Ecological diversification reveals routes of pathogen emergence in endemic Vibrio vulnificus populations

Pathogen emergence is a complex phenomenon that, despite its public health relevance, remains poorly understood. Vibrio vulnificus, an emergent human pathogen, can cause a deadly septicaemia with over 50% mortality rate. To date, the ecological drivers that lead to the emergence of clinical strains and the unique genetic traits that allow these clones to colonize the human host remain mostly unknown. We recently surveyed a large estuary in eastern Florida, where outbreaks of the disease frequently occur, and found endemic populations of the bacterium. We established two sampling sites and observed strong correlations between location and pathogenic potential. One site is significantly enriched with strains that belong to one phylogenomic cluster (C1) in which the majority of clinical strains belong. Interestingly, strains isolated from this site exhibit phenotypic traits associated with clinical outcomes, whereas strains from the second site belong to a cluster that rarely causes disease in humans (C2). Analyses of C1 genomes indicate unique genetic markers in the form of clinical-associated alleles with a potential role in virulence. Finally, metagenomic and physicochemical analyses of the sampling sites indicate that this marked cluster distribution and genetic traits are strongly associated with distinct biotic and abiotic factors (e.g., salinity, nutrients, or biodiversity), revealing how ecosystems generate selective pressures that facilitate the emergence of specific strains with pathogenic potential in a population. This knowledge can be applied to assess the risk of pathogen emergence from environmental sources and integrated toward the development of novel strategies for the prevention of future outbreaks.

The emergence of human pathogens is one of the most concerning public health topics of modern times (1–4). According to the World Health Organization, over 300 emerging infectious diseases have been reported in the 1940 to 2004 period, a trend that has continued steadily with recent outbreaks of Ebola in West Africa, Cholera in Yemen, and the global pandemic caused by COVID-19 (3–5). Even though classical molecular approaches have advanced our understanding of bacterial pathogenesis, to date, the genetic adaptations and ecological drivers that facilitate selected strains within a species to emerge as pathogens and successfully colonize the human host remain poorly understood. Given the magnitude and complexity of this urgent threat, it is critical to develop tractable organismal model systems and theoretical frameworks that allow us to dissect the molecular adaptations and environmental factors that lead to the emergence of such human pathogens.Vibrio vulnificus, an emergent human pathogen, is one of the leading causes of non-Cholera, Vibrio-associated deaths globally (6). Despite being a natural inhabitant of estuarine, coastal, and brackish waters (7), this flesh-eating bacterium has gained particular notoriety as one of the fastest killing pathogens (8, 9). Humans are typically infected with V. vulnificus through ingestion of contaminated raw seafood or by direct exposure of open wounds to seawater (6). V. vulnificus infections often result in fulminant septicaemia with an alarming mortality rate exceeding 50% (6, 10–13). The bacterium is particularly lethal in some susceptible hosts, such as immunocompromised patients or those with alcohol-associated liver cirrhosis, diabetes mellitus, or hemochromatosis (14). The annual case counts of V. vulnificus infections have steadily increased over the past 20 y in the United States (15). An upsurge in its worldwide distribution over the past three decades, in correlation with climate change, has led to disease outbreaks in regions with no history of V. vulnificus infections (16–18). Furthermore, models predict this trend to continue resulting in a steady expansion of its geographical range and the subsequent increased risk of human infections (16, 19–21).Based on a series of biochemical and phenotypic traits, V. vulnificus strains have been historically classified into three Biotypes (BT): BT1, which is mostly associated with human infections (22, 23), BT2, which is primarily pathogenic to eels (24, 25), and BT3, which is geographically restricted to Israel and possesses hybrid characteristics from BT1 and BT2 (26, 27). In contrast to Vibrio cholerae, in which all strains capable of causing cholera belong to a single clade, genomic comparisons of V. vulnificus reveal a more complex pattern in the distribution of its clinical strains (28–30). Phylogenomic analyses indicate that the population of V. vulnificus is composed of four distinct groups or clusters (Cluster 1 to 4), which largely overlap with the classical BT classification system (23, 26, 28, 31, 32). Our analyses indicate that the two largest clusters, C1 and C2, exhibit high genomic divergence and appear to be speciating (28), with clinical strains from BT1 predominantly belonging to C1 (22, 23), whereas strains from C2 are primarily associated with BT2 (6, 24, 25). C3 is highly clonal and fully overlaps with BT3, and the rare C4 contains only four nonclonal strains and belongs to BT1 (28, 31). Interestingly, despite patients showing conserved clinical symptoms, C1 clinical strains arise from different clades within the cluster, suggesting independent emergence events of this deadly pathogen (28, 31, 32). To date, the unique genetic traits that allow certain C1 strains to cause severe septicemia remain mostly unknown, posing a daunting public health risk as it hinders our ability to detect potentially pathogenic V. vulnificus (33).Recently, using a combination of bioinformatic and phenotypic analyses that surveyed more than 100 strains of V. vulnificus, we determined that V. vulnificus C1 appears to be associated with a unique ecological lifestyle or ecotype (28). Nonetheless, to date, the ecological drivers that lead to the emergence of clinical V. vulnificus C1 and their pathogenic traits remain poorly understood. In order to start untangling the complex in-situ interactions between genotypes and the environment that underlie the emergence of clinical strains, in this study, we recently surveyed a large estuary in eastern Florida, the Indian River Lagoon (IRL), where outbreaks of the disease frequently occur (7, 34). We found endemic populations of V. vulnificus in the estuary and established two sampling locations to study the environmental dynamics of this bacterium in several natural reservoirs such as water, sediment, oysters, and cyanobacteria. Interestingly, the two sampling sites show major differences in the distribution of V. vulnificus clusters. One of them, Feller’s house (Site A), appears to be significantly enriched with C1 strains, whereas in the second sampling site, Shepard Park (Site B), we mostly recovered strains from C2. Genomic analyses of these strains indicate that, despite these major differences in distribution, high recombination rates as well as frequent exchange of mobile genetic elements and virulence factors between these V. vulnificus populations occur. Microdiversity analyses of these genomes revealed unique genomic markers among C1 strains in the form of clinical-associated alleles (CAAs) with a potential direct role in virulence. The isolated V. vulnificus strains are resistant to numerous commonly used antibiotics irrespective of cluster or site of isolation. However, phenotypic analyses indicate that strains from Site A exhibit traits associated with clinical outcomes, including the ability to resist serum and catabolize sialic acid, unlike those from Site B. Finally, metagenomic and physicochemical analyses of the sampling sites indicate that this marked cluster distribution is strongly associated with distinct biotic and abiotic factors (e.g., salinity, nutrients, or biodiversity) revealing how ecosystems might generate selective pressures that facilitate the emergence of specific strains in a population with pathogenic potential.