The global emergence and spread of multi-drug resistant (MDR) strains is becoming increasingly worrisome due to the overuse of broad-spectrum antibiotics. Colistin, the last resort for treating MDR strains infections, has once again returned to the clinician’s choice. However, with the widespread use of colistin, colistin-resistant gram-negative bacteria (GNB) have subsequently emerged, including colistin-resistant Pseudomonas aeruginosa (COL-R PA). Therefore, available solutions are urgently needed to respond to this situation. Here, we inspiringly found that the combination of plumbagin and colistin had an efficiently inhibitory effect for colistin-resistant P. aeruginosa in vitro through checkerboard assay and time-kill assay. The combinatorial inhibition of biofilm formation was clearly demonstrated by crystal violet staining and scanning electron microscopy (SEM), and this combination can not only inhibited biofilm formation but also eradicated the mature biofilm. Erythrocytes hemolysis test showed that plumbagin has negligible hemolysis ability. In addition, the increased survival rate of Galleria mellonella (G. mellonella) larva confirmed this combination as same as effective in vivo. As for the mechanism of this combination, propidium iodide (PI) staining showed colistin combined with plumbagin could significantly change the membrane permeability, thus exerting synergistic antibacterial activity. In conclusion, the combination of plumbagin and colistin shows a prominently synergistic antibacterial effect in vitro and in vivo, providing a promising option for the therapy of COL-R PA infection.

IntroductionPhage is promising for the treatment of hypervirulent Klebsiella pneumoniae (hvKP) infections. Although phage resistance seems inevitable, we found that there still was optimization space in phage therapy for hvKP infection.MethodsThe clinical isolate K. pneumoniae FK1979 was used to recover the lysis phage ΦFK1979 from hospital sewage. Phage-resistant bacteria were obtained on LB agar and used to isolate phages from sewage. The plaque assay, transmission electron microscopy (TEM), multiplicity of infection test, one-step growth curve assay, and genome analysis were performed to characterize the phages. Colony morphology, precipitation test and scanning electron microscope were used to characterize the bacteria. The absorption test, spot test and efficiency of plating (EOP) assay were used to identify the sensitivity of bacteria to phages. Whole genome sequencing (WGS) was used to identify gene mutations of phage-resistant bacteria. The gene expression levels were detected by RT-qPCR. Genes knockout and complementation of the mutant genes were performed. The change of capsules was detected by capsule quantification and TEM. The growth kinetics, serum resistance, biofilm formation, adhesion and invasion to A549 and RAW 264.7 cells, as well as G. mellonella and mice infection models, were used to evaluate the fitness and virulence of bacteria.Results and discussionHere, we demonstrated that K2 capsule type sequence type 86 hvKP FK1979, one of the main pandemic lineages of hvKP with thick capsule, rapidly developed resistance to a K2-specific lysis phage ΦFK1979 which was well-studied in this work to possess polysaccharide depolymerase. The phage-resistant mutants showed a marked decrease in capsule expression. WGS revealed single nucleotide polymorphism (SNP) in genes encoding RfaH, galU, sugar glycosyltransferase, and polysaccharide deacetylase family protein in the mutants. RfaH and galU were further identified as being required for capsule production and phage sensitivity. Expressions of genes involved in the biosynthesis or regulation of capsule and/or lipopolysaccharide significantly decreased in the mutants. Despite the rapid and frequent development of phage resistance being a disadvantage, the attenuation of virulence and fitness in vitro and in vivo indicated that phage-resistant mutants of hvKP were more susceptible to the immunity system. Interestingly, the newly isolated phages targeting mutants changed significantly in their plaque and virus particle morphology. Their genomes were much larger than and significantly different from that of ΦFK1979. They possessed much more functional proteins and strikingly broader host spectrums than ΦFK1979. Our study suggests that K2-specific phage has the potential to function as an antivirulence agent, or a part of phage cocktails combined with phages targeting phage-resistant bacteria, against hvKP-relevant infections.

Excessive use of antibiotics can lead to bacterial resistance and the development of multidrug-resistant bacteria. Antibiotic misuse can also promote resistance against last-resort antibiotics.

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Nanoparticle-based antibiotic delivery systems are essential in combating antibiotic-resistant bacterial infections arising from acquired resistance and/or biofilm formation. Here, we report that the ceftazidime-decorated gold nanoparticles (CAZ_Au NPs) can effectively kill clinical ceftazidime-avibactam-resistant Enterobacteriaceae with various resistance mechanisms.