Pandemic Vibrio Cholerae Research Articles

Fine-tuning of a CRISPRi screen in the seventh pandemic Vibrio cholerae

BackgroundVibrio cholerae O1 El Tor, the etiological agent responsible for the last cholera pandemic, has become a well-established model organism for which some genetic tools are available. While CRISPRi technology has been applied to V. cholerae, improvements were necessary to upscale it and enable pooled screening by high-throughput sequencing in this bacterium.ResultsIn this study, we present a genome-wide CRISPR-dCas9 screen specifically optimized for the N16961 El Tor model strain of V. cholerae. This approach is characterized by a tight control of dCas9 expression and activity, as well as a streamlined experimental setup. Our library allows the depletion of 3,674 (98.9%) annotated genes from the V. cholerae genome. To confirm its effectiveness, we screened for genes that are essential during exponential growth in rich medium and identified 369 genes for which guides were significantly depleted from the library (log2FC < -2). Remarkably, 82% of these genes had previously been described as hypothetical essential genes in V. cholerae or in a closely related bacterium, V. natriegens.ConclusionWe thus validated the robustness and accuracy of our CRISPRi-based approach for assessing gene fitness in a given condition. Our findings highlight the efficacy of the developed CRISPRi platform as a powerful tool for high-throughput functional genomics studies of V. cholerae.

Genomic evidence of two-staged transmission of the early seventh cholera pandemic

The seventh cholera pandemic started in 1961 in Indonesia and spread across the world in three waves in the decades that followed. Here, we utilised genomic evidence to detail the first wave of the seventh pandemic. Genomes of 22 seventh pandemic Vibrio cholerae isolates from 1961 to 1979 were completely sequenced. Together with 152 publicly available genomes from the same period, they fell into seven phylogenetic clusters (CL1–CL7). By multilevel genome typing (MGT), all were assigned to MGT2 ST1 (Wave 1) except three isolates in CL7 which were typed as MGT2 ST2 (Wave 2). The Wave 1 seventh pandemic expanded in two stages, with Stage 1 (CL1–CL5) spread across Asia and Stage 2 (CL6 and CL7) spread to the Middle East and Africa. Three non-synonymous mutations, one each, in three regulatory genes, csrD (global regulator), acfB (chemotaxis), and luxO (quorum sensing) may have critically contributed to its pandemicity. The three MGT2 ST2 isolates in CL7 were the progenitors of Wave 2 and evolved from within Wave 1 with acquisition of a novel IncA/C plasmid. Our findings provide new insight into the evolution and transmission of the early seventh pandemic, which may aid future cholera prevention and control.

Molecular mechanism of plasmid elimination by the DdmDE defense system.

Seventh-pandemic Vibrio cholerae strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. In this study, we used cryogenic electron microscopy to determine the mechanistic basis for plasmid defense by DdmDE. The helicase-nuclease DdmD adopts an autoinhibited dimeric architecture. The prokaryotic Argonaute protein DdmE uses a DNA guide to target plasmid DNA. The structure of the DdmDE complex, validated by in vivo mutational studies, shows that DNA binding by DdmE triggers disassembly of the DdmD dimer and loading of monomeric DdmD onto the nontarget DNA strand. In vitro studies indicate that DdmD translocates in the 5'-to-3' direction, while partially degrading the plasmid DNA. These findings provide critical insights into the mechanism of DdmDE systems in plasmid elimination.

Genetic approach toward linkage of Iran 2012–2016 cholera outbreaks with 7th pandemic Vibrio cholerae

Vibrio cholerae, as a natural inhabitant of the marine environment is among the world-leading causes of diarrheal diseases. The present study aimed to investigate the genetic relatedness of Iran 2012–2016 V. cholerae outbreaks with 7th pandemic cholera and to further characterize the non-ST69/non-ST75 sequence types strains by whole-genome sequencing (WGS).Twenty V. cholerae isolates related to 2012, 2013, 2015 and 2016 cholera outbreaks were studied by two genotyping methods – Pulsed-field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST)–and by antimicrobial susceptibility testing. Seven sequence types (STs) and sixteen pulsotypes were detected. Sequence type 69 was the most abundant ST confirming that most (65%, 13/20) of the studied isolates collected in Iran between 2012 and 2016 belonged to the 7th pandemic clone. All these ST69 isolates (except two) exhibited similar pulsotypes. ST75 was the second most abundant ST. It was identified in 2015 and 2016. ST438, ST178, ST579 and STs of 983 and 984 (as newfound STs) each were only detected in one isolate. All strains collected in 2016 appeared as distinct STs and pulsotypes indicative of probable different originations. All ST69 strains were resistant to nalidixic acid. Moreover, resistance to nalidixic acid, trimethoprim-sulfamethoxazole and tetracycline was only observed in strains of ST69. These properties propose the ST69 as a unique genotype derived from a separate lineage with distinct resistance properties. The circulation of V. cholerae ST69 and its traits in recent years in Iran proposes the 7th pandemic strains as the ongoing causes of cholera outbreaks in this country, although the role of ST75 as the probable upcoming dominant ST should not be ignored.Genomic analysis of non-ST69/non-ST75 strains in this study showed ST579 is the most similar ST type to 7th pandemic sequence types, due to the presence of wild type-El Tor sequences of tcpA and VC-1319, VC-1320, VC-1577, VC-1578 genes (responsible for polymyxin resistance in El Tor biotype), the traits of rstC of RS1 phage in one strain of this ST type and the presence of VPI-1 and VSP-I islands in ST579 and ST178 strains. In silico analysis showed no significant presence of resistance genes/cassettes/plasmids within non-ST69/non-ST75 strains genomes. Overall, these data indicate the higher susceptibility of V. cholerae non-ST69/non-ST75 strains in comparison with more ubiquitous and more circulating ST69 and ST75 strains.In conclusion, the occurrence of small outbreaks and sporadic cholera cases due to V. cholerae ST69 in recent years in Iran shows the 7th pandemic strains as the persistent causes of cholera outbreaks in this country, although the role of ST75 as the second most contributed ST should not be ignored. The occurrence of non-ST69/non-ST75 sequence types with some virulence factors characteristics in border provinces in recent years is noteworthy, and further studies together with surveillance efforts are expected to determine their likely route of transport.

Inferring the proportion of undetected cholera infections from serological and clinical surveillance in an immunologically naive population.

Most infections with pandemic Vibrio cholerae are thought to result in subclinical disease and are not captured by surveillance. Previous estimates of the ratio of infections to clinical cases have varied widely (2 to 100 infections per case). Understanding cholera epidemiology and immunity relies on the ability to translate between numbers of clinical cases and the underlying number of infections in the population. We estimated the infection incidence during the first months of an outbreak in a cholera-naive population using a Bayesian vibriocidal antibody titer decay model combining measurements from a representative serosurvey and clinical surveillance data. 3,880 suspected cases were reported in Grande Saline, Haiti, between 20 October 2010 and 6 April 2011 (clinical attack rate 18.4%). We found that more than 52.6% (95% Credible Interval (CrI) 49.4-55.7) of the population ≥2 years showed serologic evidence of infection, with a lower infection rate among children aged 2-4 years (35.5%; 95%CrI 24.2-51.6) compared with people ≥5 years (53.1%; 95%CrI 49.4-56.4). This estimated infection rate, nearly three times the clinical attack rate, with underdetection mainly seen in those ≥5 years, has likely impacted subsequent outbreak dynamics. Our findings show how seroincidence estimates improve understanding of links between cholera burden, transmission dynamics and immunity.

Valid and accepted novel bacterial taxa derived from human clinical specimens and taxonomic revisions published in 2022.

Although some nomenclature changes have caused consternation among clinical microbiologists, the discovery of novel taxa and improving classification of existing groups of organisms is exciting and adds to our understanding of microbial pathogenesis. In this mini-review, we present an in-depth summary of novel taxonomic designations and revisions to prokaryotic taxonomy that were published in 2022. Henceforth, these bacteriology taxonomic summaries will appear annually. Several of the novel Gram-positive organisms have been associated with disease, namely, the Corynebacterium kroppenstedtii-like organisms Corynebacterium parakroppenstedtii sp. nov. and Corynebacterium pseudokroppenstedtii sp. nov. A newly described Streptococcus species, Streptococcus toyakuensis sp. nov., is noteworthy for exhibiting multi-drug resistance. Among the novel Gram-negative pathogens, Vibrio paracholerae sp. nov. stands out as an organism associated with diarrhea and sepsis and has probably been co-circulating with pandemic Vibrio cholerae for decades. Many new anaerobic organisms have been described in this past year largely from genetic assessments of gastrointestinal microbiome collections. With respect to revised taxa, as discussed in previous reviews, the genus Bacillus continues to undergo further division into additional genera and reassignment of existing species into them. Reassignment of two subspecies of Fusobacterium nucleatum to species designations (Fusobacterium animalis sp. nov. and Fusobacterium vincentii sp. nov.) is also noteworthy. As was typical of previous reviews, literature updates for selected clinically relevant organisms discovered between 2017 and 2021 have been included.

Activation of a Vibrio cholerae CBASS anti-phage system by quorum sensing and folate depletion.

To counteract infection with phage, bacteria have evolved a myriad of molecular defense systems. Some of these systems initiate a process called abortive infection, in which the infected cell kills itself to prevent phage propagation. However, such systems must be inhibited in the absence of phage infection to prevent spurious death of the host. Here, we show that the cyclic oligonucleotide based anti-phage signaling system (CBASS) accomplishes this by sensing intracellular folate molecules and only expressing this system in a group. These results enhance our understanding of the evolution of the seventh Vibrio cholerae pandemic and more broadly how bacteria defend themselves against phage infection.

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.

Altered molecular attributes and antimicrobial resistance patterns of Vibrio cholerae O1 El Tor strains isolated from the cholera endemic regions of India.

The present study aimed to document the comparative analysis of differential hypervirulent features of Vibrio cholerae O1 strains isolated during 2018 from cholera endemic regions in Gujarat and Maharashtra (Western India) and West Bengal (Eastern India). A total of 87V. cholerae O1 clinical strains from Western India and 48 from Eastern India were analysed for a number of biotypic and genotypic features followed by antimicrobial resistance (AMR) profile. A novel polymerase chain reaction was designed to detect a large fragment deletion in the Vibrio seventh pandemic island II (VSP-II) genomic region, which is a significant genetic feature of the V. cholerae strains that have caused Yemen cholera outbreak. All the strains from Western India belong to the Ogawa serotype, polymyxin B-sensitive, hemolytic, had a deletion in VSP-II (VSP-IIC) region and carried Haitian genetic alleles of ctxB, tcpA and rtxA. Conversely, 14.6% (7/48) of the strains from Eastern India belonged to the Inaba serotype, polymyxin B-resistant, nonhemolytic, harboured VSP-II other than VSP-IIC type, classical ctxB, Haitian tcpA and El Tor rtxA alleles. Resistance to tetracycline and chloramphenicol has been observed in strains from both regions. This study showed hypervirulent, polymyxin B-sensitive epidemic causing strains in India along with the strains with polymyxin B-resistant and nonhemolytic traits that may spread and cause serious disease outcomes in future. The outcomes of this study can help to improve the understanding of the hyperpathogenic property of recently circulating pandemic Vibrio cholerae strains in India. Special attention is also needed for the monitoring of AMR surveillance because V. cholerae strains are losing susceptibility to many antibiotics used as a second line of defence in the treatment of cholera.

Pandemic Vibrio cholerae acquired competitive traits from an environmental Vibrio species.

Vibrio cholerae is a human pathogen that thrives in estuarine environments. Within the environment and human host, V. cholerae uses the type VI secretion system (T6SS) to inject toxic effectors into neighboring microbes and to establish its replicative niche. V. cholerae strains encode a wide variety of horizontally shared effectors, but pandemic isolates encode an identical set of distinct effectors. Effector set retention in pandemic strains despite mobility between disparate strains suggests that horizontal acquisition of these effectors was crucial for evolving pandemic V. cholerae We attempted to locate the donor of the pandemic effectors to V. cholerae To this end, we identified potential gene transfer events of the pandemic-associated T6SS clusters between a fish pathogen, Vibrio anguillarum, and V. cholerae We supported the likelihood of interaction between these species by demonstrating that homologous effector-immunity pairs from V. cholerae and V. anguillarum can cross-neutralize one another. Thus, V. anguillarum constitutes an environmental reservoir of pandemic-associated V. cholerae T6SS effectors that may have initially facilitated competition between pre-pandemic V. cholerae and V. anguillarum for an environmental niche.

Two defence systems eliminate plasmids from seventh pandemic Vibrio cholerae.

Horizontal gene transfer can trigger rapid shifts in bacterial evolution. Driven by a variety of mobile genetic elements-in particular bacteriophages and plasmids-the ability to share genes within and across species underpins the exceptional adaptability of bacteria. Nevertheless, invasive mobile genetic elements can also present grave risks to the host; bacteria have therefore evolved a vast array of defences against these elements1. Here we identify two plasmid defence systems conserved in the Vibrio cholerae El Tor strains responsible for the ongoing seventh cholera pandemic2-4. These systems, termed DdmABC and DdmDE, are encoded on two major pathogenicity islands that are a hallmark of current pandemic strains. We show that the modules cooperate to rapidly eliminate small multicopy plasmids by degradation. Moreover, the DdmABC system is widespread and can defend against bacteriophage infection by triggering cell suicide (abortive infection, or Abi). Notably, we go on to show that, through an Abi-like mechanism, DdmABC increases the burden of large low-copy-number conjugative plasmids, including a broad-host IncC multidrug resistance plasmid, which creates a fitness disadvantage that counterselects against plasmid-carrying cells. Our results answer the long-standing question of why plasmids, although abundant in environmental strains, are rare in pandemic strains; have implications for understanding the dissemination of antibiotic resistance plasmids; and provide insights into how the interplay between two defence systems has shapedthe evolution of the most successful lineage of pandemic V. cholerae.

A fructose/H+ symporter controlled by a LacI-type regulator promotes survival of pandemic Vibrio cholerae in seawater

The bacterium Vibrio cholerae can colonize the human intestine and cause cholera, but spends much of its life cycle in seawater. The pathogen must adapt to substantial environmental changes when moving between seawater and the human intestine, including different availability of carbon sources such as fructose. Here, we use in vitro experiments as well as mouse intestinal colonization assays to study the mechanisms used by pandemic V. cholerae to adapt to these environmental changes. We show that a LacI-type regulator (FruI) and a fructose/H+ symporter (FruT) are important for fructose uptake at low fructose concentrations, as those found in seawater. FruT is downregulated by FruI, which is upregulated when O2 concentrations are low (as in the intestine) by ArcAB, a two-component system known to respond to changes in oxygen levels. As a result, the bacteria predominantly use FruT for fructose uptake under seawater conditions (low fructose, high O2), and use a known fructose phosphotransferase system (PTS, Fpr) for fructose uptake under conditions found in the intestine. PTS activity leads to reduced levels of intracellular cAMP, which in turn upregulate virulence genes. Our results indicate that the FruT/FruI system may be important for survival of pandemic V. cholerae in seawater.

Genomic contextualisation of ancient DNA molecular data from an Argentinian fifth pandemic Vibrio cholerae infection.

Specific lineages of serogroup O1 Vibrio cholerae are notorious for causing cholera pandemics, of which there have been seven since the 1800s. Much is known about the sixth pandemic (1899–1923) and the ongoing seventh pandemic (1961–present), but we know very little about the bacteriology of pandemics 1 to 5. Moreover, although we are learning about the contribution of non-O1 non-pandemic V. cholerae to cholera dynamics during the current pandemic, we know almost nothing about their role in the past. A recent ancient DNA study has presented what may be the first molecular evidence of a V. cholerae infection from the fifth cholera pandemic period (1886–1887 AD) in Argentina. Here, we place the molecular evidence from that study into the genomic context of non-pandemic V. cholerae from Latin America and elsewhere, and show that a gene fragment amplified from ancient DNA is most similar to that of V. cholerae from the Americas, and from Argentina. Our results corroborate and reinforce the findings of the original study, and collectively suggest that even in the 1880s, non-pandemic V. cholerae local to the Americas may have caused sporadic infections in Argentina, just as we know this to have happened during the seventh pandemic in Latin America.

Successive epidemic waves of cholera in South Sudan between 2014 and 2017: a descriptive epidemiological study

SummaryBackgroundBetween 2014 and 2017, successive cholera epidemics occurred in South Sudan within the context of civil war, population displacement, flooding, and drought. We aim to describe the spatiotemporal and molecular features of the three distinct epidemic waves and explore the role of vaccination campaigns, precipitation, and population movement in shaping cholera spread in this complex setting.MethodsIn this descriptive epidemiological study, we analysed cholera linelist data to describe the spatiotemporal progression of the epidemics. We placed whole-genome sequence data from pandemic Vibrio cholerae collected throughout these epidemics into the global phylogenetic context. Using whole-genome sequence data in combination with other molecular attributes, we characterise the relatedness of strains circulating in each wave and the region. We investigated the association of rainfall and the instantaneous basic reproduction number using distributed lag non-linear models, compared county-level attack rates between those with early and late reactive vaccination campaigns, and explored the consistency of the spatial patterns of displacement and suspected cholera case reports.FindingsThe 2014 (6389 cases) and 2015 (1818 cases) cholera epidemics in South Sudan remained spatially limited whereas the 2016–17 epidemic (20 438 cases) spread among settlements along the Nile river. Initial cases of each epidemic were reported in or around Juba soon after the start of the rainy season, but we found no evidence that rainfall modulated transmission during each epidemic. All isolates analysed had similar genotypic and phenotypic characteristics, closely related to sequences from Uganda and Democratic Republic of the Congo. Large-scale population movements between counties of South Sudan with cholera outbreaks were consistent with the spatial distribution of cases. 21 of 26 vaccination campaigns occurred during or after the county-level epidemic peak. Counties vaccinated on or after the peak incidence week had 2·2 times (95% CI 2·1–2·3) higher attack rates than those where vaccination occurred before the peak.InterpretationPandemic V cholerae of the same clonal origin was isolated throughout the study period despite interepidemic periods of no reported cases. Although the complex emergency in South Sudan probably shaped some of the observed spatial and temporal patterns of cases, the full scope of transmission determinants remains unclear. Timely and well targeted use of vaccines can reduce the burden of cholera; however, rapid vaccine deployment in complex emergencies remains challenging.FundingThe Bill & Melinda Gates Foundation.

Pandemic Vibrio cholerae shuts down site-specific recombination to retain an interbacterial defence mechanism

Vibrio cholerae is an aquatic microbe that can be divided into three subtypes: harmless environmental strains, localised pathogenic strains, and pandemic strains causing global cholera outbreaks. Each type has a contact-dependent type VI secretion system (T6SS) that kills neighbouring competitors by translocating unique toxic effector proteins. Pandemic isolates possess identical effectors, indicating that T6SS effectors may affect pandemicity. Here, we show that one of the T6SS gene clusters (Aux3) exists in two states: a mobile, prophage-like element in a small subset of environmental strains, and a truncated Aux3 unique to and conserved in pandemic isolates. Environmental Aux3 can be readily excised from and integrated into the genome via site-specific recombination, whereas pandemic Aux3 recombination is reduced. Our data suggest that environmental Aux3 acquisition conferred increased competitive fitness to pre-pandemic V. cholerae, leading to grounding of the element in the chromosome and propagation throughout the pandemic clade.

Emergence of Haitian variant genotype and altered drug susceptibility in Vibrio cholerae O1 El Tor-associated cholera outbreaks in Solapur, India

It is evident from previous cholera epidemics/outbreaks in India, Africa and America that isolates of the seventh pandemic Vibrio cholerae El Tor (7PET) with Haitian cholera toxin (HCT) genotype were associated with increased mortality. The present study highlights the emergence of 7PET-HCT isolates causing two cholera outbreaks in Walsang and Wagdari (Solapur, India) in 2016. Molecular analyses revealed that 7PET strains from earlier outbreaks (2010 and 2012) were progenitors of the current 7PET-HCT isolates. Isolates from the 2016 outbreaks carried qnrVC and floR genes and showed reduced susceptibility to tetracycline, ciprofloxacin and azithromycin, drugs recommended by the World Health Organization (WHO) for the treatment of cholera. Remarkably, protein profiling and mass spectrometry revealed disappearance of the outer membrane protein U (OmpU) porin in 7PET-HCT isolates from the second outbreak in 2016. Downregulation of ompU gene expression was also confirmed at the transcriptional level. Strains with downregulated OmpU showed reduced minimum inhibitory concentrations (MICs) for polymyxin B, which is a pore-forming antimicrobial agent. A multipronged approach is of utmost importance to prevent further spread of circulating 7PET-HCT strains. There is a pressing need for the formulation and implementation of international policies to closely monitor the effective use of antibiotics in order to prevent the further rise and spread of antimicrobial resistance.

Long-Read-Based Genome Sequences of Pandemic and Environmental Vibrio cholerae Strains.

The bacterium Vibrio cholerae exhibits two distinct lifestyles, one as an aquatic bacterium and the other as the etiological agent of the pandemic human disease cholera. Here, we report closed genome sequences of two seventh pandemic V. cholerae O1 El Tor strains, A1552 and N16961, and the environmental strain Sa5Y.

AlmG, responsible for polymyxin resistance in pandemic Vibrio cholerae, is a glycyltransferase distantly related to lipid A late acyltransferases

Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections. However, some bacteria have developed resistance mechanisms to survive these compounds. Current pandemic O1 Vibrio cholerae biotype El Tor is resistant to polymyxins, whereas a previous pandemic strain of the biotype Classical is polymyxin-sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to antimicrobial peptides through aminoacylation of lipopolysaccharide (LPS), expected to decrease the negatively charged surface of the V. cholerae outer membrane. This Gram-negative system bears striking resemblance to a related Gram-positive cell-wall remodeling strategy that also promotes CAMP resistance. Mutants defective in AlmEF-dependent LPS modification exhibit reduced fitness in vivo Here, we present investigation of AlmG, the hitherto uncharacterized member of the AlmEFG pathway. Evidence for AlmG glycyl to lipid substrate transferase activity is demonstrated in vivo by heterologous expression of V. cholerae pathway enzymes in a specially engineered Escherichia coli strain. Development of a minimal keto-deoxyoctulosonate (Kdo)-lipid A domain in E. coli was necessary to facilitate chemical structure analysis and to produce a mimetic Kdo-lipid A domain AlmG substrate to that synthesized by V. cholerae. Our biochemical studies support a uniquely nuanced pathway of Gram-negative CAMPs resistance and provide a more detailed description of an enzyme of the pharmacologically relevant lysophosphospholipid acyltransferase (LPLAT) superfamily.

Integrated view of Vibrio cholerae in the Americas.

Latin America has experienced two of the largest cholera epidemics in modern history; one in 1991 and the other in 2010. However, confusion still surrounds the relationships between globally circulating pandemic Vibrio cholerae clones and local bacterial populations. We used whole-genome sequencing to characterize cholera across the Americas over a 40-year time span. We found that both epidemics were the result of intercontinental introductions of seventh pandemic El Tor V. cholerae and that at least seven lineages local to the Americas are associated with disease that differs epidemiologically from epidemic cholera. Our results consolidate historical accounts of pandemic cholera with data to show the importance of local lineages, presenting an integrated view of cholera that is important to the design of future disease control strategies.

Origins of pandemic Vibrio cholerae from environmental gene pools.

Some microorganisms can transition from an environmental lifestyle to a pathogenic one1-3. This ecological switch typically occurs through the acquisition of horizontally acquired virulence genes4,5. However, the genomic features that must be present in a population before the acquisition of virulence genes and emergence of pathogenic clones remain unknown. We hypothesized that virulence adaptive polymorphisms (VAPs) circulate in environmental populations and are required for this transition. We developed a comparative genomic framework for identifying VAPs, using Vibrio cholerae as a model. We then characterized several environmental VAP alleles to show that while some of them reduced the ability of clinical strains to colonize a mammalian host, other alleles conferred efficient host colonization. These results show that VAPs are present in environmental bacterial populations before the emergence of virulent clones. We propose a scenario in which VAPs circulate in the environment and become selected and enriched under certain ecological conditions, and finally a genomic background containing several VAPs acquires virulence factors that allow for its emergence as a pathogenic clone.