Human pathogenic vibrios in the aquatic environment: effects of ocean warming

Vibrios are Gram negative marine bacteria and are still regarded by most marine microbiologists as the dominant culturable bacteria in the ocean. They can live in association with different biotic and abiotic substrates, in particular with chitinous plankton, which is considered one of the most abundant reservoir of these bacteria in the environment. Vibrio genus includes important human pathogens such as Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. There is substantial evidence that Vibrio-associated diseases are increasing worldwide with climate warming. Increased Sea Surface Temperature (SST) linked to El Niño events have been shown to pre-date increases in cholera incidence in both Asia and South America. Similarly, climate anomalies have been implicated in the expansion of the geographical and seasonal range of seafood-borne illnesses caused by V. parahaemolyticus and V. vulnificus. Moreover, in recent years, an unexpected increase in the number of bathing infections associated with warm water Vibrio species has been reported in Northern Europe countries. Although much evidence has been accumulated on the long-term effects of ocean warming on eukaryotic populations, no experimental information exists for the effects this may have on marine prokaryotic abundance and diversity. An explanation for this gap is the lack of historical data at a global scale, being most available data collected on a seasonal scale and based on point samplings. The present work was designed to overcome this gap by applying a molecular analysis to formalin-fixed Continuous Plankton Recorder (CPR) samples, collected in the North Sea during the last 50 years. CPR is the longest running marine biological monitoring program in the world, operated by SAHFOS (UK) since 1954. CPR device is towed, at a depth of ~10 m, from ships during their normal sailings. Water passes through the CPR, and plankton is filtered onto a moving band of silk (270 µm mesh size), covered by a second silk and spooled into a storage tank containing formalin. On the basis of the well established association between vibrios and plankton organisms, it is expected that CPR system significantly captures a major fraction of these bacteria providing a unique opportunity to carry out retrospective studies on Vibrio population dynamics. A set of 55 samples collected by the CPR survey in the North Sea from off the Rhine and Humber estuaries between 1961 and 2005 was analyzed. All the samples were collected in the month of August. DNA was extracted from each CPR samples, quantified and confirmed feasible for molecular analysis. Vibrios presence and relative abundance on CPR samples was assessed by applying a real-time PCR approach. The results were expressed as Vibrio Abundance Index (VAI) that was arbitrarily defined as the ratio of Vibrio spp. cells to the total number of bacterial cells assessed by real-time PCR using genus-specific and universal primers, respectively. Both the used couples of primers generate small and similar size amplicons (about 100 bp), allowing amplification to take place and avoiding age/formalin induced bias by assuming that DNA damage over time was the same for both amplified fragments. By plotting on a graph the obtained Vibrio abundance values with the SST data registered during the same period of time for each North Sea site under study, it was shown a long-term increase in relative Vibrio abundance coupled to a positive and statistically significant correlation with SST off the Rhine Estuary but not off the Humber Estuary during the past 44 years. Such different correlation results for the two areas may be related to the generally higher summer SST values recorded in the Rhine compared to the Humber area. The registered Vibrio abundance increase off the Rhine Estuary matches with the documented ecological regime shift occurred in the North Sea ecosystem in the late 1980s that affected all trophic levels, from phytoplankton to fish and that led to a warming of seawater in the southern North Sea. In order to study changes within microbial community associated to CPR samples over four decades, a pyrosequencing analyses was applied on five selected CPR samples collected off the Rhine Estuary in 1961, 1972, 1976, 1998 and 2004. The results provided evidence that bacteria belonging to the genus Vibrio, including V. cholerae, not only increased in relative abundance over the last half century in the southern North Sea, but also became dominant within the plankton-associated bacterial community of coastal marine waters. Based on this evidence, the increasing dominance of both harmless and pathogenic Vibrio spp. among the plankton - associated bacterial community of coastal marine water could be a likely event also in other areas around the world as a response to climate change. The role of temperature as a major environmental variable driving Vibrio spp. abundance has been confirmed at a local and seasonal scale. An in situ analysis, conducted during an 8 months cycle in the Goro Lagoon (Ferrara, Italy), showed that both free-living and plankton attached vibrios reached the highest concentrations during the warmest months. The role of temperature on the ecology of human pathogenic vibrios was further confirmed by another set of experiments intended to study in vitro temperature effects on V. cholerae adhesion to different plankton organisms. It was shown that the efficiency of colonization of the copepod T. fulvus by V. cholerae El Tor increases at increasing incubation temperatures, with a maximum efficiency registered at 25°C. Another set of experiments was conducted in order to study if the observed temperature-dependent V. cholerae increase in interactions with plankton was due, at least in part, to an increase in the expression of the GbpA ligand, the main adhesin involved in V. cholerae interactions with plankton organisms by specifically binding N-acetyl glucosamine. GbpA gene expression level was evaluated relative to the expression of the endogenous control gene gyrA. The results showed that GbpA expression increases at increasing temperature values (from 15°C to 25°C). In conclusion, the presented findings have shown that: i) formalin preserved CPR samples can be used for the assessment of microbial diversity, opening up a novel window for long-term and retrospective studies of the global ecology of marine bacterial communities; ii) a major change in the structure of the bacterial community associated with plankton occurred in the southern North Sea in response to increasing SST over the last four decades; iii) SST plays a major role in affecting Vibrio spp. persistence in the aquatic environment also promoting colonization of plankton surfaces; this phenomenon is in turn dependent upon an enhanced expression of bacterial ligands mediating adhesion to chitin containing substrates; iv) based on all the above, the increasing dominance of marine Vibrio spp., including pathogenic species, among plankton-associated bacterial communities of coastal water, may have occurred (and may occur) in other areas around the world as a response to climate change. Studies on vibrios in natural aquatic environments and the mechanisms driving their spread and transmission to humans are of great importance for deciphering the epidemiology of cholera and other diseases (both food borne and extra-intestinal) associated to pathogenic vibrios. Knowledge in this field represents the starting point for the development of updated control measures based on predictive models that include biological, microbiological and physico-chemical variables.