Meteorological associations of Vibrio vulnificus clinical infections in tropical settings: Correlations with air pressure, wind speed, and temperature.

V. vulnificus is one of the deadliest waterborne pathogens, yet little is known of the ecological and environmental forces that drive outbreaks. As a nationally notifiable disease, all cases of V. vulnificus diagnosed in the United States are reported to the state in which they occurred, as well as to the Centers for Disease Control (CDC) in Atlanta, Georgia. Given that the state of Florida is a 'hotspot' for V. vulnificus in the United States, we examined the prevalence and incidence of cases reported to the Florida Department of Health (2008-2020). Using a dataset comprised of 448 cases of disease caused by V. vulnificus infection, we identified meteorological variables that were associated with clinical cases and deaths. Combined with data from the National Oceanic and Atmospheric Administration (NOAA), we first utilized correlation analysis to examine the linear relationships between satellite meteorological measurements such as wind speed, air temperature, water temperature, and sea-level pressure. We then measured the correlation of those meteorological variables with coastal cases of V. vulnificus, including the outcome, survival, or death. We also constructed a series of logistic regression models to analyze the relationship between temporal and meteorological variables during months that V. vulnificus cases were reported versus months when V. vulnificus cases were not reported. We report that between 2008 and 2020, V. vulnificus cases generally increased over time, peaking in 2017. As water temperature and air temperature increased, so too did the likelihood that infection with V. vulnificus would lead to patient death. We also found that as mean wind speed and sea-level pressure decreased, the probability that a V. vulnificus case would be reported increased. In summary, we discuss the potential factors that may contribute to the observed correlations and speculate that meteorological variables may increase in their public health relevance in light of rising global temperatures.

Allelic diversity uncovers protein domains contributing to the emergence of antimicrobial resistance.

Antimicrobial resistance (AMR) remains a major threat to global health. To date, tractable approaches that decipher how AMR emerges within a bacterial population remain limited. Here, we developed a framework that exploits genetic diversity from environmental bacterial populations to decode emergent phenotypes such as AMR. OmpU is a porin that can make up to 60% of the outer membrane of Vibrio cholerae, the cholera pathogen. This porin is directly associated with the emergence of toxigenic clades and confers resistance to numerous host antimicrobials. In this study, we examined naturally occurring allelic variants of OmpU in environmental V. cholerae and established associations that connected genotypic variation with phenotypic outcome. We covered the landscape of gene variability and found that the porin forms two major phylogenetic clusters with striking genetic diversity. We generated 14 isogenic mutant strains, each encoding a unique ompU allele, and found that divergent genotypes lead to convergent antimicrobial resistance profiles. We identified and characterized functional domains in OmpU unique to variants conferring AMR-associated phenotypes. Specifically, we identified four conserved domains that are linked with resistance to bile and host-derived antimicrobial peptides. Mutant strains for these domains exhibit differential susceptibility patterns to these and other antimicrobials. Interestingly, a mutant strain in which we exchanged the four domains of the clinical allele for those of a sensitive strain exhibits a resistance profile closer to a porin deletion mutant. Finally, using phenotypic microarrays, we uncovered novel functions of OmpU and their connection with allelic variability. Our findings highlight the suitability of our approach towards dissecting the specific protein domains associated with the emergence of AMR and can be naturally extended to other bacterial pathogens and biological processes.

Vibrio Infections and the Twenty-First Century.

The Vibrionaceae is a highly diverse family of aquatic bacteria. Some members of this ubiquitous group can cause a variety of diseases in humans ranging from cholera caused by Vibrio cholerae, severe septicemia caused by Vibrio vulnificus, to acute gastroenteritis by Vibrio parahaemolyticus. Planet Earth is experiencing unprecedented changes of planetary scale associated with climate change. These environmental perturbations paired with overpopulation and pollution are increasing the distribution of pathogenic Vibrios and exacerbating the risk of causing infections. In this chapter, we discuss various aspects of Vibrio infections within the context of the twenty-first century with a major emphasis on the aforementioned pathogenic species. Overall, we believe that the twenty-first century is posed to be both one full of challenges due to the rise of these pathogens, and also a catalyst for innovative and groundbreaking discoveries.

Cholera Dynamics and the Emergence of Pandemic Vibrio cholerae.

Cholera is a severe diarrheal disease caused by the aquatic bacterium Vibrio cholerae. Interestingly, to date, only one major clade has emerged to cause pandemic disease in humans: the clade that encompasses the strains from the O1 and O139 serogroups. In this chapter, we provide a comprehensive perspective on the virulence factors and mobile genetic elements (MGEs) associated with the emergence of pandemic V. cholerae strains and highlight novel findings such as specific genomic background or interactions between MGEs that explain their confined distribution. Finally, we discuss pandemic cholera dynamics contextualizing them within the evolution of the bacterium.

Single-amplified genomes reveal most streamlined free-living marine bacteria.

Evolutionary adaptations of prokaryotes to the environment sometimes result in genome reduction. Our knowledge of this phenomenon among free-living bacteria remains scarce. We address the dynamics and limits of genome reduction by examining one of the most abundant bacteria in the ocean, the SAR86 clade. Despite its abundance, comparative genomics has been limited by the absence of pure cultures and the poor representation in metagenome-assembled genomes. We co-assembled multiple previously available single-amplified genomes to obtain the first complete genomes from members of the four families. All families showed a convergent evolutionary trajectory with characteristic features of streamlined genomes, most pronounced in the TMED112 family. This family has a genome size of ca. 1 Mb and only 1 bp as median intergenic distance, exceeding values found in other abundant microbes such as SAR11, OM43 and Prochlorococcus. This genomic simplification led to a reduction in the biosynthesis of essential molecules, DNA repair-related genes, and the ability to sense and respond to environmental factors, which could suggest an evolutionary dependence on other co-occurring microbes for survival (Black Queen hypothesis). Therefore, these reconstructed genomes within the SAR86 clade provide new insights into the limits of genome reduction in free-living marine bacteria.

Vibrio floridensis sp. nov., a novel species closely related to the human pathogen Vibrio vulnificus isolated from a cyanobacterial bloom.

A Gram-stain-negative, rod-shaped bacterial strain, designated Vibrio floridensis IRLE0018 (=NRRL B-65642=NCTC 14661), was isolated from a cyanobacterial bloom along the Indian River Lagoon (IRL), a large and highly biodiverse estuary in eastern Florida (USA). The results of phylogenetic, biochemical, and phenotypic analyses indicate that this isolate is distinct from species of the genus Vibrio with validly published names and is the closest relative to the emergent human pathogen, Vibrio vulnificus. Here, we present the complete genome sequence of V. floridensis strain IRLE0018 (4 535 135 bp). On the basis of the established average nucleotide identity (ANI) values for the determination of different species (ANI <95 %), strain IRLE0018, with an ANI of approximately 92 % compared with its closest relative, V. vulnificus, represents a novel species within the genus Vibrio. To our knowledge, this represents the first time this species has been described. The results of genomic analyses of V. floridensis IRLE0018 indicate the presence of antibiotic resistance genes and several known virulence factors, however, its pathogenicity profile (e.g. survival in serum, phagocytosis avoidance) reveals limited virulence potential of this species in contrast to V. vulnificus.

Thanks, but no thanks: Cholera pathogen keeps incoming DNA at bay.

Bacterial pathogens must maintain a delicate balance between acquiring novel genetic material and preserving their well-engrained cell physiology. In the recent study by Jaskólska et al., an astonishing example of this sophisticated phenomenon was found in Vibrio cholerae,with the pandemic strain encoding systems within two horizontally acquired pathogenicity islands that degrade incoming DNA.

Molecular mechanisms and drivers of pathogen emergence.

Pathogen emergence (PE) is a complex phenomenon with major public health implications. Over the past decades, numerous underlying mechanisms facilitating the emergence of pathogenic bacteria have been elucidated. In this review, we highlight the diverse molecular and environmental drivers associated with PE, with an emphasis on the interplay of canonical gene transfer mechanisms and the increasingly appreciated role of genetic variations, providing a more coherent picture of this process. Given the interactive and multifactorial nature of PE, we contend that the development of approaches that embrace the integration of these factors is indispensable in order to truly comprehend this complex phenomenon and develop strategies to mitigate this threat.

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.

JMM Profile: Vibrio cholerae: an opportunist of human crises.

Vibrio cholerae O1 is the aetiological agent of the severe diarrhoeal disease cholera. Annually, there are an estimated 1-4 million cholera cases worldwide and over 140 000 deaths. The primary mode of disease transmission is through the consumption of water or food contaminated with the bacterium. Although cholera patients can be treated effectively using rehydration therapy, the disease remains a major scourge in areas with limited access to clean water and proper sanitation. Its continued prevalence highlights the failure of socioeconomic policies leading to wealth disparities, fragile and dated public infrastructure, and lack of appropriate health surveillance.

Cholera dynamics: lessons from an epidemic.

Cholera is a severe diarrhoeal disease that spreads rapidly and affects millions of people each year, resulting in tens of thousands of deaths. The disease is caused by Vibrio cholerae O1 and is characterized by watery diarrhoea that can be lethal if not properly treated. Cholera had not been reported in South America from the late 1800s until 1991, when it was introduced in Peru, wreaking havoc in one of the biggest epidemics reported to date. Within a year, the disease had spread to most of the Latin American region, resulting in millions of cases and thousands of deaths in all affected countries. Despite its aggressive entry, cholera virtually disappeared from the continent after 1999. The progression of the entire epidemic was well documented, making it an ideal model to understand cholera dynamics. In this review, we highlight how the synergy of socioeconomic, political and ecological factors led to the emergence, rapid spread and eventual disappearance of cholera in Latin America. We discuss how measures implemented during the cholera epidemic drastically changed its course and continental dynamics. Finally, we synthesize our findings and highlight potential lessons that can be learned for efficient and standardized cholera management programmes during future outbreaks in non-endemic areas.

Dysbiosis in marine aquaculture revealed through microbiome analysis: reverse ecology for environmental sustainability.

The increasing demand for products for human consumption is leading to the fast-growing expansion of numerous food sectors such as marine aquaculture (mariculture). However, excessive input of nutrients and pollutants modifies marine ecosystems. Here, we applied a metagenomic approach to investigate these perturbations in samples from marine farms of gilthead seabream cultures. Results revealed dysbiosis and functional imbalance within the net cage with a unique structure, with little interference with samples from the fish microbiota or those collected far away from the coast. Remarkably, below the cage the prokaryotic community was highly similar to the marine microbiome of photic offshore samples. We recovered 48 novel metagenome-assembled genomes. Metagenomic recruitment revealed a significant change in the microbial community which was dominated by several Proteobacteria orders (Sphingomonadales, Pseudomonadales, Caudobacterales and Rhizobiales). Genomic potential for bioremediation processes, including nitrate removal through aerobic denitrification, and degradation of aromatic compounds and other toxic products were enriched in these microbes. The detrimental side effects were the increased number of antimicrobial resistance genes and the presence of potentially emergent pathogens. Knowledge of this metabolic diversity and the microbes involved in ecological balance recovery can be used to reduce the environmental impact of these practices.

Improved Method for Transformation of Vibrio vulnificus by Electroporation.

Vibrio vulnificus, an emergent human pathogen, causes fulminant septicemia with a mortality rate of over 50%. Unlike for other pathogenic Vibrio species, the factors to conclusively indicate the virulence potential of V. vulnificus strains remain largely unknown. Understanding the pathogenesis of this bacterium at a molecular level is severely hindered by inefficiencies in transformation, for instance, due to the presence of a periplasmic nuclease, Vvn. Currently, successful transformation of V. vulnificus is nearly impossible due to lack of mobilizable plasmids for the bacterium, requiring (i) very high DNA concentrations, (ii) plasmid linearization, (iii) development of novel V. vulnificus-derived plasmids, or (iv) time-consuming conjugation-based methods. To overcome these limitations, we describe a rapid, efficient, and reproducible electroporation protocol to effectively transform widely available plasmids, with different copy numbers and antibiotic resistances, into phylogenetically distant strains of V. vulnificus. Cells are made competent in high concentrations of sucrose devoid of cations and recovered from electroporation using a high-salinity recovery medium. Compared to existing methods for transformation of V. vulnificus, significantly higher efficiencies are obtained using this improved protocol. Rapid and effective transformations can markedly improve molecular analyses of V. vulnificus leading to a greater understanding of its virulence potential. This is crucial to develop rapid detection methods which have the potential to prevent future outbreaks. The electroporation protocol described here may be particularly useful for optimizing transformation of other nuclease-producing bacteria. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent cells Basic Protocol 2: Transformation of cells by electroporation.

Synergistic role of abiotic factors driving viable but non-culturable Vibrio cholerae.

Vibrio cholerae O1, a natural inhabitant of estuarine environments, is found in a dormant, viable but non-culturable (VBNC) state during interepidemic periods. Although the individual roles of abiotic factors affecting VBNC formation have been extensively studied, their interplay in driving this phenomenon remains largely unaddressed. Here, we identified that major abiotic factors synergize with low nutrient conditions governing entry of cells into the VBNC state. Specifically, V. cholerae cells exposed to a combination of alkaline pH and high salinity under aeration at low temperatures (VBNC-inducing conditions) synergize to facilitate rapid entry into VBNC, whereas the opposite conditions prevented entry into the state. The major virulence regulator ToxR, and the stringent response protein RelA played opposing roles, repressing and facilitating VBNC entry respectively. Further, VBNC-inducing conditions negated the effects of ToxR and RelA, facilitating rapid formation of VBNC cells. In summary, this study highlights the synergy between critical abiotic factors and identified ToxR and RelA as two associated regulators, allowing for the persistence of V. cholerae in aquatic environments. Insights obtained in this study will help better understand environmental survival non-sporulating bacteria and transmission of facultative bacterial pathogens.

Direct transmission via households informs models of disease and intervention dynamics in cholera.

While several basic properties of cholera outbreaks are common to most settings-the pathophysiology of the disease, the waterborne nature of transmission, and others-recent findings suggest that transmission within households may play a larger role in cholera outbreaks than previously appreciated. Important features of cholera outbreaks have long been effectively modeled with mathematical and computational approaches, but little is known about how variation in direct transmission via households may influence epidemic dynamics. In this study, we construct a mathematical model of cholera that incorporates transmission within and between households. We observe that variation in the magnitude of household transmission changes multiple features of disease dynamics, including the severity and duration of outbreaks. Strikingly, we observe that household transmission influences the effectiveness of possible public health interventions (e.g. water treatment, antibiotics, vaccines). We find that vaccine interventions are more effective than water treatment or antibiotic administration when direct household transmission is present. Summarizing, we position these results within the landscape of existing models of cholera, and speculate on its implications for epidemiology and public health.

Hepatitis C virus modelled as an indirectly transmitted infection highlights the centrality of injection drug equipment in disease dynamics.

The hepatitis C virus (HCV) epidemic often occurs through the persistence of injection drug use. Mathematical models have been useful in understanding various aspects of the HCV epidemic, and especially, the importance of new treatment measures. Until now, however, few models have attempted to understand HCV in terms of an interaction between the various actors in an HCV outbreak-hosts, viruses and the needle injection equipment. In this study, we apply perspectives from the ecology of infectious diseases to model the transmission of HCV among a population of injection drug users. The products of our model suggest that modelling HCV as an indirectly transmitted infection-where the injection equipment serves as an environmental reservoir for infection-facilitates a more nuanced understanding of disease dynamics, by animating the underappreciated actors and interactions that frame disease. This lens may allow us to understand how certain public health interventions (e.g. needle exchange programmes) influence HCV epidemics. Lastly, we argue that this model is of particular importance in the light of the modern opioid epidemic, which has already been associated with outbreaks of viral diseases.

Evolutionary Model of Cluster Divergence of the Emergent Marine Pathogen Vibrio vulnificus: From Genotype to Ecotype.

Vibrio vulnificus, an opportunistic pathogen, is the causative agent of a life-threatening septicemia and a rising problem for aquaculture worldwide. The genetic factors that differentiate its clinical and environmental strains remain enigmatic. Furthermore, clinical strains have emerged from every clade of V. vulnificus In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species from an evolutionary and ecological point of view. Genome comparisons and bioinformatic analyses of 113 V. vulnificus isolates indicate that the population of V. vulnificus is made up of four different clusters. We found evidence that recombination and gene flow between the two largest clusters (cluster 1 [C1] and C2) have drastically decreased to the point where they are diverging independently. Pangenome and phenotypic analyses showed two markedly different lifestyles for these two clusters, indicating commensal (C2) and bloomer (C1) ecotypes, with differences in carbohydrate utilization, defense systems, and chemotaxis, among other characteristics. Nonetheless, we identified frequent intra- and interspecies exchange of mobile genetic elements (e.g., antibiotic resistance plasmids, novel "chromids," or two different and concurrent type VI secretion systems) that provide high levels of genetic diversity in the population. Surprisingly, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together. We propose an evolutionary model of V. vulnificus that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections and emergence.IMPORTANCEVibrio vulnificus is an emergent marine pathogen and is the cause of a deadly septicemia. However, the genetic factors that differentiate its clinical and environmental strains and its several biotypes remain mostly enigmatic. In this work, we investigated the underlying genomic properties and population dynamics of the V. vulnificus species to elucidate the traits that make these strains emerge as a human pathogen. The acquisition of different ecological determinants could have allowed the development of highly divergent clusters with different lifestyles within the same environment. However, we identified strains from both clusters in the mucosa of aquaculture species, indicating that manmade niches are bringing strains from the two clusters together, posing a potential risk of recombination and of emergence of novel variants. We propose a new evolutionary model that provides a perspective that could be broadly applicable to other pathogenic vibrios and facultative bacterial pathogens to pursue strategies to prevent their infections.

Catabolism of mucus components influences motility of Vibrio cholerae in the presence of environmental reservoirs.

Vibrio cholerae O1, the etiological agent of cholera, is a natural inhabitant of aquatic ecosystems. Motility is a critical element for the colonization of both the human host and its environmental reservoirs. In this study, we investigated the molecular mechanisms underlying the chemotactic response of V. cholerae in the presence of some of its environmental reservoirs. We found that, from the several oligosaccharides found in mucin, two specifically triggered motility of V. cholerae O1: N-acetylneuraminic acid (Neu5Ac) and N-acetylglucosamine (GlcNAc). We determined that the compounds need to be internally catabolized in order to trigger motility of V. cholerae. Interestingly, the catabolism of Neu5Ac and GlcNAc converges and the production of one molecule common to both pathways, glucosamine-6-phosphate (GlcN-6P), is essential to induce motility in the presence of both compounds. Mutants unable to produce GlcN-6P show greatly reduced motility towards mucin. Furthermore, we determined that the production of GlcN-6P is necessary to induce motility of V. cholerae in the presence of some of its environmental reservoirs such as crustaceans or cyanobacteria, revealing a molecular link between the two distinct modes of the complex life cycle of V. cholerae. Finally, cross-species comparisons revealed varied chemotactic responses towards mucin, GlcNAc, and Neu5Ac for environmental (non-pathogenic) strains of V. cholerae, clinical and environmental isolates of the human pathogens Vibrio vulnificus and Vibrio parahaemolyticus, and fish and squid isolates of the symbiotic bacterium Vibrio fischeri. The data presented here suggest nuance in convergent strategies across species of the same bacterial family for motility towards suitable substrates for colonization.

Environmental role of pathogenic traits in Vibrio cholerae.

Vibrio cholerae is a natural inhabitant of aquatic ecosystems. Some strains of V. cholerae can colonize the human host and cause cholera, a profuse watery diarrhea. The major pathogenicity factors and virulence regulators of V. cholerae are either encoded in mobile genetic elements acquired in the environment (e.g. pathogenicity islands or lysogenic phages) or in the core genome. Several lines of evidence indicate that the emergence of numerous virulence traits of V. cholerae occurred in its natural environment due to biotic and abiotic pressures. Here, we discuss the connection between the human host and the potential ecological role of these virulent traits. Unraveling these connections will help us understand the emergence of this organism and other facultative bacterial pathogens.