The Development of Bacteriophage Resistance in Vibrio alginolyticus Depends on a Complex Metabolic Adaptation Strategy

Abstract

Lytic bacteriophages have been well documented to play a pivotal role in microbial ecology due to their complex interactions with bacterial species, especially in aquatic habitats. Although the use of phages as antimicrobial agents, known as phage therapy, in the aquatic environment has been increasing, recent research has revealed drawbacks due to the development of phage-resistant strains among Gram-negative species. Acquired phage resistance in marine Vibrios has been proven to be a very complicated process utilizing biochemical, metabolic, and molecular adaptation strategies. The results of our multi-omics approach, incorporating transcriptome and metabolome analyses of Vibrio alginolyticus phage-resistant strains, corroborate this prospect. Our results provide insights into phage-tolerant strains diminishing the expression of phage receptors ompF, lamB, and btuB. The same pattern was observed for genes encoding natural nutrient channels, such as rbsA, ptsG, tryP, livH, lysE, and hisp, meaning that the cell needs to readjust its biochemistry to achieve phage resistance. The results showed reprogramming of bacterial metabolism by transcript regulations in key-metabolic pathways, such as the tricarboxylic acid cycle (TCA) and lysine biosynthesis, as well as the content of intracellular metabolites belonging to processes that could also significantly affect the cell physiology. Finally, SNP analysis in resistant strains revealed no evidence of amino acid alterations in the studied putative bacterial phage receptors, but several SNPs were detected in genes involved in transcriptional regulation. This phenomenon appears to be a phage-specific, fine-tuned metabolic engineering, imposed by the different phage genera the bacteria have interacted with, updating the role of lytic phages in microbial marine ecology.

Perturbation of Quorum Sensing after the Acquisition of Bacteriophage Resistance Could Contribute to Novel Traits in Vibrio alginolyticus

“…Bacteriophage-resistant mutants were obtained through an experimental procedure involving overnight exposure to Athena1 at a multiplicity of infection (MOI) of 100 as described in Skliros et al [ 9 ]. Briefly, three separate cultures of V. alginolyticus were grown in LB medium and inoculated with each phage during the exponential phase (OD 600 : ~0.2) at a MOI of 100.…”

“…The phage-resistant colonies were further propagated by re-plating, and one colony from each plate was selected as a phage-resistant biological replicate and preserved at −80 °C with 20% glycerol for subsequent analysis. For φ St2 bacteriophage, resistant strains developed earlier [ 9 ] were used.…”

“…To study gene expression, wild-type (phage-sensitive) and phage-resistant bacterial colonies were retrieved from −80 °C stocks and grown overnight at 25 °C with vigorous shaking in LB broth. In the present study, the resistant strains that were generated by Skliros et al [ 9 ] for φ St2 were also used. Bacterial cells were recultured until an LCD state (OD 600 : 0.2) was achieved, and at the same time, a small aliquot was retrieved to re-validate their phage-resistant status with a spot assay.…”

“…Bacteriophage resistance can involve well-documented molecular mechanisms within the cell [ 7 ] or a metabolic adaptation strategy triggered by genomic mutations, commonly observed in Gram-negative bacteria [ 8 ]. Our previous study demonstrated that aquatic species, such as vibrios, can enhance their tolerance towards lytic bacteriophages, by downregulating the expression pattern of membrane transporters [ 9 ]. This study revealed the cellular and metabolic cost of the acquired phage resistance at genomic, transcriptomic, and metabolomic levels, subsequently impacting various phenotypic traits in V. alginolyticus in a phage-specific manner.…”

Exposure of Helicobacter pylori to clarithromycin in vitro resulting in the development of resistance and triggers metabolic reprogramming associated with virulence and pathogenicity

“…A key component of cellular regulatory system is metabolites produced by bacteria that interacts with the surrounding host microenvironment [ 4 , 5 ]. Several studies have shown that in Staphylococcus aureus and Vibrio alginolyticus , phenotypic changes, including antimicrobial resistance, can be associated with metabolomic reprogramming [ 6 , 7 ]. Antibiotic resistance has been demonstrated to correlate with alterations in bacterial metabolic pathway, such as folate biosynthesis pathway which is associated with changing bacterial susceptibility to antibiotics (phenotypic resistance) [ 8 ].…”