pneumoniae pathogenicity and its drug-resistance, developing novel anti-infective strategies or compounds is urgent. In this study, the anti-infective effect of shionone was explored. A minimum inhibitory concentration (MIC) assay and growth curve determination were performed to evaluate the effect of the tetracyclic triterpenoid compound shionone against S. pneumoniae. Hemolysis tests, western blotting, oligomerization inhibition assays, and molecular docking were carried out to explore the anti-infective mechanism of shionone. Moreover, the protective effect of shionone was also confirmed in a mousepneumonia model. The results showed that the excellent hemolytic inhibitory activity of shionone was observed at less than 8 µg/mL. Meanwhile, shionone could disturb the oligomerization of pneumolysin (PLY) but did not interfere with PLY expression at less than 4 µg/mL. Molecular docking suggested that shionone targeted the ASP-59, ILE-60, THR-57, PHE-344, and ASN-346 amino acid sites to reduce S. pneumoniae pathogenicity. Furthermore, shionone alleviated lung histopathologic injury and decreased lung bacterial colonization in vivo. The above results showed that shionone could bind to the PLY active pocket under the concentrations of 8 µg/mL and neutralize PLY hemolysis activity to reduce S. pneumoniae pathogenicity in vitro and in vivo.

Polyphosphate Kinase Is Required for the Processes of Virulence and Persistence in Acinetobacter baumannii.

Acinetobacter baumannii, one of the most successful bacteria causing severe nosocomial infection, was identified as a top-priority pathogen by the WHO. Thus, genetic manipulations to clarify the potential targets for fighting A. baumannii resistance and virulence are vital. Polyphosphate (polyP) kinase (PPK) is conserved in nearly all bacteria and is responsible for polyP formation, which is associated with bacterial pathogenicity and antibiotic resistance. In this study, ppk1-deficient (Δppk1::Apr), ppk1-complemented (Δppk1::Apr/PJL02-ppk1), and wild-type strains of A. baumannii ATCC 17978 were used to determine the influence of PPK1 on A. baumannii virulence and persistence mainly by polyP quantification, surface motility, biofilm formation, and bacterial persistence assays. Our work found that PPK1 is indispensable for polyP formation in vivo and that the motility of the PPK1-deficient strain was significantly impaired due to the lack of a pilus-like structure typically present compared with the complemented and wild-type strains. The deficiency of PPK1 also inhibited the biofilm formation of A. baumannii and decreased bacterial persistence under stimuli of high-concentration ampicillin (Amp) treatment, H2O2 stress, heat shock, and starvation stress. Furthermore, ppk1-deficient bacterium-infected mice showed a significantly reduced bacterial load and a decreased inflammatory response. However, complementation with PPK1 effectively rescued the impaired virulence and persistence of ppk1-deficient A. baumannii. In addition, metabonomic analysis revealed that PPK1 was associated with glycerophospholipid metabolism and fatty acid biosynthesis. Taken together, our results suggest that targeting PPK1 to control A. baumannii pathogenicity and persistence is a feasible strategy to fight this pathogen. IMPORTANCE A. baumannii was identified as a top-priority pathogen by the WHO due to its antibiotic resistance. Meanwhile, the pathogenicity of A. baumannii mediated by several vital virulence factors also cannot be ignored. Here, the role of PPK1 in A. baumannii was also explored. We found that the motility ability and biofilm formation of a PPK1-deficient strain were significantly impaired. Furthermore, PPK1 was essential for its persistence maintenance to resist stimuli of high-concentration Amp treatment, H2O2 stress, heat shock, and starvation stress. Metabonomic analysis revealed that PPK1 was associated with glycerophospholipid metabolism and fatty acid biosynthesis. In addition, ppk1-deficient bacterium-infected mice showed significantly reduced bacterial loads and a decreased inflammatory responses in vivo. Together, our results suggest that PPK1 is vital for A. baumannii pathogenicity and persistence.

A Dual-Action Molecule Suppresses S. aureus Infection as an Inhibitor Targeting Hla Pore Formation and TLR2 Signaling.

Antibiotic resistance is the greatest challenge for the treatment of Staphylococcus aureus (S. aureus) infection under the global antibiotic resistance crisis. With the bottleneck period of the development of new antibiotics, novel alternative agents are urgently in need. In this study, the small molecule amentoflavone is identified as a dual-action inhibitor of Hla, a pore-forming virulence determinant particularly important for S. aureus pathogenicity and Toll-like receptor 2 (TLR2) signaling, which triggers inflammation response upon recognizing pathogen-associated molecular patterns. Amentoflavone treatment conferred effective protection against S. aureus pneumonia through this dual-action mechanism. Mechanically, amentoflavone effectively inhibited Hla pore formation, thereby reducing Hla-mediated cytotoxicity and tissue damage; at the same time, amentoflavone suppressed TLR2-mediated inflammatory response by blocking the interaction between TLR2 and its adapter myeloid differentiation primary response gene 88 (MyD88). Surprisingly, TLR2 signaling induced by synthetic bacterial TLR2 agonists and other heat-killed gram-positive bacteria is also blocked by amentoflavone. In summary, these results presented amentoflavone as a potential antibiotic alternative that curbed S. aureus infection by simultaneously suppressing host-damaging virulence determinants derived from bacteria and the detrimental effect of excessive inflammation derived from the host rather than bacteria viability.

Synergistic Effect of Lithocholic Acid with Gentamicin against Gram-Positive Bacteria but Not against Gram-Negative Bacteria.

Listeria monocytogenes (L. monocytogenes) is an important Gram-positive food-borne pathogen that severely threatens public health. A checkerboard microdilution method was performed to evaluate the synergistic effect of lithocholic acid (LCA) with Gentamicin (Genta) against L. monocytogenes. BacLight LIVE/DEAD staining, scanning electron microscopy and biofilm inhibition assays were further used to explore the bactericidal effect and antibiofilm effect of this combination on L. monocytogenes. Additionally, the synergistic effects of LCA derivatives with Genta were also evaluated against L. monocytogenes, S.aureus and S. suis. The results indicated that a synergistic bactericidal effect was observed for the combined therapy of LCA at the concentration without affecting bacteria viability, with Genta. Additionally, LCA in combination with Genta had a synergistic effect against Gram-positive bacteria (L. monocytogenes, S. aureus and S. suis) but not against Gram-negative bacteria (E. coli, A. baumannii and Salmonella). BacLight LIVE/DEAD staining and scanning electron microscopy analysis revealed that the combination of LCA with Genta caused L. monocytogenes membrane injury, leading to bacteria death. We found that 8 µg/mL LCA treatment effectively improved the ability of Genta to eradicate L. monocytogenes biofilms. In addition, we found that chenodeoxycholic acid, as a cholic acid derivative, also improved the bactericidal effect of Genta against Gram-positive bacteria. Our results indicate that LCA represents a broad-spectrum adjuvant with Genta for infection caused by L. monocytogenes and other Gram-positive pathogens.

Dryocrassin ABBA ameliorates Streptococcus pneumoniae-induced infection in vitro through inhibiting Streptococcus pneumoniae growth and neutralizing pneumolysin activity.

To explore the role of dryocrassin ABBA (ABBA) in the prevention and treatment of Streptococcus pneumoniae (S. pneumoniae) infections in vitro, a minimal inhibitory concentration test, growth curve assay, hemolysis assay, BacLight LIVE/DEAD staining experiments, oligomerization inhibition assay, time-killing test, LDH release detection assay and cytotoxicity test were performed to evaluate the efficacy of ABBA against S. pneumoniae infections in vitro. The results indicated that ABBA treatment exists bactericidal effect on S. pneumoniae at a concentration of less than 8 µg/ml. Furthermore, ABBA was effective at inhibiting the oligomerization of pneumolysin (PLY) from reducing its hemolytic activity. Meanwhile, ABBA could ameliorate cell injury by neutralizing the biological activity of PLY without cytotoxicity. In summary, ABBA was a leading compound against S. pneumoniae infections through bactericidal effect and neutralizing PLY activity.

A Natural Dietary Flavone Myricetin as an α-Hemolysin Inhibitor for Controlling Staphylococcus aureus Infection.

Staphylococcus aureus, an important agent for lethal bacterial infections, can cause a broad spectrum of diseases in various host species. The emergence of multidrug-resistant and highly virulent strains has raised increasing concerns about the novel therapeutic strategies or agents available for treating S. aureus infection. The critical role of Hla, an essential virulence determinant, in the pathogenicity of S. aureus renders this toxin an attractive target for effective therapeutic applications. Here, we have identified myricetin as an effective inhibitor of Hla that simultaneously inhibits Hla production and neutralizes Hla activity without affecting bacterial growth. Myricetin treatment reduced the oligomerization of Hla and Hla-mediated biofilm formation. The addition of myricetin to the coinfection system of host cells and S. aureus significantly decreased cell injury and downregulated the inflammatory response in cells. Furthermore, S. aureus-infected mice that received myricetin showed alleviated tissue damage in the lung. Our results indicated that myricetin inhibits S. aureus virulence by targeting Hla and downregulates the inflammatory response in host cells. Overall, in addition to traditional antibiotics with antibacterial activity, myricetin may represent a potential candidate, and strategy for S. aureus infection.