Quercetin targets SarA of methicillin-resistant Staphylococcus aureus to mitigate biofilm formation.

IMPORTANCE: Anti-biofilm is an important strategy against Staphylococcus aureus chronic infection. SarA is a positive regulator of biofilm formation in S. aureus. In this study, we identified the SarA inhibitor quercetin using computer simulation screening. Previous studies have shown that quercetin inhibits biofilm; however, the underlying mechanism remains unknown. This study revealed the inhibitory effect of quercetin on the SarA protein. We also isolated the SarA protein and confirmed its interaction with quercetin in vitro. Besides, the inhibitory effect of quercetin on the transcription and translation levels of the SarA protein was also determined. The effects of quercetin on S. aureus biofilm inhibition and biofilm components were consistent with the changes in the transcription level of biofilm-related genes regulated by SarA. In summary, our study revealed the mechanism by which quercetin affects biofilm formation by inhibiting the transcriptional regulator SarA of S. aureus.

Study on the inhibitory effect of quercetin combined with gentamicin on the formation of Pseudomonas aeruginosa and its bioenvelope.

OBJECTIVE: The potential effects of quercetin and gentamicin combination on the bacteriostatic activity and biofilm formation of Pseudomonas aeruginosa (PA) were examined, and the findings provided a theoretical basis for the development of quercetin as a new biofilm inhibitor. METHODS: The minimum inhibitory concentration (MIC) of eight PAs was determined by microdilution method and the partial inhibitory concentration index (FICI) of the combined drug was analyzed by micro-dilution method. Thereafter, the lowest film inhibitory concentration (MBIC) of quercetin and gentamicin alone and in combination was evaluated by crystal violet staining. Finally, scanning electron microscopy (SEM) and laser confocal microscopy (CLSM) were used to decipher the inhibitory effect of the combination on biofilm formation. OUTCOME: The antibacterial activity of quercetin alone was relatively weak, but after combination with gentamicin, the antibacterial activity was significantly enhanced, as evident by FICI of 0.28 and 0.53 and manifested as synergistic or additive effect, which indicated that quercetin can enhance gentamicin antibacterial activity. The results of crystal violet staining revealed that quercetin and gentamicin alone exhibited a similar biofilm formation inhibitory effect, but the inhibitory effect was substantially weaker, and the antibiofilm activity was stronger and exhibited a dose-dependent response after the combination of the two with 1/2FICI. The results of scanning electron microscopy and laser confocal microscopy also showed that the treatment of PA biofilm after combining quercetin and gentamicin with 1/2FICI could completely destroy the spatial structure of the complete biofilm, significantly reduce the thickness of bacteria, and markedly reduce the proportion of viable bacteria in the membrane. CONCLUSION: The combination of quercetin and gentamicin can effectively inhibit the formation of PA as well as its biofilm, and exhibit synergistic and additive effects.

Insights into the Effects and Mechanism of Andrographolide-Mediated Recovery of Susceptibility of Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

The frequent resistance associated with ß-lactam antibiotics and the high frequency of mutations in ß-lactamases constitute a major clinical challenge that can no longer be ignored. Andrographolide (AP), a natural active compound, has been shown to restore susceptibility to ß-lactam antibiotics. Fluorescence quenching and molecular simulation showed that AP quenched the intrinsic fluorescence of ß-lactamase BlaZ and stably bound to the residues in the catalytic cavity of BlaZ. Of note, AP was found to reduce the stability of the cell wall (CW) in methicillin-resistant Staphylococcus aureus (MRSA), and in combination with penicillin G (PEN), it significantly induced CW roughness and dispersion and even caused its disintegration, while the same concentration of PEN did not. In addition, transcriptome sequencing revealed that AP induced a significant stress response and increased peptidoglycan (PG) synthesis but disrupted its cross-linking, and it repressed the expression of critical genes such as mecA, blaZ, and sarA. We also validated these findings by quantitative reverse transcription-PCR (qRT-PCR). Association analysis using the GEO database showed that the alterations caused by AP were similar to those caused by mutations in the sarA gene. In summary, AP was able to restore the susceptibility of MRSA to ß-lactam antibiotics, mainly by inhibiting the ß-lactamase BlaZ, by downregulating the expression of critical resistance genes such as mecA and blaZ, and by disrupting CW homeostasis. In addition, restoration of susceptibility to antibiotics could be achieved by inhibiting the global regulator SarA, providing an effective solution to alleviate the problem of bacterial resistance. IMPORTANCE Increasingly, alternatives to antibiotics are being used to mitigate the rapid onset and development of bacterial resistance, and the combination of natural compounds with traditional antibiotics has become an effective therapeutic strategy. Therefore, we attempted to discover more mechanisms to restore susceptibility and effective dosing strategies. Andrographolide (AP), as a natural active ingredient, can mediate recovery of susceptibility of MRSA to ß-lactam antibiotics. AP bound stably to the ß-lactamase BlaZ and impaired its hydrolytic activity. Notably, AP was able to downregulate the expression of critical resistance genes such as mecA, blaZ, and sarA. Meanwhile, it disrupted the CW cross-linking and homeostasis, while the same concentration of penicillin could not. The multiple inhibitory effect of AP resensitizes intrinsically resistant bacteria to ß-lactam antibiotics, effectively prolonging the use cycle of these antibiotics and providing an effective solution to reduce the dosage of antibiotics and providing a theoretical reference for the prevention and control of MRSA.

Research progress on the association between mastitis and gastrointestinal microbes in dairy cows and the effect of probiotics.

Mastitis in dairy cows affects milk quality and thereby constrains the development of the dairy industry. A clear understanding of the pathogenesis of mastitis can help its treatment. Mastitis is caused by the invasion of pathogenic bacteria into the mammary gland through the mammary ducts. However, recent studies suggested that an endogenous entero-mammary pathway in dairy cattle might also be playing an important role in regulating mastitis. Also, probiotic intervention regulating host gut microbes has become an interesting tool to control mastitis. This review discusses the association of gastrointestinal microbes with mastitis and the mechanism of action of probiotics in dairy cows to provide new ideas for the management of mastitis in large-scale dairy farms.

Analysis of virulence genes, drug resistance detection, and pathogenicity in Enterococcus from farm animals.

This study aimed to investigate the presence of eight virulence genes (ace, asa1, esp, efaA, gelE, cylA, agg, fsr) in Enterococcus from a variety of animals and to explore the drug resistance and pathogenicity. This could provide a theoretical basis for clinical treatment of Enterococcus infections. Anal swabs from pigs, chickens, cattle, and dogs in farms and pet hospitals were collected for Enterococcus isolation and identification. Eight virulence genes were detected (PCR method), and drug resistance was assessed (drug-sensitive paper method). The strains containing different virulence genes were then divided into EV1, EV2, and EV3 groups. The LD50 and pathogenicity was examined by intra-peritoneal injection to infect mice. Differences were found in the detection rates of virulence genes in Enterococcus from the different animals. The highest overall detection rate was for the esp gene (78.0%), and the lowest for the cylA gene (15.5%). Eight genes were detected most frequently in Enterococcus from dogs and least frequently from cattle. Among the Enterococcus strains from four variety of animals, drug resistance was highest against sulfamethoxazole (100%), cefotaxime (>97%), and cefotaxitin (>93%). Drug resistance was lowest against vancomycin (0%), levofloxacin (<12%) and ciprofloxacin (<13%). The LD50 for each of the three groups was EV1LD50=8.71×109CFU， EV2LD50=2.34×1010CFU，and EV3LD50=9.33×1010CFU. The Enterococcus12LD50 dose group caused significant clinical symptoms in mice, with pathological effects on the heart, liver, lungs, and kidneys, and particularly on the urinary system. The abundance of Enterococcus virulence genes, drug resistance, and pathogenicity vary among different animal origins, and the pathology caused by Enterococcus requires effective treatment protocols based on species and regional characteristics.

Inhibitory Effect of Andrographis paniculata Lactone on Staphylococcus aureus α-Hemolysin.

We investigated the effect of andrographolide (AP) on the hemolytic capacity of Staphylococcus aureus (S. aureus) isolated from our region. AP is a labdane diterpenoid isolated from the stem and leaves of Andrographis paniculata. The hla gene from 234 S. aureus strains and the quality control standard strain ATCC29213 in dairy cows in some areas of Ningxia was analyzed. Evolutionary analysis, homology modeling, and functional enrichment annotation of α-hemolysin Hla detected from our region were performed through bioinformatics. The hemolytic ability of S. aureus isolates from the region was examined using the hemolysis test, and the effect of AP on S. aureus was quantified. Moreover, the effect of AP on the transcript levels of hla and genes highly related to hla (i.e., clfA and fnbA) was examined through fluorescence quantitative PCR. The mode of action of AP on the detected Hla was analyzed through molecular docking and dynamic simulation. The results showed that S. aureus in our region has a high rate of hla carriage. The hemolytic activity of strains NM98 and XF10 was significant, and ATCC29213 also exhibited some hemolytic activity. AP could inhibit the expression of Hla and its related proteins by downregulating hla, clfA, and fnbA transcript levels, which in turn attenuated the S. aureus hemolytic activity. Meanwhile, the AP molecule can form three hydrogen bonds with residues ASN105, SER106, and THR155 of Hla protein; bind with PRO103 through alkyl intermolecular forces; and form carbon hydrogen bonds with LYS154, reflecting that the AP molecule has a comparatively ideal theoretical binding activity with Hla protein. Among them, PRO103 and LYS154 are highly conserved in Hla protein molecules and play pivotal roles in the biological functions of Hla, and their binding may affect these functions. Their binding may also prevent the conformational transition of Hla from a monomer to an oligomer, thus inhibiting Hla hemolytic activity. This study offers a molecular basis for use of AP as an antivirulence drug and new ideas for developing novel drugs against S. aureus infection.

Potential Mechanisms of Quercetin Influence the ClfB Protein During Biofilm Formation of Staphylococcus aureus.

This study aimed to establish the mode of binding between Quercetin (QEN) and an essential protein called ClfB in forming biofilm in Staphylococcus aureus (S. aureus). In this study, the raw data of GSE163153 were analyzed for quality control, alignment, and gene counts, and the differential analysis detected the key differentially expressed genes (DEGs) assisting in the formation of the S. aureus biofilm. Then, the protein-protein interaction (PPI) and gene function enrichment analyses of the target genes, identified a gene called clfB to be closely related to biofilm formation. ClfB was structurally characterized, molecularly docked, and kinetically simulated to unravel the mode of binding of QEN to ClfB. Meanwhile, the growth curve and transmission electron microscopy methods examined the effect of QEN on the S. aureus growth. Results indicated that the clfB gene was increasingly expressed during biofilm formation and was involved in cell adhesion, pathogenicity, and infection. We identified 5 amino acid sites of ClfB (D272, R331, I379, K391, E490) as potential sites for binding QEN, which would indirectly influence the changes in the functional sites N234, D270, Y273, F328, inhibiting the formation of biofilm. Meanwhile, 128 µg/ml of QEN could significantly inhibit the S. aureus biofilm formation. This manuscript serves as a molecular foundation for QEN as an antibacterial drug providing a new perspective for developing antibacterial drugs.

The effect of EBN combined with integrated hierarchical accountability nursing on patients with severe pneumonia.

OBJECTIVE: To explore the effects of evidence-based nursing (EBN) combined with integrated hierarchical accountability nursing on patients with severe pneumonia (SP). METHODS: 72 SP patients admitted to our hospital from March 2019 to March 2020 were recruited as the study cohort and randomly divided into control group (36 patients) or research group (36 patients). The control group underwent conventional nursing, and the research group underwent EBN combined with integrated hierarchical accountability nursing plus. The patients' respiratory function, inflammatory factor levels, hospital stay durations, mechanical ventilation times, complication rates, and nursing satisfaction levels were compared between the two groups. RESULTS: Before the nursing, there were no significant differences in the FVC, TLC, MVV, or VC levels between the two groups (P>0.05). After the nursing, the FVC, TLC, MVV, and VC levels in the research group were all lower than they were in the control group (all P<0.05). Before the nursing, the WBC, CRP, and PCT levels in the two groups were similar (P>0.05). After the nursing, the WBC, CRP, and PCT levels in the research group were significantly lower than they were in the control group (P<0.05). The hospital stay durations and mechanical ventilation times in the research group were shorter than they were in the control group (P<0.05). The complication rate in the research group was lower than it was in the control group (5.56% vs. 27.78% P<0.05). The nursing satisfaction level in the research group was higher than it was in the control group (97.22% vs. 77.78% P<0.05). CONCLUSION: EBN combined with integrated hierarchical accountability nursing has a good application effect on patients with SP. It can significantly improve patients' respiratory function and inflammatory factor levels, reduce the duration of patient hospital stays, reduce their mechanical ventilation times, and complication rate, and enhance their satisfaction with the nursing. Thus, it is worthy of further promotion.

[Effects of MTA, iRoot SP and AH Plus on proliferation and differentiation of human periodontal ligament stem cells].

PURPOSE: To explore the effects of MTA, iRoot SP and AH Plus on periodontal ligament stem cells. METHODS: The periodontal ligament stem cells were cloned by limiting dilution culture method. The effects of MTA, iRoot SP and AH Plus on proliferation and apoptosis of periodontal ligament stem cells were detected by MTT and Annexin-V-FITC/PI double staining. Alizarin and qRT-PCR were used to evaluate the effect of MTA, iRoot SP and AH plus on osteogenesis of periodontal ligament stem cells. SPSS 21.0 software package was used for statistical analysis. RESULTS: MTA showed mild toxicity at 24 and 48 hours, AH Plus showed mild toxicity at 24 h. iRoot SP was the least (P<0.05) compared to MTA and AH Plus. The effect of three kinds of materials on apoptosis of periodontal ligament stem cells gradually decreased with the prolongation of time. Compared with the control group, the three kinds of materials were toxic at 3 d, the toxicity of MTA was the strongest and the toxicity of iRoot SP was the lowest(P<0.05). Mineralization nodules in MTA and iRoot SP group were significantly higher than those in AH Plus and control group. The expression of OC, RUNX2, COL1A and ALP gene was higher at 7, 14, 21 d than in the control group and the expression of iRoot SP mineralization was the greatest(P<0.05). CONCLUSIONS: The hardened iRoot SP is non-toxic to human periodontal ligament stem cells. Osteogenic ability and mineralization capacity of hardened iRoot SP on human periodontal ligament stem cells are better than MTA.

Mutation signatures in germline mitochondrial genome provide insights into human mitochondrial evolution and disease.

Variations in mitochondrial DNA (mtDNA) have been fundamental for understanding human evolution and are causative for a plethora of inherited mitochondrial diseases, but the mutation signatures of germline mtDNA and their value in understanding mitochondrial pathogenicity remain unknown. Here, we carried out a systematic analysis of mutation patterns in germline mtDNA based on 97,566 mtDNA variants from 45,494 full-length sequences and revealed a highly non-stochastic and replication-coupled mutation signature characterized by nucleotide-specific mutation pressure (G > T>A > C) and position-specific selection pressure, suggesting the existence of an intensive mutation-selection interplay in germline mtDNA. We provide evidence that this mutation-selection interplay has strongly shaped the mtDNA sequence during evolution, which not only manifests as an oriented alteration of amino acid compositions of mitochondrial encoded proteins, but also explains the long-lasting mystery of CpG depletion in mitochondrial genome. Finally, we demonstrated that these insights may be integrated to better understand the pathogenicity of disease-implicated mitochondrial variants.