Vibrio splendidus persister cells induced by host coelomic fluids show a similar phenotype to antibiotic‐induced counterparts

Abstract

Persister cells are dormant variants of regular cells that are multidrug tolerant and have heterogeneous phenotypes; these cells are a potential threat to hosts because they can escape the immune system or antibiotic treatments and reconstitute infectious. Skin ulcer syndrome (SUS) frequently occurs in the sea cucumber (Apostichopus japonicus), and Vibrio splendidus is one of the main bacterial pathogens of SUS. This study found that the active cells of V. splendidus became persister cells more readily in the presence of A. japonicus coelomic fluids. We showed that the A. japonicus coelomic fluids plus antibiotics induce 100-fold more persister cells in V. splendidus compared with antibiotics alone via nine sets of experiments including assays for antibiotic resistance, metabolic activity, and single-cell phenotypes. Furthermore, the coelomic fluidsinduced persister cells showed similar phenotypes as the antibiotic-induced persister cells. Further investigation showed that guanosine pentaphosphate/tetraphosphate (henceforth ppGpp) and SOS response pathway involved in the formation of persister cells as determined using real-time RT-PCR. In addition, single-cell observations showed that, similar to the antibiotic-induced V. splendidus persister cells, the coelomic fluids-induced persister cells have five resuscitation phenotypes: no growth, expansion, elongation, elongation and then division, and elongation followed by death/disappearance. In addition, dark foci formed in the majority of persister cells for both the antibiotic-induced and coelomic fluids-induced persister cells. Our results highlight that the pathogen V. splendidus might escape from the host immune system by entering the persister state during the process of infection due to exposure to coelomic fluids.

Selection of the Amino Acid and Saccharide That Increase the Tetracycline Susceptibility of Vibrio splendidus

“…For amino acids, Lglutamic acid revived V. splendidus persister cells used for the shortest time, and for saccharides, D-galactose, maltose, mannose, and sorbitol woke up V. splendidus persister cells better than D-fructose, N-acetyl-D-glucosamine, and D-glucose. The different numbers of colonies emerged on the specific carbon source probably attributed to the high heterogeneity of V. splendidus persister cells in dormancy (14), which resulted in their heterogeneous waking up on different carbon sources, probably due to different metabolism of various carbon sources in V. splendidus. Consequently, the results of this study indicated that (1) V. splendidus persister cells woke up responding to nutrient incentives similar to P. aeruginosa and E. coli (17,18,36) (2) L-glutamic acid is the best amino acid to revive V. splendidus persister cells, which is specific to this bacterial species; this further strengthened the concept that different bacterial persister cells preferred to use different carbon sources to wake up (17,18).…”

“…V. splendidus persister cells were prepared by lysing active cells with high concentration of tetracycline 400 µg•ml −1 , according to our previous study (14). Briefly, 10 ml of overnight V. splendidus culture was mixed with 40 ml 2216E containing 2,000 µl of tetracycline stock solution, and the culture was incubated 4 h in a shaker with a shaking speed of 150 rpm.…”

“…Unlike resistant cells, the genes of the persister cells did not alter (6,7), but they are a little potion of bacterial cells with a transient state of hypometabolic or dormancy that can help bacteria to survive the exterior stresses, such as antibiotic treatment and nutrient deficiency (8)(9)(10)(11). Till now, this phenotype has been commonly found in Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Vibrio splendidus and Archaea (12)(13)(14) during in vitro growth as well as infection in the host.…”

“…splendidus is a gram-negative bacterium that is ubiquitously presented in marine ecosystems (26), and it is a significant opportunistic bacterial pathogen which causes infection of marine shellfish and sea cucumber Apostichopus japonicus (27)(28)(29)(30). Our previous study showed that V. splendidus could form persister cells (14). To further know more about V. splendidus persister cells, the carbon sources that could wake up V. splendidus persister cells were selected in this study, and furthermore, a strategy for completely eliminating V. splendidus was proposed.…”

Insights into Carbapenem Resistance in Vibrio Species: Current Status and Future Perspectives

“…Like the carbapenem resistant A. baumannii and P. aeruginosa, Vibrio spp. are also proven capable of assuming the spherical form and entering into dormancy under hostile conditions [ 93 ]. They can exist as persister cells as part of their antimicrobial resistance potential (see Section 5.4 ).…”