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Complete Genome Sequences of a Clinical Isolate and an Environmental Isolate of Vibrio parahaemolyticus

DOI:10.1128/genomeA.00216-15
Authors:
Catharina Lüdeke at HU Institut für Hygiene und Umwelt, Hamburg, Germany
Catharina Lüdeke
  • HU Institut für Hygiene und Umwelt, Hamburg, Germany
Nguyet Kong at University of California, Davis
Nguyet Kong
Bart C Weimer at University of California, Davis
Bart C Weimer
Markus Fischer at University of Hamburg
Markus Fischer
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Abstract

Vibrio parahaemolyticus is the leading cause of seafood-borne infections in the United States. We report complete genome sequences for two V. parahaemolyticus strains isolated in 2007, CDC_K4557 and FDA_R31 of clinical and oyster origin, respectively. These two sequences might assist in the investigation of differential virulence of this organism. FOOTNOTES Address correspondence to Jessica L. Jones, jessica.jones{at}fda.hhs.gov. Citation Lüdeke CHM, Kong N, Weimer BC, Fischer M, Jones JL. 2015. Complete genome sequences of a clinical isolate and an environmental isolate of Vibrio parahaemolyticus. Genome Announc 3(2):e00216-15. doi:10.1128/genomeA.00216-15. Received 13 February 2015. Accepted 18 February 2015. Published 26 March 2015. Copyright © 2015 Lüdeke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
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Complete Genome Sequences of a Clinical Isolate and an
Environmental Isolate of Vibrio parahaemolyticus
Catharina H. M. Lüdeke,
a,b
Nguyet Kong,
c,d
Bart C. Weimer,
c,d
Markus Fischer,
b
Jessica L. Jones
a
Gulf Coast Seafood Laboratory, Division of Seafood Science and Technology, Food and Drug Administration, Dauphin Island, Alabama, USA
a
; Hamburg School of Food
Science, University of Hamburg, Hamburg, Germany
b
; School of Veterinary Medicine
c
and 100K Pathogen Genome Project,
d
University of California, Davis, California, USA
Vibrio parahaemolyticus is the leading cause of seafood-borne infections in the United States. We report complete genome se-
quences for two V. parahaemolyticus strains isolated in 2007, CDC_K4557 and FDA_R31 of clinical and oyster origin, respec-
tively. These two sequences might assist in the investigation of differential virulence of this organism.
Received 13 February 2015 Accepted 18 February 2015 Published 26 March 2015
Citation Lüdeke CHM, Kong N, Weimer BC, Fischer M, Jones JL. 2015. Complete genome sequences of a clinical isolate and an environmental isolate of Vibrio
parahaemolyticus. Genome Announc 3(2):e00216-15. doi:10.1128/genomeA.00216-15.
Copyright © 2015 Lüdeke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Address correspondence to Jessica L. Jones, jessica.jones@fda.hhs.gov.
Vibrio parahaemolyticus is a halophilic Gram-negative bacte-
rium naturally occurring in estuarine environments (1).
Through consumption of raw or undercooked seafood, or contact
with contaminated seawater, V. parahaemolyticus can cause infec-
tions in humans; gastroenteritis is typical, but rarely, wound
and/or sepsis infections occur. V. parahaemolyticus is the leading
cause of seafood-borne gastroenteritis in the United States (2),
and there has been an increase in reported illnesses in the last two
decades (3).
V. parahaemolyticus can carry the thermostable direct hemoly-
sin (tdh) and tdh-related hemolysin (trh) genes, which are gener-
ally associated with pathogenicity and are used in outbreak inves-
tigations and assessing risk (4,5). Clinical isolates more frequently
carry the tdh and/or trh genes than environmental isolates (6).
However, in recent studies, clinical isolates lacking both of these
genes have been identified (7). Tissue culture studies have re-
vealed that the presence of tdh had no effect on cytotoxicity (8,9).
Hence, additional virulence factors likely exist for V. parahaemo-
lyticus.
We sequenced two V. parahaemolyticus isolates, CDC_K4557
and FDA_R31, to better understand the pathogenic potential of
these isolates and eventually improve risk assessment.
CDC_K4557 was isolated from the stool of a patient in Louisiana
in 2007 and submitted to the Centers for Disease Control and
Prevention (CDC). FDA_R31 was isolated by the Food and Drug
Administration (FDA) from an oyster sample harvested in Loui-
siana in 2007. As the clinical isolate is tdh
⫺
trh
⫺
and the oyster
isolate is tdh
⫹
trh
⫹
by PCR, these strains are ideal for identifying
new and/or additional virulence markers.
The genomes were sequenced within the University of Califor-
nia at Davis 100K Pathogen Genome Project using the PacBio RSII
sequencing platform (Pacific Biomarkers, Menlo Park, CA, USA).
High-molecular-weight gDNA was extracted from overnight cul-
tures grown on Trypticase soy agar, lysed with an enzyme cocktail,
purified with the QIAamp DNA minikit (Qiagen, Valencia, CA,
USA), and analyzed on a 2200 TapeStation system with the
Genomic DNA ScreenTape (Agilent Technologies, Santa Clara,
CA, USA) assay for integrity of high molecular weight gDNA (10).
After evaluation of gDNA size and quantity, 10
␮
g was used for
fragmentation using the Covaris g-TUBE device (Covaris,
Woburn, MA, USA) following the manufacturer’s instructions
(11). The fragmented gDNA was used for library construction
with the PacBio SMRTbell 10kb Library preparation kit, which
was normalized to 1 to 5
␮
g input. Libraries were sequenced
utilizing PacBio RSII and C2 chemistry with 100⫻coverage per
the manufacturer’s instructions. For each isolate, the genomic
sequence single-pass reads were de novo assembled using the
Hierarchical Genome Assembly Process (HGAP) version 1.4
software (Pacific Biosciences) and were then annotated using
the NCBI Prokaryotic Genomes Automatic Annotation Pipe-
line (http://www.ncbi.nlm.nih.gov/genome/annotation_prok)
(12). Through the annotation process, 4,771 and 4,937 genes for
the clinical and oyster isolates, respectively, as well as 4,579 and
4,731 coding regions were identified. The presence or absence of
the tdh and trh genes was confirmed in both isolates.
Nucleotide sequence accession numbers. The closed genome
sequences of the two V. parahaemolyticus isolates are available
in GenBank under the accession numbers CP006004 and
CP006005 for chromosomes I and II of FDA_R31, respectively,
and CP006008 and CP006007 for CDC_K4557. The versions de-
scribed in this paper are the first versions.
ACKNOWLEDGMENTS
This project was supported by an appointment to the Research Fellowship
Program for the Center for Food Safety and Applied Nutrition adminis-
tered by the Oak Ridge Associated Universities through a contract with
the FDA. The 100K Pathogen Genome Project was supported by the FDA
and Agilent Technologies to produce these sequences.
We thank Whitney Ng and Kao Thao for their effort in the isolate
logistics.
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In this study, 77 clinical and 67 oyster Vibrio parahaemolyticus isolates from North America were examined for biochemical profiles, serotype, and the presence of potential virulence factors (tdh, trh, and type III secretion system [T3SS] genes). All isolates were positive for oxidase, indole, and glucose fermentation, consistent with previous reports. The isolates represented 35 different serotypes, 9 of which were shared by clinical and oyster isolates. Serotypes associated with pandemic strains (O1:KUT, O1:K25, O3:K6, and O4:K68) were observed for clinical isolates, and 7 (9%) oyster isolates belonged to serotype O1:KUT. Of the clinical isolates, 27% were negative for tdh and trh, while 45% contained both genes. Oyster isolates were preferentially selected for the presence of tdh and/or trh; 34% contained both genes, 42% had trh but not tdh, and 3% had tdh but not trh. All but 1 isolate (143/144) had at least three of the four T3SS1 genes examined. The isolates lacking both tdh and trh contained no T3SS2α or T3SS2β genes. All clinical isolates positive for tdh and negative for trh possessed all T3SS2α genes, and all isolates negative for tdh and positive for trh possessed all T3SS2β genes. The two oyster isolates containing tdh but not trh possessed all but the vopB2 gene of T3SS2α, as reported previously. In contrast to the findings of previous studies, all strains examined that were positive for both tdh and trh also carried T3SS2β genes. This report identifies the serotype as the most distinguishing feature between clinical and oyster isolates. Our findings raise concerns about the reliability of the tdh, trh, and T3SS genes as virulence markers and highlight the need for more-detailed pathogenicity investigations of V. parahaemolyticus.
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Foodborne Illness Acquired in the United States—Major Pathogens
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  • Jan 2011
Estimates of foodborne illness can be used to direct food safety policy and interventions. We used data from active and passive surveillance and other sources to estimate that each year 31 major pathogens acquired in the United States caused 9.4 million episodes of foodborne illness (90% credible interval [CrI] 6.6–12.7 million), 55,961 hospitalizations (90% CrI 39,534–75,741), and 1,351 deaths (90% CrI 712–2,268). Most (58%) illnesses were caused by norovirus, followed by nontyphoidal Salmonella spp. (11%), Clostridium perfringens (10%), and Campylobacter spp. (9%). Leading causes of hospitalization were nontyphoidal Salmonella spp. (35%), norovirus (26%), Campylobacter spp. (15%), and Toxoplasma gondii (8%). Leading causes of death were nontyphoidal Salmonella spp. (28%), T. gondii (24%), Listeria monocytogenes (19%), and norovirus (11%). These estimates cannot be compared with prior (1999) estimates to assess trends because different methods were used. Additional data and more refined methods can improve future estimates.
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Bacterial and Viral Pathogens in Live Oysters: 2007 United States Market Survey
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Two samples of market oysters, primarily from retail establishments, were collected twice each month in each of nine states during 2007. Samples were shipped refrigerated overnight to five U.S. Food and Drug Administration laboratories on a rotating basis and analyzed by most probable number (MPN) for total and pathogenic Vibrio parahaemolyticus and V. vulnificus numbers and for the presence of toxigenic V. cholerae, Salmonella spp., norovirus (NoV), and hepatitis A virus (HAV). Levels of indicator organisms, including fecal coliforms (MPN), Escherichia coli (MPN), male-specific bacteriophage, and aerobic plate counts, were also determined. V. parahaemolyticus and V. vulnificus levels were distributed seasonally and geographically by harvest region and were similar to levels observed in a previous study conducted in 1998-1999. Levels of pathogenic V. parahaemolyticus were typically several logs lower than total V. parahaemolyticus levels regardless of season or region. Pathogenic V. parahaemolyticus levels in the Gulf and Mid-Atlantic regions were about two logs greater than the levels observed in the Pacific and North Atlantic regions. Pathogens generally associated with fecal pollution were detected sporadically or not at all (toxigenic V. cholerae, 0%; Salmonella, 1.5%; NoV, 3.9%; HAV, 4.4%). While seasonal prevalences of NoV and HAV were generally greater in oysters harvested from December to March, the low detection frequency obscured any apparent seasonal effects. Overall, there was no relationship between the levels of indicator microorganisms and the presence of enteric viruses. These data provide a baseline that can be used to further validate risk assessment predictions, determine the effectiveness of new control measures, and compare the level of protection provided by the U.S. shellfish sanitation system to those in other countries.
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The National Center for Biotechnology Information's Protein Clusters Database
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Rapid increases in DNA sequencing capabilities have led to a vast increase in the data generated from prokaryotic genomic studies, which has been a boon to scientists studying micro-organism evolution and to those who wish to understand the biological underpinnings of microbial systems. The NCBI Protein Clusters Database (ProtClustDB) has been created to efficiently maintain and keep the deluge of data up to date. ProtClustDB contains both curated and uncurated clusters of proteins grouped by sequence similarity. The May 2008 release contains a total of 285 386 clusters derived from over 1.7 million proteins encoded by 3806 nt sequences from the RefSeq collection of complete chromosomes and plasmids from four major groups: prokaryotes, bacteriophages and the mitochondrial and chloroplast organelles. There are 7180 clusters containing 376 513 proteins with curated gene and protein functional annotation. PubMed identifiers and external cross references are collected for all clusters and provide additional information resources. A suite of web tools is available to explore more detailed information, such as multiple alignments, phylogenetic trees and genomic neighborhoods. ProtClustDB provides an efficient method to aggregate gene and protein annotation for researchers and is available at http://www.ncbi.nlm.nih.gov/sites/entrez?db=proteinclusters.
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Increasing Rates of Vibriosis in the United States, 1996-2010: Review of Surveillance Data From 2 Systems
Article
The Centers for Disease Control and Prevention monitors vibriosis through 2 surveillance systems: the nationwide Cholera and Other Vibrio Illness Surveillance (COVIS) system and the 10-state Foodborne Diseases Active Surveillance Network (FoodNet). COVIS conducts passive surveillance and FoodNet conducts active surveillance for laboratory-confirmed Vibrio infections. We summarized Vibrio infections (excluding toxigenic V. cholerae O1 and O139) reported to COVIS and FoodNet from 1996 through 2010. For each system, we calculated incidence rates using US Census Bureau population estimates for the surveillance area. From 1996 to 2010, 7700 cases of vibriosis were reported to COVIS and 1519 to FoodNet. Annual incidence of reported vibriosis per 100,000 population increased from 1996 to 2010 in both systems, from 0.09 to 0.28 in COVIS and from 0.15 to 0.42 in FoodNet. The 3 commonly reported Vibrio species were V. parahaemolyticus, V. vulnificus, and V. alginolyticus; both surveillance systems showed that the incidence of each increased. In both systems, most hospitalizations and deaths were caused by V. vulnificus infection, and most patients were white men. The number of cases peaked in the summer months. Surveillance data from both COVIS and FoodNet indicate that the incidence of vibriosis increased from 1996 to 2010 overall and for each of the 3 most commonly reported species. Epidemiologic patterns were similar in both systems. Current prevention efforts have failed to prevent increasing rates of vibriosis; more effective efforts will be needed to decrease rates.
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Vibrio parahaemolyticus cell biology and pathogenicity determinants
Article
  • Jul 2011
Vibrio parahaemolyticus is a significant cause of gastroenteritis worldwide. Characterization of this pathogen has revealed a unique repertoire of virulence factors that allow for colonization of the human host and disease. The following describes the known pathogenicity determinants while establishing the need for continued research.
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Bacteria in bivalve shellfish with special reference to the oyster
Article
Kueh, C.S.W. & Chan, K-Y. 1985. Bacteria in bivalve shellfish with special reference to the oyster. Journal of Applied Bacteriology, 59, 41–47. The bacterial flora of the Pacific oyster Crassostrea gigas, the sea mussel Perna viridis and the arkshell clam Scapharca cornea differed considerably from that of seawater in both numbers and generic composition. The numbers of heterotrophic bacteria in the bivalve shellfish, including the anaerobes and spore-forming bacteria, were greater than that in the surrounding water. Pseudomonas spp. were the dominant organisms, comprising over one third of the 321 strains characterized after isolation from the bivalves and seawater. Other bacteria isolated from the shellfish included Vibrio, Acinetobacter, and Aeromonas spp., whereas the seawater flora consisted mainly of coliform organisms, coryneform bacteria and Flavobacterium/ Cytophaga spp. Bacteria associated with the deposit-feeding clams were higher in density and more distinct in generic composition as compared with those in the suspension-feeding oysters and mussels. Over 90% of the coliform and heterotrophic bacteria in oysters were found in organs associated with the digestive tract. Coliforms were mainly found in the stomach while heterotrophs were present in both stomach and the lower intestine. The results suggest that the stomach flora of oysters are mainly derived from the external environment and, through a process of selection and multiplication, that it may be gradually replaced by a more indigenous population which dominates the lower digestive tract.
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Environmental Investigations of Vibrio parahaemolyticus in Oysters after Outbreaks in Washington, Texas, and New York (1997 and 1998)
Article
  • Dec 2000
Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York. Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V. parahaemolyticus in environmental shellfish samples. V. parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas. However, only two samples (one each from Washington and Texas) were found to harbor total V. parahaemolyticus densities exceeding the level of concern of 10,000 g−1. Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g−1). Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7°C) and V. parahaemolyticus levels (100 to 1,000 g−1) during the summer. Salinity varied from 14.9 to 29.3 ppt. A slight but significant (P < 0.05) negative correlation (−0.25) was observed between V. parahaemolyticus density and salinity. Based on our data, findings of more than 10,000 g−1 total V. parahaemolyticus or >10 g−1 tdh- and/ortrh-positive V. parahaemolyticus in environmental oysters should be considered extraordinary.
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