ß-sitosterol inhibits trimethylamine production by regulating the gut microbiota and attenuates atherosclerosis in ApoE-/- mice.

The intestinal microbial metabolite trimethylamine (TMA), which is activated by flavin monooxygenase (FMO) to produce trimethylamine-N-oxide (TMAO), has been implicated in the pathogenesis of atherosclerosis (AS), leading to the development of therapeutic strategies for AS. This study aimed to investigate whether ß-sitosterol can inhibit TMA production in ApoE-/- mice by reshaping the gut microbial structure. 16S rRNA sequencing of the gut microbiota showed that ß-sitosterol has beneficial effects on intestinal flora function, especially the inhibition of bacteria genera that contain the gene cholintrimethylamine lyase, which is responsible for the major pathway for TMA production. In parallel, ß-sitosterol effectively reduced the TMA, FMO3, and TMAO levels while ameliorating the atherosclerotic plaques of AS mice. Moreover, ß-sitosterol could alleviate cholesterol metabolism and the inflammatory response, and improve the antioxidant defense capacity. These studies offer new insights into the mechanisms responsible for the antiatherosclerotic effects of ß-sitosterol, which targets the microbiota-metabolism-immunity axis as a possible therapy for AS.

Identification of choline-degrading bacteria from healthy human feces and used for screening of trimethylamine (TMA)-lyase inhibitors.

Gut microbiota-based choline metabolism produces trimethylamine (TMA), which is then further converted to the atherosclerosis-promoting metabolite trimethylamine-N-oxide (TMAO) by hepatic flavin-containing monooxygenases (FMOs) and TMAO plays an essential role in cardiovascular disease (CVD). Many Chinese herbal medicines had been used for the treatment of CVD. This study aimed to screen choline-degrading bacteria from healthy human feces and establish a platform in silico and in vitro approaches for screening TMA-lyase inhibitors from Chinese herbal medicines. Choline-degrading bacteria were screened from healthy human feces in basic salt medium using culture method. The isolated strains were identified as Klebsiella pneumoniae based on 16S rRNA and the presence of CutC gene. Structure of CutC choline lyase was obtained from the RCSB Protein Data Bank database, and the modeled structure was docked with natural compounds of Chinese herbal medicines origin using MOE. Further, we investigated the inhibitory effects of selected compounds by picric acid-toluene method using K. pneumoniae as bioassay indicator. We found that TMA level was significantly decreased when treated with ß-sitosterol and resveratrol. This study initially demonstrates the inhibitory effect of ß-sitosterol and resveratrol on the gut microbiota responsible for choline metabolism to TMA and sets up an inhibitor-screening platform for further experiments. It can be used as a model to evaluate herbal drug sources and their effects on the gut microbiota for cardiovascular disease.

Convergence of carbapenem resistance and hypervirulence in a highly-transmissible ST11 clone of K. pneumoniae: An epidemiological, genomic and functional study.

Co-occurrence of hypervirulence and KPC-2 carbapenem resistant phenotypes in a highly-transmissible ST11 clone ofKlebsiella pneumoniae has elicited deep concerns from public health stand point. To address this puzzle, we conducted a large-scale epidemiological, clinical and genomic study of K. pneumonia ST11 clones with both hypervirulence and carbapenem resistance in two tertiary hospitals in Zhejiang province. Most of the patients (15/23) were diagnosed with exclusively carbapenem-resistant K. pneumoniae (CRKP) infections. Ten death cases were reported, some of which are due to the failure of antibiotic therapies. As a result, we identified one new rare sequence types (ST449) to KPC-2-producing CRKP, in addition to the dominant ST11. These clinical isolates of K. pneumoniae are multi-drug resistant and possess a number of virulence factors. Experimental infections of wax moth larvae revealed the presence of hypervirulence at varied level, suggesting the complexity in bacterial virulence factors. However, plasmid curing assays further suggested that the rmpA2-virulence plasmid is associated with, but not sufficient for neither phenotypic hypermucoviscosity nor virulence of K. pneumoniae. Intriguingly, all the rmpA2 genes were found to be inactive due to genetic deletion. In total, we reported 21 complete plasmid sequences comprising 13 rmpA2-positive virulence plasmids and 8 blaKPC-2-harboring resistance plasmids. In addition to the prevalent pLVKP-like virulence plasmid variants (~178kb), we found an unexpected diversity among KPC-2-producing plasmids whose dominant form is IncFII-IncR type (~120kb), rather than the previously anticipated version of ~170kb. These findings provide an updated snapshot of convergence of hypervirulence and carbapenem resistance in ST11 K. pneumoniae.

Isolation and preliminary screening of potentially probiotic Weissella confusa strains from healthy human feces by culturomics.

PURPOSE: The objective of this study was to isolate and identify strains of bacteria from feces of healthy human and screen potential probiotic candidate by using culturomics method combined with the matrix-assisted laser desorption/ionization-time of flight mass spectrum (MALDI-TOF MS) and 16S rRNA gene sequencing. METHODS AND RESULTS: 31 strains were isolated and purified from human feces by culturomics method, and identified by MALDI-TOF MS and 16S rRNA gene sequencing. Then the obtained strains were tested for haemolytic activity, antibiotic susceptibility, acid and bile salts tolerance, antimicrobial activity, morphological and physiological characteristics. Three potential probiotic candidate strains named YM5Y, YM5S1 and YM5S2 were selected and identified as Weissella confusa. CONCLUSION: Our results suggest the culturomics approach could be used to isolate and screen human fecal strains which could eventually be used for the development of novel probiotics. In addition, the isolated strains of W. confusa can act as potential probiotics and should be explored further for their potential application.

Tea polyphenols inhibits biofilm formation, attenuates the quorum sensing-controlled virulence and enhances resistance to Klebsiella pneumoniae infection in Caenorhabditis elegans model.

Bacteria cells can communicate with each other via quorum sensing (QS) system. Various physiological characteristics including virulence factors and biofilm formation are controlled by QS. So interrupting the bacterial communication is an alternative strategy instead of antibiotics for control bacterial infection. The aim of this study was to investigate the effects of tea polyphenols (TPs) on quorum sensing and virulence factors of Klebsiella pneumoniae. In vitro study showed that the anti-QS activity of tea polyphenols against Chromobacterium violaceum in violacein production. At sub-MICs, TPs inhibited the motility, reduced protease and exopolysaccharide (EPS) production and also biofilm formation in K. pneumoniae. In addition, in vivo study showed that tea polyphenols at 200 µg/mL and 400 µg/mL increased the survival rate of Caenorhabditis elegans to 73.3% and 82.2% against K. pneumonia infection. Our findings suggest that tea polyphenols can act as an effective QS inhibitor and can serve as a novel anti-virulence agent for the management of bacterial pathogens.

In-vitro antibacterial effect of tea polyphenols combined with common antibiotics on multidrug-resistant Klebsiella pneumoniae.

BACKGROUND: To investigate the bactericidal effect of tea polyphenols on multidrug resistant Klebsiella pneumoniae and its antibacterial efficacy in combination with common antibiotics, and to explore its mechanism. METHODS: The VITEK 2 Compact automatic microbial identification system was used to identify 20 strains of Klebsiella pneumoniae isolated from clinical isolates. The KB method was used to detect sensitivity of common drugs such as imipenem, piperacillin, piperacillin/tazobactam and cefepime, cefotaxime and ceftazidime; agar dilution method was used for detection of minimum inhibitory concentration (MIC) of tea polyphenols; the variation of the diameter of the antibacterial ring after different concentrations (0.25 MIC, 0.5 MIC) of tea polyphenols in combination with commonly used antibacterial drugs was detected to judge the feasibility of the inhibition of the multidrug-resistant Klebsiella pneumoniae by the combination of tea polyphenols and commonly used antibacterial drugs. Congo red and crystal violent staining was used to detect the impact on bacterial biofilm formation and extracellular mucus-like substance (slime). RESULTS: The K-B values of 20 strains of Klebsiella pneumoniae against common antibiotics were 6-14 mm; the MIC of tea polyphenols against 20 strains of multi-drug resistant Klebsiella pneumoniae was 1024 ug/mL; 0.5 MIC tea polyphenols can increase the diameter of the bacteriostatic ring of imipenem, piperacillin, piperacillin/tazobactam, cefepime, cefotaxime and ceftazidime by 3-28 mm. Tea polyphenols at sub-inhibitory concentrations significantly inhibited the production of Klebsiella pneumoniae biofilm and extracellular mucus (slime). CONCLUSIONS: Tea polyphenols could be used in combination with commonly used antibiotics (imipenem, piperacillin, piperacillin/tazobactam, cefepime, cefotaxime and ceftazidime) against multidrug-resistant Klebsiella pneumoniae. It has a synergistic bactericidal effect and affects the formation of bacterial outer membrane and the production of extracellular slime-like substances (slime).

Tea polyphenols as an antivirulence compound Disrupt Quorum-Sensing Regulated Pathogenicity of Pseudomonas aeruginosa.

Green tea, a water extract of non-fermented leaves of Camellia sinensis L., is one of the nonalcoholic beverages in China. It is becoming increasingly popular worldwide, because of its refreshing, mild stimulant and medicinal properties. Here we examined the quorum sensing inhibitory potentials of tea polyphenols (TP) as antivirulence compounds both in vitro and in vivo. Biosensor assay data suggested minimum inhibitory concentrations (MICs) of TP against selected pathogens were 6.25 ~ 12.5 mg/mL. At sub-MIC, TP can specifically inhibit the production of violacein in Chromobacterium violaceum 12472 with almost 98% reduction at 3.125 mg/mL without affecting its growth rate. Moreover, TP exhibited inhibitory effects on virulence phenotypes regulated by QS in Pseudomonas aeruginosa. The total proteolytic activity, elastase, swarming motility and biofilm formation were reduced in a concentration-dependent manner. In vivo, TP treatment resulted in the reduction of P. aeruginosa pathogenicity in Caenorhabditis elegans. When its concentration was 3.125 mg/mL, the survival rate reached 63.3%. In the excision wound infection model, the wound contraction percentage in treatment groups was relatively increased and the colony-forming units (CFU) in the wound area were significantly decreased. These results suggested that TP could be developed as a novel non-antibiotic QS inhibitor without killing the bacteria but as an antivirulence compound to control bacterial infection.