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.

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.

Factors and Mechanisms Influencing Conjugation In Vivo in the Gastrointestinal Tract Environment: A Review.

The emergence and spread of antibiotic resistance genes (ARGs) have imposed a serious threat on global public health. Horizontal gene transfer (HGT) via plasmids is mainly responsible for the spread of ARGs, and conjugation plays an important role in HGT. The conjugation process is very active in vivo and its effect on the spreading of ARGs may be underestimated. In this review, factors affecting conjugation in vivo, especially in the intestinal environment, are summarized. In addition, the potential mechanisms affecting conjugation in vivo are summarized from the perspectives of bacterial colonization and the conjugation process.

Metformin Reverses tmexCD1-toprJ1- and tet(A)-Mediated High-Level Tigecycline Resistance in K. pneumoniae.

Tigecycline (TIG) is one of the last effective options against multidrug resistance bacteria. Recently, the RND (resistance-nodulation-division) efflux pump gene cluster, tmexCD1-toprJ1, and the tetracycline-efflux pump tet(A) mutation were reported to mediate high level resistance to TIG in clinically important pathogens, weakening the efficacy of TIG. In this study, we report the potent synergistic effect of the antidiabetic drug metformin in combination with TIG against tet(A) mutant and tmexCD1-toprJ1 positive K. pneumoniae. The fractional inhibitory concentration index (FICI) of TIG and metformin were less than 0.05 for all the tested isolates. The time-kill curve assay showed that the combination of TIG and metformin exhibited much better antimicrobial effect than TIG alone. The synergistic effect was also confirmed in vivo using a well-studied Galleria mellonella larvae model. Mechanistic studies demonstrated that metformin disrupted the important component of proton motive force, the electric potential (Δψ) and the function of efflux pump, thereby increasing the intracellular concentration of TIG. This finding revealed that metformin might be a possible adjuvant of TIG for combating with superbugs carrying the tet(A) mutant and tmexCD1-toprJ1 genes.