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Enhanced antibacterial effect of a novel Friunavirus phage vWU2001 in combination with colistin against carbapenem-resistant Acinetobacter baumannii

ABSTRACT: The emergence of carbapenem-resistant Acinetobacter baumannii has been increasingly reported, leading to more challenges in treating its infections. With the development of phage therapy and phage-antibiotic combinations, it is possible to improve the treatment of bacterial infections. In the present study, a vB_AbaP_WU2001 (vWU2001 for short) phage-specific CRAB was isolated and the genome size is 40,792 bp in length. The novel phage vWU2001 belongs to the Autographiviridae family and the order Caudovirales. Shotgun proteomics identified 289 proteins. The broad host range phage vWU2001 displayed a high adsorption rate, short latent period, large burst size and good stability. The phage could reduce preformed biofilms and inhibit biofilm formation. The combination of phage vWU2001 and colistin had significantly higher bacterial growth inhibition activity than that of phage, or colistin alone. The efficacy of the combined treatment was also evaluated in Galleria mellonella. The evaluation of its therapeutic potential revealed that the combination of phage and colistin showed a significantly greater increase in G. mellonella survival and clearance of bacterial number compared to that of phage or colistin alone, indicating that the combination was synergistic against CRAB. The results demonstrated that phage vWU2001 has the potential to be developed as an antibacterial agent.

ORGANISM(S): Acinetobacter Baumannii

SUBMITTER: Sittiruk Roytrakul

PROVIDER: PXD028206 | JPOST Repository | Sat Sep 04 00:00:00 BST 2021

REPOSITORIES: jPOST

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Dataset's files

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			Action	DRS
	Cutoff%20AB_1.mzXML	Mzxml
	Cutoff%20AB_2.mzXML	Mzxml
	Cutoff%20AB_3.mzXML	Mzxml
	Cutoff%2520AB_1_RC6_01_8848.mgf	Mgf
	Cutoff%2520AB_2_RC6_01_8849.mgf	Mgf

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Project description:Carbapenem-resistant Acinetobacter baumannii (CRAB) is a Priority 1 (Critical) pathogen urgently requiring new antibiotics. Polymyxins are a last-line option against CRAB-associated infections. This transcriptomic study utilized a CRAB strain to investigate mechanisms of bacterial killing with polymyxin B, colistin, colistin B and colistin/sulbactam combination therapy. After 4 h of 2 mg/L polymyxin monotherapy, all polymyxins exhibited common modes of action which primarily involved disruption to amino acid and fatty acid metabolism. Of the three monotherapies, polymyxin B induced the greatest number of differentially expressed genes (DEGs), including for genes involved with fatty acid metabolism. Gene disturbances with colistin and colistin B were highly similar (89% common genes for colistin B), though effects on gene expression were generally lower (0-1.5-fold in most cases) with colistin B. Colistin alone (2 mg/L) or combined with sulbactam (64 mg/L) resulted in rapid membrane disruption as early as 1 h. Transcriptomic analysis of this combination revealed the effects were driven by colistin and included disturbances in fatty acid synthesis and catabolism and inhibition of nutrient uptake. Combination therapy produced substantially higher fold changes in 72% of DEGs shared with monotherapy, resulting in substantially greater reductions in fatty acid biosynthesis and increases in biofilm, cell wall and phospholipid synthesis. This indicates synergistic bacterial killing with the colistin/sulbactam combination results from a systematic increase in perturbation of many genes associated with bacterial metabolism. These mechanistic insights enhance our understanding of bacterial responses to polymyxin mono- and combination therapy and will assist to optimize polymyxin use in patients. Carbapenem-resistant Acinetobacter baumannii (CRAB) is a Priority 1 (Critical) pathogen urgently requiring new antibiotics. Polymyxins are a last-line option against CRAB-associated infections. This transcriptomic study utilized a CRAB strain to investigate mechanisms of bacterial killing with polymyxin B, colistin, colistin B and colistin/sulbactam combination therapy. After 4 h of 2 mg/L polymyxin monotherapy, all polymyxins exhibited common modes of action which primarily involved disruption to amino acid and fatty acid metabolism. Of the three monotherapies, polymyxin B induced the greatest number of differentially expressed genes (DEGs), including for genes involved with fatty acid metabolism. Gene disturbances with colistin and colistin B were highly similar (89% common genes for colistin B), though effects on gene expression were generally lower (0-1.5-fold in most cases) with colistin B. Colistin alone (2 mg/L) or combined with sulbactam (64 mg/L) resulted in rapid membrane disruption as early as 1 h. Transcriptomic analysis of this combination revealed the effects were driven by colistin and included disturbances in fatty acid synthesis and catabolism and inhibition of nutrient uptake. Combination therapy produced substantially higher fold changes in 72% of DEGs shared with monotherapy, resulting in substantially greater reductions in fatty acid biosynthesis and increases in biofilm, cell wall and phospholipid synthesis. This indicates synergistic bacterial killing with the colistin/sulbactam combination results from a systematic increase in perturbation of many genes associated with bacterial metabolism. These mechanistic insights enhance our understanding of bacterial responses to polymyxin mono- and combination therapy and will assist to optimize polymyxin use in patients.

Project description:Among both healthy and immunocompromised patient populations, pneumonia is a leading cause of death worldwide. Yet, despite structural vulnerability resulting in recurrent exposure to pathogens, the lungs’ mucosal immunity successfully suppresses most infections. We recently reported that these innate defenses can be substantially augmented by inhalational exposure to a crude bacterial lysate, protecting broadly against respiratory pathogens, including lethal pneumonia caused by bacteria, fungi or viruses. The phenomenon of inducible resistance is associated with rapid pathogen killing in the lungs and persists in the absence of the typical leukocytes of innate immunity. Rather, the respiratory epithelium appears to be the predominant effector. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors crucial to host defense through the sensing of pathogen associated molecular patterns. Given the importance of TLRs to mucosal immunity, the presence of numerous pathogen associated molecular patterns in the bacterial lysate, and the induction of many TLR-dependent genes following lysate treatment, we hypothesized that induced resistance follows simultaneous stimulation of multiple TLRs. To test this, we challenged mice deficient in TLR/IL1R adaptor proteins and found that resistance could not be induced in mice lacking MyD88. Having identified this phenomenon to be MyD88-dependent, we sought to determine whether the protective phenomenon could be recapitulated by treatment with synthetic TLR agonists. Mice were treated with aerosolized TLR ligands, alone and in combination, prior to infection with virulent pathogens. While limited protection against pneumonia was afforded by the individual TLR ligands, we discovered that the synergistic combination of diacylated lipopetide TLR2/6 agonist Pam2CSK4 and CpG oligodeoxynucleotide TLR9 agonist ODN2395 induced profound resistance against all tested pathogens. This combination also induced greater than additive pathogen killing in the lungs of challenged mice, and we found that the combination could effectively induce pathogen killing by respiratory epithelial cells in vitro. In order to better understand the mechanisms underlying the inducible pathogen killing by this unique combination of TLR agonists, we performed microarray analysis of murine MLE-15 respiratory epithelial cells following 4 h treatment with PBS (sham treatment), Pam2CSK4 alone, ODN2395 alone, or the combination of both agonists, using Illumina Sentrix MouseRef-8 v2 BeadChips. This allows for assessment of differential gene expression, not only between treated and untreated, but between single and combination treated. The intent of the experiment is to gain insight into the transcriptionally-regulated means by which TLR2/6 and TLR9 signaling pathways synergistically interact. Keywords: Differential expression, epithelium, in vitro

Project description:Several groups have shown that through evolution experiments, tolerance evolved rapidly under cyclic antibiotic treatment which then promote the emergence of resistance. In other words, intermittent antibiotic exposure will “train” the bacteria to become tolerant to the drug, and then boost the chances for them to become fully resistant. This evolutionary trajectory of evolving tolerance and resistance not only occurs in vitro, but also in clinical patients with MRSA blood infections. Despite the fact that repetitive antibiotic treatment will lead to tolerance and resistance development in bacterial populations, the difference on the evolutionary path between those treated with a single drug and drug combination remains unaddressed. In this study, we monitored the development of tolerance in MRSA by treating them with either daptomycin alone for 2 weeks (Scheme 1), or daptomycin combined with rifampin for two weeks (Scheme 2), in a cyclic manner. In addition, we also investigate another scenario where we treat MRSA cells with daptomycin alone for one week, and subsequently treat them with daptomycin and rifampin combination in the second week (Scheme 3). At the end of the evolution experiment, we observed that the population from Scheme 1 developed tolerance towards daptomycin, the population from Scheme 2 developed both tolerance and resistance towards daptomycin, while the population from Scheme 3 developed tolerance after a week of treatment, but then the tolerance phenotype diminished at the end of the second week due to the drug combination treatment. We subjected them to whole genome sequencing and identified single point mutations that are responsible for the observed phenotype. Through proteomics, we compared the proteome profile of the population evolved from scheme 1 (S1D14), scheme 2 (S2D14) and scheme 3 (S3D14), and we observed that these three populations employ distinct mechanisms to survive antibiotic treatment, suggesting that the treatment strategy for these populations should be tailored depending on the treatment conditions.

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Enhanced antibacterial effect of a novel Friunavirus phage vWU2001 in combination with colistin against carbapenem-resistant Acinetobacter baumannii

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