Predatory bacteria as natural modulators of Vibrio parahaemolyticus and Vibrio vulnificus in seawater and oysters.

Start searching for selective advantage and becoming predation dynamics.

Vibrio parahaemoliticus is one of the principal sources of seafood-associated human illness worldwide. Eating contaminated raw seafood or shellfish (for example oysters) can lead a diarrheal disease. Different serotypes were identified during last decade pandemic episodes and V. parahaemoliticus serotype O3:K6 was one of the most diffused. Although high infecting dose of vibrios is required to lead outbreak, not only the number of ingested organisms are important but also the presence of virulence factors in them. Virulence factors are genes able to encode toxins, enzymes or regulatory proteins that can enhance the infectivity and the pathogenicity of the strain that show them.

Vibrio genus is widely spread as symbiont and pathogen but is also a prey of other organisms. It’s known that Vibrio can be predated by a class of gram- bacteria: Bdellovibrio and like organisms (BALOs). This group is phylogenetically diverse and has complex life cycles with both host-dependent and host-independent replication. BALOs exploit and kill hosts attacking on them and using host cell content as nutrient reservoir for replication.

Richards et al. in this article evaluate whether rpoS and toxRS genes confer advantages in uptake or colonization of vibrios in Eastern oysters Crassostrea virginica or survival of vibrios in seawater. The rpoS and toxRS genes are involved in alternative stress response and virulence regulation respectively so are considered integrally involved in Vibrio survival and virulence. Authors hypothesized that knockout mutant for these genes (ΔrpoS and ΔtoxRS) could have different colonization or survival success in respect to wildtype O3:K6. They also evaluate growth and persistence of V. parahaemolyticus O1:KUT (strand for K untypeable) and V. vulnificus both associated with outbreaks of seafood-associated illness or wound infections.

They set separate experiments to count number of each O3:K6, ΔrpoS and ΔtoxRS strain at 0, 24, 48, and 72h after same inoculation quantity in: (A) tanks of natural seawater (NSW) with oyster and live microalgae (to feed oysters); (B) in oyster directly (kept and fed in same conditions of A). The result was a decrease of more than 96% after 48h and values of less 1% after 72h in A; and after a peak of accumulation at 24h a following decrease of 91-97% at 72h in B for every vibrios strain inoculated. Since there were no significant differences between three strains, authors proposed that toxRS and rpoS genes not give any selective advantages in colonization and persistence in oysters.

To evaluate whether this failure to thrive was unique to this species and serotype (O3:K6), they test in the same way (A and B design) V. parahaemolyticus O1:KUT and V. vulnificus comparing uptake and persistence in seawater and oysters. Authors obtained same failure to survival: significant decrease number after 24h and negligible level after 72h in A; significant decrease after 72h in B. This result showed the inability of all these pathogens to persist in NSW or oysters.

At this point to determine if Vibrio level decrease in seawater was due to the filtering process of oysters, O3:K6 was inoculated in NSW (without oysters and added microalgae). The result was again a significant decrease to negligible level after 72h. Indeed some factor in the seawater was responsible for vibrios reduction.

A different interesting result was obtained testing the same inoculation of O3:K6 in autoclaved seawater (ASW) and NSW. There was an increase of 1000-fold for the former and a decrease of 47-fold for the second, this last result was coherent with all the previous results.

This step gives the idea of a heat-labile inhibitor. Authors devised a plaque assay in order to quantify VPB and to obtain scanning microscopy images of them. Furthermore was set the same counting experiment (0-24-48-72h for A and B) monitoring the number of O3:K6 and VPB. Here they found an interesting prey-predator relationship (FIG 1-A) in NSW but not in oysters because methods to detect and count VPB within oysters are not yet available. Microscopy scanning images were compared between O3:K6 plaque isolates (FIG 1-B) and Bdellovibrio bacterivorus and Bacterivorax stolpii cultured in E.coli host cell (both of them BALOs model species). This gave a good visual similarity allowing authors to identify BALOs in a variety of morphology near to Bd. bacterivorus-, Ba. stolpii- and M. aeruginosavorus-like cells.

 FIG 1 A) Comparison of mean counts of V. parahaemoliticus (Vp) O3:K6 and Vibrio predatory bacteria (VPB) over time in natural seawater. B) Image from scanning microscopy of VPB (BALO) entering a V. parahaemoliticus host cell.

At this point, authors assayed for VPB samples of NSW from 5 different sites on Atlantic and Pacific US coast and monthly in 4 sites in Delaware from October to March. They found the higher level of VPB in the site with higher salinity and average temperature, a marshland. Authors propose a possible association of VPB with high-productivity marshes and high salinity, although insufficient information were available to infer this correlation both with salinity and temperature. However they support this hypothesis citing other evidences of disappearance of V.vulnificus from costal NSW and oyster in North Carolina after 2 years of hard drought.

In conclusion in this article authors start wanting to determine if toxRS or rpoS gene were involved in colonization and persistence of V. parahaemoliticus O3:K6 in oysters giving a selective advantage to different strains. Step by step they excluded strain-specific and species-specific features arriving to the identification of a biotic factor as responsible for Vibrio decrease in seawater. Started as a lab molecular experiment, it become an inter-specific predation assay with also a screening of field samples. This work seems to me like some of the pioneering works done for discover etiologic causes of bacterial disease in past centuries. This article give useful details to better understand pandemic Vibrio pathogen outbreak, suggest that VPB should play an important role in Vibrio decline and maybe climatic conditions limit potential spread of this pathogen. In my opinion more investigation on the ecology of VPB should be done. Maybe revealing their presence in other pathogen bacteria it should discover more prey and predators. And VPB also being bacteria, what’s happens to them with antibiotics pollution?

Richards, G. P., Fay, J. P., Dickens, K. A., Parent, M. A., Soroka, D. S., & Boyd, E. F. (2012). Predatory bacteria as natural modulators of Vibrio parahaemolyticus and Vibrio vulnificus in seawater and oysters. Applied and environmental microbiology, 78(20), 7455-7466.