Daniela Ceccarelli

Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors

Vibrio parahaemolyticus, autochthonous to estuarine, marine, and coastal environments throughout the world, is the causative agent of food-borne gastroenteritis. More than 80 serotypes have been described worldwide, based on antigenic properties of the somatic (O) and capsular (K) antigens. Serovar O3:K6 emerged in India in 1996 and subsequently was isolated worldwide, leading to the conclusion that the first V. parahaemolyticus pandemic had taken place. Most strains of V. parahaemolyticus isolated from the environment or seafood, in contrast to clinical strains, do not produce a thermostable direct hemolysin (TDH) and/or a TDH-related hemolysin (TRH). Type 3 secretion systems (T3SSs), needle-like apparatuses able to deliver bacterial effectors into host cytoplasm, were identified as triggering cytotoxicity and enterotoxicity. Type 6 secretion systems (T6SS) predicted to be involved in intracellular trafficking and vesicular transport appear to play a role in V. parahaemolyticus virulence. Recent advances in V. parahaemolyticus genomics identified several pathogenicity islands (VpaIs) located on either chromosome in both epidemic and pandemic strains and comprising additional colonization factors, such as restriction-modification complexes, chemotaxis proteins, classical bacterial surface virulence factors, and putative colicins. Furthermore, studies indicate strains lacking toxins and genomic regions associated with pathogenicity may also be pathogenic, suggesting other important virulence factors remain to be identified. The unique repertoire of virulence factors identified to date, their occurrence and distribution in both epidemic and pandemic strains worldwide are described, with the aim of highlighting the complexity of V. parahaemolyticus pathogenicity as well as its dynamic genome.

Acquisition and Evolution of SXT-R391 Integrative Conjugative Elements in the Seventh-Pandemic Vibrio cholerae Lineage

ABSTRACT SXT-R391 Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements able to confer multidrug resistance and other adaptive features to bacterial hosts, including Vibrio cholerae, the causative agent of cholera. ICEs are arranged in a mosaic genetic structure composed of a conserved backbone interspersed with variable DNA clusters located in conserved hot spots. In this study, we investigated ICE acquisition and subsequent microevolution in pandemic V. cholerae. Ninety-six ICEs were retrieved from publicly available sequence databases from V. cholerae clinical strains and were compared to a set of reference ICEs. Comparative genomics highlighted the existence of five main ICE groups with a distinct genetic makeup, exemplified by ICEVchInd5, ICEVchMoz10, SXT, ICEVchInd6, and ICEVchBan11. ICEVchInd5 (the most frequent element, represented by 70 of 96 elements analyzed) displayed no sequence rearrangements and was characterized by 46 single nucleotide polymorphisms (SNPs). SNP analysis revealed that recent inter-ICE homologous recombination between ICEVchInd5 and other ICEs circulating in gammaproteobacteria generated ICEVchMoz10, ICEVchInd6, and ICEVchBan11. Bayesian phylogenetic analyses indicated that ICEVchInd5 and SXT were independently acquired by the current pandemic V. cholerae O1 and O139 lineages, respectively, within a period of only a few years. IMPORTANCE SXT-R391 ICEs have been recognized as key vectors of antibiotic resistance in the seventh-pandemic lineage of V. cholerae, which remains a major cause of mortality and morbidity on a global scale. ICEs were acquired only recently in this clade and are acknowledged to be major contributors to horizontal gene transfer and the acquisition of new traits in bacterial species. We have reconstructed the temporal dynamics of SXT-R391 ICE acquisition and spread and have identified subsequent recombination events generating significant diversity in ICEs currently circulating among V. cholerae clinical strains. Our results showed that acquisition of SXT-R391 ICEs provided the V. cholerae seventh-pandemic lineage not only with a multidrug resistance phenotype but also with a powerful molecular tool for rapidly accessing the pan-genome of a large number of gammaproteobacteria. SXT-R391 ICEs have been recognized as key vectors of antibiotic resistance in the seventh-pandemic lineage of V. cholerae, which remains a major cause of mortality and morbidity on a global scale. ICEs were acquired only recently in this clade and are acknowledged to be major contributors to horizontal gene transfer and the acquisition of new traits in bacterial species. We have reconstructed the temporal dynamics of SXT-R391 ICE acquisition and spread and have identified subsequent recombination events generating significant diversity in ICEs currently circulating among V. cholerae clinical strains. Our results showed that acquisition of SXT-R391 ICEs provided the V. cholerae seventh-pandemic lineage not only with a multidrug resistance phenotype but also with a powerful molecular tool for rapidly accessing the pan-genome of a large number of gammaproteobacteria.

Non-O1/non-O139 Vibrio cholerae carrying multiple virulence factors and V. cholerae O1 in the Chesapeake Bay, Maryland.

ABSTRACT Non-O1/non-O139 Vibrio cholerae inhabits estuarine and coastal waters globally, but its clinical significance has not been sufficiently investigated, despite the fact that it has been associated with septicemia and gastroenteritis. The emergence of virulent non-O1/non-O139 V. cholerae is consistent with the recognition of new pathogenic variants worldwide. Oyster, sediment, and water samples were collected during a vibrio surveillance program carried out from 2009 to 2012 in the Chesapeake Bay, Maryland. V. cholerae O1 was detected by a direct fluorescent-antibody (DFA) assay but was not successfully cultured, whereas 395 isolates of non-O1/non-O139 V. cholerae were confirmed by multiplex PCR and serology. Only a few of the non-O1/non-O139 V. cholerae isolates were resistant to ampicillin and/or penicillin. Most of the isolates were sensitive to all antibiotics tested, and 77 to 90% carried the El Tor variant hemolysin gene hlyA ET, the actin cross-linking repeats in toxin gene rtxA, the hemagglutinin protease gene hap, and the type 6 secretion system. About 19 to 21% of the isolates carried the neuraminidase-encoding gene nanH and/or the heat-stable toxin (NAG-ST), and only 5% contained a type 3 secretion system. None of the non-O1/non-O139 V. cholerae isolates contained Vibrio pathogenicity island-associated genes. However, ctxA, ace, or zot was present in nine isolates. Fifty-five different genotypes showed up to 12 virulence factors, independent of the source of isolation, and represent the first report of both antibiotic susceptibility and virulence associated with non-O1/non-O139 V. cholerae from the Chesapeake Bay. Since these results confirm the presence of potentially pathogenic non-O1/non-O139 V. cholerae, monitoring for total V. cholerae, regardless of serotype, should be done within the context of public health.

A Review of SHV Extended-Spectrum β-Lactamases : Neglected Yet Ubiquitous

β-lactamases are the primary cause of resistance to β-lactams among members of the family Enterobacteriaceae. SHV enzymes have emerged in Enterobacteriaceae causing infections in health care in the last decades of the Twentieth century, and they are now observed in isolates in different epidemiological settings both in human, animal and the environment. Likely originated from a chromosomal penicillinase of Klebsiella pneumoniae, SHV β-lactamases currently encompass a large number of allelic variants including extended-spectrum β-lactamases (ESBL), non-ESBL and several not classified variants. SHV enzymes have evolved from a narrow- to an extended-spectrum of hydrolyzing activity, including monobactams and carbapenems, as a result of amino acid changes that altered the configuration around the active site of the β -lactamases. SHV-ESBLs are usually encoded by self-transmissible plasmids that frequently carry resistance genes to other drug classes and have become widespread throughout the world in several Enterobacteriaceae, emphasizing their clinical significance.

IncA/C Conjugative Plasmids Mobilize a New Family of Multidrug Resistance Islands in Clinical Vibrio cholerae Non-O1/Non-O139 Isolates from Haiti

ABSTRACT Mobile genetic elements play a pivotal role in the adaptation of bacterial populations, allowing them to rapidly cope with hostile conditions, including the presence of antimicrobial compounds. IncA/C conjugative plasmids (ACPs) are efficient vehicles for dissemination of multidrug resistance genes in a broad range of pathogenic species of Enterobacteriaceae. ACPs have sporadically been reported in Vibrio cholerae, the infectious agent of the diarrheal disease cholera. The regulatory network that controls ACP mobility ultimately depends on the transcriptional activation of multiple ACP-borne operons by the master activator AcaCD. Beyond ACP conjugation, AcaCD has also recently been shown to activate the expression of genes located in the Salmonella genomic island 1 (SGI1). Here, we describe MGIVchHai6, a novel and unrelated mobilizable genomic island (MGI) integrated into the 3′ end of trmE in chromosome I of V. cholerae HC-36A1, a non-O1/non-O139 multidrug-resistant clinical isolate recovered from Haiti in 2010. MGIVchHai6 contains a mercury resistance transposon and an integron In104-like multidrug resistance element similar to the one of SGI1. We show that MGIVchHai6 excises from the chromosome in an AcaCD-dependent manner and is mobilized by ACPs. Acquisition of MGIVchHai6 confers resistance to β-lactams, sulfamethoxazole, tetracycline, chloramphenicol, trimethoprim, and streptomycin/spectinomycin. In silico analyses revealed that MGIVchHai6-like elements are carried by several environmental and clinical V. cholerae strains recovered from the Indian subcontinent, as well as from North and South America, including all non-O1/non-O139 clinical isolates from Haiti. IMPORTANCE Vibrio cholerae, the causative agent of cholera, remains a global public health threat. Seventh-pandemic V. cholerae acquired multidrug resistance genes primarily through circulation of SXT/R391 integrative and conjugative elements. IncA/C conjugative plasmids have sporadically been reported to mediate antimicrobial resistance in environmental and clinical V. cholerae isolates. Our results showed that while IncA/C plasmids are rare in V. cholerae populations, they play an important yet insidious role by specifically propagating a new family of genomic islands conferring resistance to multiple antibiotics. These results suggest that nonepidemic V. cholerae non-O1/non-O139 strains bearing these genomic islands constitute a reservoir of transmissible resistance genes that can be propagated by IncA/C plasmids to V. cholerae populations in epidemic geographical areas as well to pathogenic species of Enterobacteriaceae. We recommend future epidemiological surveys take into account the circulation of these genomic islands. Vibrio cholerae, the causative agent of cholera, remains a global public health threat. Seventh-pandemic V. cholerae acquired multidrug resistance genes primarily through circulation of SXT/R391 integrative and conjugative elements. IncA/C conjugative plasmids have sporadically been reported to mediate antimicrobial resistance in environmental and clinical V. cholerae isolates. Our results showed that while IncA/C plasmids are rare in V. cholerae populations, they play an important yet insidious role by specifically propagating a new family of genomic islands conferring resistance to multiple antibiotics. These results suggest that nonepidemic V. cholerae non-O1/non-O139 strains bearing these genomic islands constitute a reservoir of transmissible resistance genes that can be propagated by IncA/C plasmids to V. cholerae populations in epidemic geographical areas as well to pathogenic species of Enterobacteriaceae. We recommend future epidemiological surveys take into account the circulation of these genomic islands.

Vibrio ecology, pathogenesis, and evolution

This Research Topic brings together 24 articles that highlight the most recent research findings concerning the biology of the genus Vibrio and covers pathogenicity and host interaction, genome plasticity and evolution, and the dynamics of factors influencing the ecology of vibrios. Vibrio comprises one of the most diverse marine bacterial genera (Gomez-Gil et al., 2014), and its diversity is emphasized in two of the articles opening this set of Research Topic papers. Sawabe et al. (2013) present a molecular phylogeny of 86 Vibrio species based on genome sequencing that provides insight into Vibrio biodiversity and evolutionary history. In a study of more than 300 Vibrio genome sequences, Lukjancenko and Ussery (2014) conclude that the Vibrio pan-genome comprises 17,000 gene families, differentially present and/or expressed in any given species. A remarkable feature of all Vibrio species is an highly plastic genome, a feature examined in five papers. The two chromosomes are shaped by horizontal gene transfer involving, among others, antibiotic resistance, virulence, and niche adaptation (Rowe-Magnus et al., 2001; Kirkup et al., 2010). V. vulnificus biotype 3 is a notable example. Efimov et al. (2013) suggest biotype 3 evolved from biotype 1 by acquisition of unique genes from other bacterial species, such as Shewanella, sharing the same ecological niche. Carraro et al. (2014) employ molecular and functional characterization of pVCR94, to identify the role of IncA/C plasmids in antibiotic resistance in a Rwandan V. cholerae isolate. A retrospective analysis of epidemic V. cholerae in Angola is reported by Valia et al. (2013), showing unexpected genomic variability among variants, highlighting the role of genomic islands, phages, and integrative conjugative elements in the genetic diversity of V. cholerae in a single epidemic. Rivas et al. (2013) describe acquisition by Photobacterium damselae subsp. damselae of virulence plasmid pPHDD1 that encodes pore-forming toxins and hemolysins which play a key role in virulence for both fish and humans. A review by Rapa and Labbate (2013) describes the role of integrons in Vibrio species for which gene cassettes comprise approximately 1–3% of the entire genome and are very likely involved in bacterial adaptation and evolution. Nine of the manuscripts analyze Vibrio pathogenicity, disease development, specificity, and adaptation in both human and animal hosts. Tan et al. (2014) deciphered the biosynthetic network of the siderophore vulnibactin, essential in iron uptake from host proteins, the importance of which in V. vulnificus pathogenicity has been clinically demonstrated. Inhibition/resistance mechanisms developed by V. salmonicida, the causative agent of hemorrhagic septicemia in Atlantic salmon, is described by Bjelland et al. (2013), who show that it overcomes the salmon innate immune system to a point where the infection overwhelms the host. The role in bacterial virulence of diverse extracellular proteolytic enzymes secreted by human pathogenic Vibrio species is the focus of a review by Miyoshi (2013). The engagement of type VI secretion systems by V. cholerae is suggested as a means of intra- and inter-species predation and nutrient acquisition, inducing rapid multiplication in the human host (Pukatzki and Provenzano, 2013). The bioluminescent marine bacterium V. campbellii is used by Wang et al. (2013) to analyze the pyomelanin-pigmented phenotype, known to provide Vibrio species with greater UV and oxidative stress resistance and enhanced intestine colonization. The relationship between pathogenicity and motility in Vibrio species and the contribution of flagella to adhesion and biofilm formation are discussed by Zhu et al. (2013). The largely unexplored V. fluvialis mechanisms of pathogenesis, survival and fitness are reviewed by Ramamurthy et al. (2014). Twenty new Vibrio species associated with molluscans are described and their pathogenic potential for molluscs elucidated by Romalde et al. (2014). The exquisite bacteria–host interaction between V. fisheri and its squid host, Euprymna scolopes, is described in detail, as are the molecular pathways of biofilm formation, motility, and chemotaxis (Norsworthy and Visick, 2013). The capacity of Vibrio species to persist in the aquatic environment, their ecology and association with abiotic and biotic factors, as well as environmental surveillance for public health (Lipp et al., 2002; Grimes et al., 2009; Johnson, 2013) comprise a section in the Research Topic that opens with a review by Lutz et al. (2013) elucidating complex mechanisms enabling V. cholerae to withstand starvation, temperature fluctuation, salinity variation, and predation. Haley et al. (2014) report water temperature increase can be correlated with rise of a diverse population of V. parahaemolyticus, some of which carry pandemic markers, in water and plankton along the Georgian coast of the Black Sea. V. parahaemolyticus and V. vulnificus populations associated with oyster, sediment, and surface water related to a hurricane event in the Chesapeake Bay are concluded to be influenced by wave energy and sediment resuspension (Shaw et al., 2014). Canesi et al. (2013) show the serum of Mytilus galloprovincialis promotes phagocytosis and killing by hemocytes of both V. cholerae O1 and non-O1/non-O139 in edible bivalves. Chakraborty et al. (2013) evaluate a sensitive and specific dipstick test to detect toxigenic V. cholerae in water, validating a simple, inexpensive method for use in areas at risk of cholera. Three articles addressing Vibrio environmental diversity and dynamics complete this Research Topic. Mansergh and Zehr (2014) suggest that the natural shift of Vibrio populations in Monterey Bay is affected by larger oceanographic conditions (flow velocities and wind patterns), rather than individual environmental factors. Meta-analysis of environmental variables and Vibrio association with plants, algae, zooplankton, and animals are reviewed by Takemura et al. (2014). As a final point concerning environmental distribution, Constantin De Magny et al. (2014) propose temporal shifts, zooplankton community variability, and occurrence of V. cholerae in the aquatic environment are related to cholera dynamics. These factors, analyzed by metagenomics, permit greater understanding of community structure, function, and competition. In summary, the collection of manuscripts provided in this Research Topic offers a comprehensive exploration of Vibrio biology, from the single gene to the bacterial community, elucidating Vibrio molecular pathways and evolutionary history. This special issue shows the significant progress achieved in understanding the complex biology of the genus Vibrio and should both stimulate discussion and offer a challenge to researchers in microbial ecology and evolution.

Distribution of virulence genes in clinical and environmental Vibrio cholerae strains in Bangladesh.

ABSTRACT Vibrio cholerae, an environmental organism, is a facultative human pathogen. Here, we report the virulence profiles, comprising 18 genetic markers, of 102 clinical and 692 environmental V. cholerae strains isolated in Bangladesh between March 2004 and January 2006, showing the variability of virulence determinants within the context of public health.

A new integrative conjugative element detected in Haitian isolates of Vibrio cholerae non-O1/non-O139

The presence of SXT/R391-related integrating conjugative elements (ICEs) in Vibrio cholerae O1 and non-O1/non-O139 isolated from clinical and environmental samples in Haiti in 2010 was studied. The main finding of this work was the identification of the novel ICEVchHai2 among closely related V. cholerae non-O1/non-O139 clinical strains. The mosaic structure of this element confirms the role of ICEs as efficient recombination systems whereby new genetic material can be acquired and exchanged, according V. cholerae strains new accessory functions.

Vibrio cholerae O1 epidemic variants in Angola: a retrospective study between 1992 and 2006

Cholera is still a major public health concern in many African countries. In Angola, after a decade of absence, cholera reemerged in 1987, spreading throughout the country until 1996, with outbreaks recurring in a seasonal pattern. In 2006 Angola was hit by one of the most severe outbreaks of the last decade, with ca. 240,000 cases reported. We analyzed 21 clinical strains isolated between 1992 and 2006 from several provinces throughout the country: Benguela, Bengo, Luanda, Cuando Cubango, and Cabinda. We used two multiplex PCR assays to investigate discriminatory mobile genetic elements (MGE) [Integrative Conjugative Elements (ICEs), VSP-II, GI12, GI14, GI15, K, and TLC phages] and we compared the profiles obtained with those of different reference V. cholerae O1 variants (prototypical, altered, and hybrid), responsible for the ongoing 7th pandemic. We also tested the strains for the presence of specific VSP-II variants and for the presence of a genomic island (GI) (WASA-1), correlated with the transmission of seventh pandemic cholera from Africa to South America. Based on the presence/absence of the analyzed genetic elements, five novel profiles were detected in the epidemic strains circulating in the 1990s. The most frequent profiles, F and G, were characterized by the absence of ICEs and the three GIs tested, and the presence of GI WASA-1 and the WASA variant of the VSP-II island. Our results identified unexpected variability within the 1990s epidemic, showing different rearrangements in a dynamic part of the genome not present in the prototypical V. cholerae O1 N16961. Moreover the 2006 strains differed from the current pandemic V. cholerae O1 strain. Taken together, our results highlight the role of horizontal gene transfer (HGT) in diversifying the genetic background of V. cholerae within a single epidemic.

Reduced Susceptibility to Extended-Spectrum β-Lactams in Vibrio cholerae Isolated in Bangladesh

β-lactams are antibiotic molecules able to inhibit cell wall biosynthesis. Among other mechanisms, resistance in Gram-negative bacteria is mostly associated with production of β-lactamase enzymes able to bind and hydrolyze the β-lactam ring. Extended-spectrum β-lactamases extend this ability also to third- and fourth-generation cephalosporins, as well as to carbapenems and monobactams. Vibrio cholerae is the causative agent of epidemic cholera and a public health burden for developing countries like Bangladesh. Although appropriate oral or intravenous rehydration is the therapy of choice for cholera, severe infections and V. cholerae-associated septicemia are treated with antimicrobial drugs, including doxycycline, erythromycin, azithromycin, ciprofloxacin, and/or third-generation cephalosporins. In the years after the introduction of antibiotics in clinical practice, V. cholerae developed resistance to commonly used drugs worldwide mostly through gene acquisition via horizontal gene transfer. Reduced susceptibility of V. cholerae to third-generation cephalosporins has been occasionally documented. However, carbapenemase-producing V. cholerae has been reported at higher rates than resistance to extended-spectrum β-lactams, mainly associated with blaNDM-1 emergence and successful plasmid dissemination. Recent findings suggest limited β-lactam resistance is present in V. cholerae O1 isolates collected during ecological and epidemiological surveillance in Bangladesh. However, a trend to intermediate-susceptibility insurgence was observed. Horizontal gene transfer of β-lactam resistance from enteric pathogens to environmental microorganisms should not be underrated, given the ability of V. cholerae to acquire new genetic information.