Gramine sensitizes Klebsiella pneumoniae to tigecycline killing.

BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters. METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect. RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1). CONCLUSION: This study provides the first evidence of gramine's therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.

IS6 family insertion sequences promote optrA dissemination between plasmids varying in transfer abilities.

Plasmids are the primary vectors for intercellular transfer of the oxazolidinone and phenicol cross-resistance gene optrA, while insertion sequences (ISs) are mobile genetic elements that can mobilize plasmid-borne optrA intracellularly. However, little is known about how the IS-mediated intracellular mobility facilitates the dissemination of the optrA gene between plasmid categories that vary in transfer abilities, including non-mobilizable, mobilizable, and conjugative plasmids. Here, we performed a holistic genomic study of 52 optrA-carrying plasmids obtained from searches guided by the Comprehensive Antibiotic Resistance Database. Among the 132 ISs identified within 10 kbp from the optrA gene in the plasmids, IS6 family genes were the most prevalent (86/132). Homologous gene arrays containing IS6 family genes were shared between different plasmids, especially between mobilizable and conjugative plasmids. All these indicated the central role of IS6 family genes in disseminating plasmid-borne optrA. Thirty-three of the 52 plasmids were harbored by Enterococcus faecalis found mainly in humans and animals. By Nanopore sequencing and inverse PCR, the potential of the enterococcal optrA to be transmitted from a mobilizable plasmid to a conjugative plasmid mediated by IS6 family genes was further confirmed in Enterococcus faecalis strains recovered from the effluents of anaerobic digestion systems for treating chicken manure. Our findings highlight the increased intercellular transfer abilities and dissemination risk of plasmid-borne optrA gene caused by IS-mediated intracellular mobility, and underscore the importance of routinely monitoring the dynamic genetic contexts of clinically important antibiotic resistance genes to effectively control this critical public health threat. KEY POINTS: • IS6 was prevalent in optrA-plasmids varying in intercellular transfer abilities. • Enterococcal optrA-plasmids were widespread among human, animal, and the environment. • IS6 elevated the dissemination risk of enterococcal optrA-plasmids.

Gigantol restores the sensitivity of mcr carrying multidrug-resistant bacteria to colistin.

BACKGROUND: The emergence and wide spread of plasmid-mediated colistin resistance gene (mcr-1) and its mutants have immensely limited the efficacy of colistin in treating multidrug-resistant (MDR) Gram-negative bacterial infections. The development of synergistic combinations of antibiotics with a natural product that coped with the resistance of MDR bacteria was an economic strategy to restore antibiotics activity. Herein, we investigated gigantol, a bibenzyl phytocompound, for restoring in vitro and in vivo, the sensitivity of mcr-positive bacteria to colistin. METHODS: The synergistic activity of gigantol and colistin against multidrug-resistant Enterobacterales was studied via checkerboard assay and time-killing curve. Subsequently, the transcription and protein expression levels of mcr-1 gene were determined by RT-PCR and Western blots. The interaction of gigantol and MCR-1 was simulated via molecular docking and verified via site-directed mutagenesis of MCR-1. Hemolytic activity and cytotoxicity assay were used to evaluate the safety of gigantol. Finally, the in vivo synergistic effect was evaluated via two animal infection models. RESULTS: Gigantol restored the activity of colistin against mcr-positive bacteria E.coli B2 (MIC from 4 µg/ml to 0.25 µg/ml), Salmonella 15E343 (MIC from 8 µg/ml to 1 µg/ml), K. pneumoniae 19-2-1 (MIC from 32 µg/ml to 2 µg/ml) carrying mcr-1, mcr-3, mcr-8, respectively. Mechanistic studies revealed that gigantol down-regulated the expression of genes involved in LPS-modification, reduced the MCR-1 products and inhibited the activity of MCR-1 by binding to amino acid residues Tyr287 and Pro481 in its D-glucose-binding pocket. Safety evaluation showed that the addition of gigantol relieves the hemolysis caused by colistin. Compared with monotherapy, the combination of gigantol and colistin significantly improved the survival rate of Gallgallella mellonella larvae and mice infected by E.coli B2. Moreover, there was a considerable decrease in the bacterial load present in the viscera of mice. CONCLUSION: Our results confirmed that gigantol was a potential colistin adjuvant, and could be used to tackle multi-drug resistant Gram-negative pathogen infections combined with colistin.

[Chemical constituents and pharmacological effects of Dictamni Cortex: a review].

Dictamni Cortex, the dried root bark of Dictamnus dasycarpus, has many chemical constituents, such as alkaloids, limonoids, flavonoids, sesquiterpenoids, glycosides, and steroids.It has the effects of anti-inflammation, anti-fungi, anti-arteriosclerosis, stopping bleeding, anti-cancer, neuroprotection, and antioxidation.The chemical constituents of Dictamni Cortex are the important material basis for its medicinal effects.This paper reviewed the chemical constituents and pharmacological activities of Dictamni Cortex and analyzed the research trend and present research progress on this medicinal, with a view to its further development and utilization.

Sodium dehydroacetate confers broad antibiotic tolerance by remodeling bacterial metabolism.

Antibiotic tolerance has been a growing crisis that is seriously threatening global public health. However, little is known about the exogenous factors capable of triggering the development of antibiotic tolerance, particularly in vivo. Here we uncovered that an previously approved food additive termed sodium dehydroacetate (DHA-S) supplementation remarkably impaired the activity of bactericidal antibiotics against various bacterial pathogens. Mechanistic studies indicated that DHA-S induced glyoxylate shunt and reduced bacterial cellular respiration by inhibiting the enzymatic activity of α-ketoglutarate dehydrogenase (α-KGDH). Furthermore, DHA-S mitigated oxidative stress imposed by bactericidal antibiotics and enhanced the function of multidrug efflux pumps. These actions worked together to induce bacterial tolerance to antibiotic killing. Interestingly, the addition of five exogenous amino acids, particularly cysteine and proline, effectively reversed antibiotic tolerance elicited by DHA-S both in vitro and in mouse models of infection. Taken together, these findings advance our understanding of the potential risks of DHA-S in the treatment of bacterial infections, and shed new insights into the relationships between antibiotic tolerance and bacterial metabolism.

Melatonin overcomes MCR-mediated colistin resistance in Gram-negative pathogens.

Background: Emergence, prevalence and widely spread of plasmid-mediated colistin resistance in Enterobacteriaceae strongly impairs the clinical efficacy of colistin against life-threatening bacterial infections. Combinations of antibiotics and FDA-approved non-antibiotic agents represent a promising means to address the widespread emergence of antibiotic-resistant pathogens. Methods: Herein, we investigated the synergistic activity between melatonin and antibiotics against MCR (mobilized colistin resistance)-positive Gram-negative pathogens through checkerboard assay and time-killing curve. Molecular mechanisms underlying its mode of action were elucidated. Finally, we assessed the in vivo efficacy of melatonin in combination with colistin against drug-resistant Gram-negative bacteria. Results: Melatonin, which has been approved for treating sleep disturbances and circadian disorders, substantially potentiates the activity of three antibiotics, particularly colistin, against MCR-expressing pathogens without enhancing its toxicity. This is evidence that the combination of colistin with melatonin enhances bacterial outer membrane permeability, promotes oxidative damage and inhibits the effect of efflux pumps. In three animal models infected by mcr-1-carrying E. coli, melatonin dramatically rescues colistin efficacy. Conclusion: Our findings revealed that melatonin serves as a promising colistin adjuvant against MCR-positive Gram-negative pathogens.

[Clinical examples of professor LI Zhi-dao's "tonifying three qi" acupuncture method].

Professor LI Zhi-dao, according to acupoint selection of syndrome differentiation in TCM basic theory, concluded a new therapy, namely "tonifying three qi" that is mainly based on three acupoints in the Conception Vessel. This method is consisted of Danzhong (CV 17), Zhongwan (CV 12) and Qihai (CV 6) in the Conception Vessel, which could successively nourish clear qi, stomach qi and original qi. In clinic, according to the severity of symptoms of three qi, the acupoints are selected flexibly, which could respectively treat deficiency of heart-lung qi, deficiency of stomach-spleen qi and deficiency of original qi. Some examples are also given in the article.

[Comparison of the effect of palonosetron versus tropisetron in prevention of vomiting in patients receiving high dose cisplatin-based chemotherapy].

OBJECTIVE: To evaluate the efficacy and toxicity of palonosetron for prevention of vomiting induced by high dose cisplatin-based chemotherapy. METHODS: One-hundred and twenty-eight patients received tropisetron 5 mg plus dexamethasone 10 mg at the first cycle or palonosetron 0.25 mg plus dexamethasone 10 mg, respectively, each administered 30 min before the initiation of high dose cisplatin-based chemotherapy. To observe the remission rate of acute emetic episodes and delayed emetic episodes, adverse effects and daily food-intake in the patients after the chemotherapy. RESULTS: The complete response (CR) rates for acute vomiting were not significantly different between the tropisetron and palonosetron cycles (75.8% vs. 79.7%, P>0.05). The complete control rate of delayed vomiting in the palonosetron cycle was significantly higher than that in the tropisetron cycle (70.3% vs. 50.8%, P<0.01). The food-intake decrease rate of palonosetron cycle was 18.8%, significantly lower than the 53.1% of the tropisetron cycle (P<0.05). The toxicity in the two cycles was similar and no grade 3-4 toxicity was observed. CONCLUSIONS: Palonosetron is superior to tropisetron with a lower remission rate of delayed emesis induced by high dose cisplatin-based chemotherapy and with tolerable toxicity. Moreover, the apparent emesis control of palonosetron treatment seems to provide an adequate food-intake in these patients.