Comparative analysis of quantitative methodologies for Vibrionaceae biofilms.

Multiple symbiotic and free-living Vibrio spp. grow as a form of microbial community known as a biofilm. In the laboratory, methods to quantify Vibrio biofilm mass include crystal violet staining, direct colony-forming unit (CFU) counting, dry biofilm cell mass measurement, and observation of development of wrinkled colonies. Another approach for bacterial biofilms also involves the use of tetrazolium (XTT) assays (used widely in studies of fungi) that are an appropriate measure of metabolic activity and vitality of cells within the biofilm matrix. This study systematically tested five techniques, among which the XTT assay and wrinkled colony measurement provided the most reproducible, accurate, and efficient methods for the quantitative estimation of Vibrionaceae biofilms.

Interactions of Vibrio spp. with Zooplankton.

Members of the genus Vibrio are known to interact with phyto- and zooplankton in aquatic environments. These interactions have been proven to protect the bacterium from various environmental stresses, serve as a nutrient source, facilitate exchange of DNA, and to serve as vectors of disease transmission. This review highlights the impact of Vibrio-zooplankton interactions at the ecosystem scale and the importance of studies focusing on a wide range of Vibrio-zooplankton interactions. The current knowledge on chitin utilization (i.e., chemotaxis, attachment, and degradation) and the role of these factors in attachment to nonchitinous zooplankton is also presented.

Micro-fabricated polydimethyl siloxane (PDMS) surfaces regulate the development of marine microbial biofilm communities.

This study explored an antifouling (AF) concept based on deployment of microfabricated polydimethyl siloxane (PDMS) surfaces with 1-10 µm periodicity corrugated topographies in temperate marine waters. The effect of the surfaces on the development of microbial biofilms over 28 days and during different seasons, including both summer and winter, was examined using confocal laser scanning microscopy (CLSM) as well as terminal restriction fragment (T-RF) analysis for phylogenetic fingerprinting. The microscale topography significantly impacted biofilm development by altering the attachment pattern and reducing microcolony formation on the 1, 2 and 4 µm PDMS surfaces. Also, field deployments over 28 days showed a significant reduction in biovolume on the 4 and 10 µm PDMS surfaces despite altered environmental conditions. The microfabricated PDMS surfaces further significantly impacted on the community composition of the biofilms, as revealed by changes in T-RF profiles, at different stages of development. Moreover, altered biofilm resistance was demonstrated by exposing pre-established biofilms on 10 µm micro-fabricated surfaces to enhanced flagellate predation by a heterotrophic protist, Rhynchomonas nasuta. Pronounced changes in the overall marine microbial biofilm development as well as community composition warrant exploring substratum modification for marine AF applications.

The rise of pathogens: predation as a factor driving the evolution of human pathogens in the environment.

Bacteria in the environment must survive predation from bacteriophage, heterotrophic protists, and predatory bacteria. This selective pressure has resulted in the evolution of a variety of defense mechanisms, which can also function as virulence factors. Here we discuss the potential dual function of some of the mechanisms, which protect against heterotrophic protists, and how predation pressure leads to the evolution of pathogenicity. This is in accordance with the coincidental evolution hypothesis, which suggests that virulence factors arose as a response to other selective pressures, for example, predation rather than for virulence per se. In this review we discuss some of those environmental factors that may be associated with the rise of pathogens in the marine environment. In particular, we will discuss the role of heterotrophic protists in the evolution of virulence factors in marine bacteria. Finally, we will discuss the implications for expansion of current pathogens and emergence of new pathogens.

Predation response of Vibrio fischeri biofilms to bacterivorus protists.

Vibrio fischeri proliferates in a sessile, stable community known as a biofilm, which is one alternative survival strategy of its life cycle. Although this survival strategy provides adequate protection from abiotic factors, marine biofilms are still susceptible to grazing by bacteria-consuming protozoa. Subsequently, grazing pressure can be controlled by certain defense mechanisms that confer higher biofilm antipredator fitness. In the present work, we hypothesized that V. fischeri exhibits an antipredator fitness behavior while forming biofilms. Different predators representing commonly found species in aquatic populations were examined, including the flagellates Rhynchomonas nasuta and Neobodo designis (early biofilm feeders) and the ciliate Tetrahymena pyriformis (late biofilm grazer). V. fischeri biofilms included isolates from both seawater and squid hosts (Euprymna and Sepiola species). Our results demonstrate inhibition of predation by biofilms, specifically, isolates from seawater. Additionally, antiprotozoan behavior was observed to be higher in late biofilms, particularly toward the ciliate T. pyriformis; however, inhibitory effects were found to be widespread among all isolates tested. These results provide an alternative explanation for the adaptive advantage and persistence of V. fischeri biofilms and provide an important contribution to the understanding of defensive mechanisms that exist in the out-of-host environment.

Environmental reservoirs and mechanisms of persistence of Vibrio cholerae.

It is now well accepted that Vibrio cholerae, the causative agent of the water-borne disease cholera, is acquired from environmental sources where it persists between outbreaks of the disease. Recent advances in molecular technology have demonstrated that this bacterium can be detected in areas where it has not previously been isolated, indicating a much broader, global distribution of this bacterium outside of endemic regions. The environmental persistence of V. cholerae in the aquatic environment can be attributed to multiple intra- and interspecific strategies such as responsive gene regulation and biofilm formation on biotic and abiotic surfaces, as well as interactions with a multitude of other organisms. This review will discuss some of the mechanisms that enable the persistence of this bacterium in the environment. In particular, we will discuss how V. cholerae can survive stressors such as starvation, temperature, and salinity fluctuations as well as how the organism persists under constant predation by heterotrophic protists.

Quantification of individual flagellate - bacteria interactions within semi-natural biofilms.

Here we present a new approach to quantify food-web interactions within semi-natural biofilms by combining the establishment of biofilms from natural rivers in flow cells with video microscopy. In a first application of this approach, we focused on the surface-gliding heterotrophic flagellates (HF) Neobodo designis, Rhynchomonas nasuta and Planomonas sp. It was shown that the three HF generally ingested single biofilm-associated bacteria whereas bacteria within microcolonies were attacked but not ingested. However, grazing strategies differed considerably. While the kinetoplastids N. designis and R. nasuta displayed long search and short handling times, Planomonas sp. showed the opposite grazing characteristics. The latter behaviour resulted in a high relative predation success of 80% (precent of attacked prey ingested), whereas the relative predation success of the two kinetoplastids was only 20%. However, the two contrasting strategies resulted in similar ingestion rates for Planomonas sp. and N. designis of 0.5 to 0.6 ingestions flagellates(-1) minute(-1), respectively. Our results showed distinct differences in the feeding behaviour of three flagellates having similar life forms and provide direct evidence that microcolony formation in biofilms protects bacteria from grazing by HF in situ. The new approach provides individual-based insights into the complex food web interactions within biofilms.

In situ grazing resistance of Vibrio cholerae in the marine environment.

Previous laboratory experiments revealed that Vibrio cholerae A1552 biofilms secrete an antiprotozoal factor that prevents Rhynchomonas nasuta from growing and thus prevents grazing losses. The antiprotozoal factor is regulated by the quorum-sensing response regulator, HapR. Here, we investigate whether the antiprotozoal activity is ecologically relevant. Experiments were conducted in the field as well as under field-like conditions in the laboratory to assess the grazing resistance of V. cholerae A1552 and N16961 (natural frameshift mutation in hapR) biofilms to R. nasuta and Cafeteria roenbergensis. In laboratory experiments exposing the predators to V. cholerae grown in seawater containing high and low glucose concentrations, we determined that V. cholerae biofilms showed increased resistance towards grazing by both predators as glucose levels decreased. The relative resistance of the V. cholerae strains to the grazers under semi-field conditions was similar to that observed in situ. Therefore, the antipredator defense is environmentally relevant and not lost when biofilms are grown in an open system in the marine environment. The hapR mutant still exhibited some resistance to both predators and this suggests that V. cholerae may coordinate antipredator defenses by a combination of density-dependent regulation and environmental sensing to protect itself from predators in its natural habitat.