Katherine García

Dynamics of Clinical and Environmental Vibrio parahaemolyticus Strains during Seafood-Related Summer Diarrhea Outbreaks in Southern Chile

ABSTRACT Seafood consumption-related diarrhea became prevalent in Chile when the pandemic strain of Vibrio parahaemolyticus serotype O3:K6 reached a region in the south of Chile (Region de los Lagos) where approximately 80% of the countrys seafood is produced. In spite of the large outbreaks of clinical infection, the load of V. parahaemolyticus in shellfish of this region is relatively low. The pandemic strain constitutes a small but relatively stable group of a diverse V. parahaemolyticus population, composed of at least 28 genetic groups. Outbreaks in Region de los Lagos began in 2004 and reached a peak in 2005 with 3,725 clinical cases, all associated with the pandemic strain. After 2005, reported cases steadily decreased to a total of 477 cases in 2007. At that time, 40% of the clinical cases were associated with a pandemic strain of a different serotype (O3:K59), and 27% were related to V. parahaemolyticus isolates unrelated to the pandemic strain. In the results published here, we report that in the summer of 2008, when reported cases unexpectedly increased from 477 to 1,143, 98% of the clinical cases were associated with the pandemic strain serotype O3:K6, a change from 2007. Nevertheless, in 2009, when clinical cases decreased to 441, only 64% were related to the pandemic strain; the remaining cases were related to a nonpandemic tdh- and trh-negative strain first identified in shellfish in 2006. Overall, our observations indicate that the pandemic strain has become a relatively stable subpopulation and that when the number of diarrhea cases related to the pandemic strain is low, previously undetected V. parahaemolyticus pathogenic strains become evident.

Rise and fall of pandemic Vibrio parahaemolyticus serotype O3:K6 in southern Chile

Seafood consumption-related diarrhoea increased drastically in Chile when the pandemic strain of Vibrio parahaemolyticus serotype O3:K6 reached Region de Los Lagos, where most of Chiles seafood is produced. Outbreaks peaked in 2005 with 3725 clinical cases in this region and gradually decreased to fewer than 10 cases in 2010 and 2011. We show here that the pandemic strain concurrently vanished from mussels; we also report further environmental data. Integration of the 2010/2011 data with those obtained since 2004 suggests that after its arrival in southern Chile, the pandemic strain grew in mussels, likely facilitated by a minor rise in surface seawater temperature and by warming of the mussels in the intertidal region due to frequent sunny days. However, since these environmental parameters probably equally affected the pandemic strain and more than 30 V. parahaemolyticus DNA restriction clusters that inhabit local shellfish, a selective effect of bacteriophages is proposed. Lytic bacteriophage VP93 may have favoured the growth of the pandemic strain versus similar phage-sensitive strains, as shown here in a particular case. However, the pandemic strains decline may have been promoted by temperate phage VP58.5, which kills the pandemic strain and increases the UV sensitivity of lysogenized phage-resistant cells.

Enhancement of UV Light Sensitivity of a Vibrio parahaemolyticus O3:K6 Pandemic Strain Due to Natural Lysogenization by a Telomeric Phage

ABSTRACT The Vibrio parahaemolyticus O3:K6 pandemic clonal strain was first observed in southern Chile in 2004 and has since caused approximately 8,000 seafood-related diarrhea cases in this region. The massive proliferation of the original clonal population offers a unique opportunity to study the evolution of a bacterial pathogen in its natural environment by detection and characterization of emerging bacterial variants. Here, we describe a group of pandemic variants characterized by the presence of a 42-kb extrachromosomal DNA that can be recovered by alkaline extraction. Upon treatment with mitomycin C, these variants lyse with production of a myovirus containing DNA of equal size to the plasmid but which cannot be recovered by alkaline extraction. Plasmid and phage DNAs show similar restriction patterns corresponding to enzyme sites in a circular permutation. Sequenced regions showed 81 to 99% nucleotide similarity to bacteriophage VHML of Vibrio harveyi. Altogether these observations indicate that the 42-kb plasmid corresponds to a prophage, consisting of a linear DNA with terminal hairpins of a telomeric temperate phage with a linear genome. Bacteria containing the prophage were 7 to 15 times more sensitive to UV radiation, likely due to phage induction by UV irradiation as plasmid curing restored the original sensitivity. The enhanced UV sensitivity could have a significant role in reducing the survival and propagation capability of the V. parahaemolyticus pandemic strain in the ocean.

Potential probiotic yeasts isolated from the fish gut protect zebrafish (Danio rerio) from a Vibrio anguillarum challenge

Due to the negative consequences associated with the use of antibiotics, researchers, and food producers have studied alternatives, such as probiotics, for the control of fish diseases. The probiotic properties of yeasts in aquaculture have been scarcely considered. The present study investigated the probiotic properties of local yeast strains for aquaculture application in the protection of bacterial diseases. Yeast strains (n = 15), previously isolated from the intestinal gut of healthy salmonids, yellowtail, and croaker, were evaluated for their protection of zebrafish larvae following a Vibrio anguillarum challenge. We developed an infection model on zebrafish larvae with V. anguillarum, observing rapid mortality (≥50%) 5 days post-immersion challenge. Infection of Tg(Lyz:DsRed)nz50 larvae with fluorescent-marked V. anguillarum showed the oro-intestinal as the natural route of infection concomitant with an inflammatory response of the larvae reflected by neutrophil migration outside the hematopoietic tissue. Thirteen of 15 strains increased the percentage of larvae survival after the V. anguillarum challenge, although no yeast showed in vitro anti-V. anguillarum activity. In a subset of yeasts, we explored yeast–larvae interactions using fluorescent yeast and evaluated larvae colonization by culture analysis. All fluorescent yeasts were located in the gastrointestinal tract until 5 days post-inoculation (dpi). Yeasts reached 103 CFU/larvae at 0 dpi, although the persistence until 5 dpi of the viable yeast in the gut was different among the strains. These results reveal that some yeasts isolated from the gut of fish could be potential probiotics, reducing the mortality associated to V. anguillarum challenge, and suggest that gut colonization could be involved in the protective effect. Future studies should elucidate other mechanisms involved in yeast protection and verify the beneficial effects of probiotic use in commercial fish species.

Role of Non-coding Regulatory RNA in the Virulence of Human Pathogenic Vibrios

In recent decades, the identification of small non-coding RNAs in bacteria has revealed an important regulatory mechanism of gene expression involved in the response to environmental signals and to the control of virulence. In the family Vibrionaceae, which includes several human and animal pathogens, small non-coding RNAs (sRNAs) are closely related to important processes including metabolism, quorum sensing, virulence, and fitness. Studies conducted in silico and experiments using microarrays and high-throughput RNA sequencing have led to the discovery of an unexpected number of sRNAs in Vibrios. The present review discusses the most relevant reports regarding the mechanisms of action of sRNAs and their implications in the virulence of the main human pathogens in the family Vibrionaceae: Vibrio parahaemolyticus, V. vulnificus and V. cholerae.

Protective yeasts control V. anguillarum pathogenicity and modulate the innate immune response of challenged zebrafish (Danio rerio) larvae

We investigated mechanisms involved in the protection of zebrafish (Danio rerio) larvae by two probiotic candidate yeasts, Debaryomyces hansenii 97 (Dh97) and Yarrowia lypolitica 242 (Yl242), against a Vibrio anguillarum challenge. We determined the effect of different yeast concentrations (104–107 CFU/mL) to: (i) protect larvae from the challenge, (ii) reduce the in vivo pathogen concentration and (iii) modulate the innate immune response of the host. To evaluate the role of zebrafish microbiota in protection, the experiments were performed in conventionally raised and germ-free larvae. In vitro co-aggregation assays were performed to determine a direct yeast-pathogen interaction. Results showed that both yeasts significantly increased the survival rate of conventionally raised larvae challenged with V. anguillarum. The concentration of yeasts in larvae tended to increase with yeast inoculum, which was more pronounced for Dh97. Better protection was observed with Dh97 at a concentration of 106 CFU/mL compared to 104 CFU/mL. In germ-free conditions V. anguillarum reached higher concentrations in larvae and provoked significantly more mortality than in conventional conditions, revealing the protective role of the host microbiota. Interestingly, yeasts were equally (Dh97) or more effective (Yl242) in protecting germ-free than conventionally-raised larvae, showing that protection can be exerted only by yeasts and is not necessarily related to modulation of the host microbiota. Although none of the yeasts co-aggregated with V. anguillarum, they were able to reduce its proliferation in conventionally raised larvae, reduce initial pathogen concentration in germ-free larvae and prevent the upregulation of key components of the inflammatory/anti-inflammatory response (il1b, tnfa, c3, mpx, and il10, respectively). These results show that protection by yeasts of zebrafish larvae challenged with V. anguillarum relates to an in vivo anti-pathogen effect, the modulation of the innate immune system, and suggests that yeasts avoid the host-pathogen interaction through mechanisms independent of co-aggregation. This study shows, for the first time, the protective role of zebrafish microbiota against V. anguillarum infection, and reveals mechanisms involved in protection by two non-Saccharomyces yeasts against this pathogen.

Insight Into the Origin and Evolution of the Vibrio parahaemolyticus Pandemic Strain

A strain of Vibrio parahaemolyticus that emerged in 1995 caused the first known pandemic involving this species. This strain comprises clonal autochthonous ocean-dwelling bacteria whose evolution has occurred in the ocean environment. The low sequence diversity in this population enabled the discovery of information on its origin and evolution that has been hidden in bacterial clones that have evolved over a long period. Multilocus sequencing and microarray analysis, together with phylogenetic analysis, of pandemic and pre-pandemic isolates has suggested that the founder clone was an O3:K6 non-pathogenic strain that initially acquired a toxRS/new region and subsequently acquired at least seven novel genomic islands. Sequencing and comparison of whole genomes later confirmed these early observations, and it confirmed that most of the genetic changes occurred via gene conversion involving horizontally transmitted DNA. The highly clonal population rapidly diversified, especially in terms of antigenicity, and 27 serotypes have already been reported. Comparisons of the core genomes derived from the founder clone indicate that there are only a few hundred single-nucleotide variations between isolates. However, when the whole genome is considered (the core plus non-core genome and from any clonal frame), the amount of DNA with a different clonal frame can reach up to 4.2% and the number of single-nucleotide variations can reach several hundred thousand. Altogether, these and previous observations based on multilocus sequence typing, microarray analysis, and whole-genome sequencing indicate the large contribution made by DNA with different clonal genealogy to genome diversification. The evidence also indicates that horizontal gene transfer (HGT) caused the emergence of new pathogens. Furthermore, the extent of HGT seems to depend on the vicissitudes of the life of each bacterium, as exemplified by differences in thousands of base pairs acquired by HGT among almost identical genetic isolates.

Exploring the Genomic Traits of Non-toxigenic Vibrio parahaemolyticus Strains Isolated in Southern Chile

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis worldwide. As reported in other countries, after the rise and fall of the pandemic strain in Chile, other post-pandemic strains have been associated with clinical cases, including strains lacking the major toxins TDH and TRH. Since the presence or absence of tdh and trh genes has been used for diagnostic purposes and as a proxy of the virulence of V. parahaemolyticus isolates, the understanding of virulence in V. parahaemolyticus strains lacking toxins is essential to detect these strains present in water and marine products to avoid possible food-borne infection. In this study, we characterized the genome of four environmental and two clinical non-toxigenic strains (tdh-, trh-, and T3SS2-). Using whole-genome sequencing, phylogenetic, and comparative genome analysis, we identified the core and pan-genome of V. parahaemolyticus of strains of southern Chile. The phylogenetic tree based on the core genome showed low genetic diversity but the analysis of the pan-genome revealed that all strains harbored genomic islands carrying diverse virulence and fitness factors or prophage-like elements that encode toxins like Zot and RTX. Interestingly, the three strains carrying Zot-like toxin have a different sequence, although the alignment showed some conserved areas with the zot sequence found in V. cholerae. In addition, we identified an unexpected diversity in the genetic architecture of the T3SS1 gene cluster and the presence of the T3SS2 gene cluster in a non-pandemic environmental strain. Our study sheds light on the diversity of V. parahaemolyticus strains from the southern Pacific which increases our current knowledge regarding the global diversity of this organism.

Genealogy of the Genome Components in the Highly Homogeneous Pandemic Vibrio parahaemolyticus Population

Bacterial genomes evolve through two different mechanisms: 1) changes in or occasional loss of ancestral genes, which preserve the founder clonal genealogy or frame, and 2) sporadic gains of new genes via horizontal gene transfer, which introduces DNA with a different genealogy or clonal frame. Evolution has led to the emergence of a pathogenic strain pandemic of Vibrio parahaemolyticus, which has propagated globally, causing large outbreaks of seafood-associated diarrhea. The low sequence diversity of the pathogenic strain of Vibrio parahaemolyticus genome provides a model that can reveal evolutionary mechanisms that are hidden in bacteria with a greater diversity. Here, we assess the clonal genealogy of the genome components of Vibrio parahaemolyticus and identify recombinant segments in 31 isolates obtained worldwide. By comparing whole genomes using a procedure that accounts for at least 98% of the reads obtained from any isolate after high throughput sequencing, we determined that the fraction of the whole genome retaining the founder clonal frame varied from 96.7%-100% of the accountable reads. The fraction in chromosomes with other genealogies varied from 0%-3.3% and in extra-chromosomal elements from 0%-4.2%, with the relative impact of mutation and recombination varying greatly between isolates. The likely causes for this variation are proposed.

Inactivated E. coli transformed with plasmids that produce dsRNA against infectious salmon anemia virus hemagglutinin show antiviral activity when added to infected ASK cells

Infectious salmon anemia virus (ISAV) has caused great losses to the Chilean salmon industry, and the success of prevention and treatment strategies is uncertain. The use of RNA interference (RNAi) is a promising approach because during the replication cycle, the ISAV genome must be transcribed to mRNA in the cytoplasm. We explored the capacity of E. coli transformed with plasmids that produce double-stranded RNA (dsRNA) to induce antiviral activity when added to infected ASK cells. We transformed the non-pathogenic Escherichia coli HT115 (DE3) with plasmids that expressed highly conserved regions of the ISAV genes encoding the nucleoprotein (NP), fusion (F), hemagglutinin (HE), and matrix (M) proteins as dsRNA, which is the precursor of the RNAi mechanism. The inactivated transformed bacteria carrying dsRNA were tested for their capacity to silence the target ISAV genes, and the dsRNA that were able to inhibit gene expression were subsequently tested for their ability to attenuate the cytopathic effect (CPE) and reduce the viral load. Of the four target genes tested, inactivated E. coli transformed with plasmids producing dsRNA targeting HE showed antiviral activity when added to infected ASK cells.