Vibrio outbreak and surveillance: Maryland's collaborative approach

Transcription

1 Vibrio outbreak and surveillance: Maryland's collaborative approach Catey Dominguez, Ph.D Developmental Scientist Maryland Department of Health Core Sequencing James Pettengill PhD Biostatistics and Bioinformatics Staff Center for Food Safety and Applied Nutrition GenomeTrakr Meeting

2 Vibrio parahaemolyticus: an introduction

3 Vibrio parahaemolyticus Common source of foodborne illness: Consuming undercooked or contaminated seafood Detected in feces or wounds Gram negative and motile Halophilic Temperate grower (>15 o C) Found world-wide 3 Public Health Image Library (PHIL)

4 Prevalence of V. parahaemolyticus infections in the Americas Prevalent in costal regions Pandemic clone O3:K6 Sequence type (ST)3 Originating from Southeast Asia Common STs in Americas: ST3 ST36 ST631 4 Pandemic Vibrio parahaemolyticus O3:K6 on the American Continent. Velaquez-Roman, J., et al. Front. Cell. Infect. Microbiol., Jan 2014.

5 5 Distribution and dynamics of epidemic and pandemic Vibrio parahaemolyticus virulence factors. Ceccarelli, D., et al. Front. Cell. Infect. Microbiol., 11 December Global incidence of pandemic V. parahaemolyticus Most prevalent in countries that consume seafood Coastal regions Pandemic spread: Product of global food trade Ship ballast water Aquaculture Ocean warming More temperate habitats available Diminished sea ice, new currents and migration patterns

6 Modes of toxicity Toxigenic vs non-toxigenic Hemolysins: Thermoliable hemolysin (TLH) Present in all parahaemolyticus Species-specific used for species ID Thermostable direct hemolysin (TDH) TDH-related hemolysin (TRH) Type 3 secretion systems (T3SS) 6 Vibrio parahaemolyticus cell biology and pathogenicity determinants. Broberg, C., et al. Microbes and Infect., November 2011.

7 Maryland and Vibrio Maryland: major oyster and crab-producing state MD V. PARAHAEMOLYTICUS SEQUENCE TYPE DISTRIBUTION STs of less than 1% Vibriosis is a reportable condition in MD and is routinely tested All results of confirmatory testing are used for epidemiological monitoring MD V. PARAHAEMOLYTICUS SPECIMEN TYPE (N=104) UNK PFGE historically the most discriminatory tool for cluster analysis Support outbreak investigation Environmental 19% Clinical 81% Moving toward WGS; wgmlst and SNV/SNP 2014 sequencing all V. parahaemolyticus isolates 2018 sequencing all Vibrio isolates 7

8 General Vibrio workflow Clinical source or parahaemolyticus isolate V. parahaemolyticus Phenotypic species identification chromid/chromagar/tcbs plates Species confirmation MALDI-TOF PCR: species (tlh) and toxigenicity (tdh, tlh) PFGE and/or WGS; wgmlst and SNV/SNP chromid Vibrio TCBS agar (thiosulfate citrate bile salts sucrose) Relate patterns with epidemiological data 8

9 Past work and collaboration with the FDA: V. parahaemolyticus outbreak 2010 Two clinical isolates found to be related: ST8 with similar PFGE patterns Identified aquaculture (oyster) as a possible source 11 of 479 V. parahaemolyticus strains isolated were determined to be potentially pathogenic by PCR (trh) (2.3%) WGS of potentially pathogenic isolates: Identified 4 as ST8 and PFGE matches (0.84% of isolated strains) All ST8 sequences were submitted for wgmlst analysis by FDA 9 A Nonautochthonous U.S. Strain of Vibrio parahaemolyticus Isolated from Chesapeake Bay Oysters Caused the Outbreak in Maryland in Haendiges, J., et al. Appl Environ Microbiol, Jun 2016.

10 Past work and collaboration with the FDA: wgmlst analysis identified a cluster comprising of sequences from food (oyster) and clinical isolates 10 A Nonautochthonous U.S. Strain of Vibrio parahaemolyticus Isolated from Chesapeake Bay Oysters Caused the Outbreak in Maryland in Haendiges, J., et al. Appl Environ Microbiol, Jun 2016.

11 Vibrio parahaemolyticus investigation 2018

12 MD clinical isolates and related exposure: Venezuelan crab meat Outbreak first noticed by local health department Identified two cases of vibriosis Both consumed crab meat purchased at the same grocery store Epidemiological tracking of common food sources for expanded group (April 18 - July ) All individuals consumed a form of imported Venezuelan ready-to-eat crab meat Fresh and pre-cooked but not pasteurized Potentially contaminated during processing MD press release issued July FDA and CDC press release issued July brands containing Venezuelan crab meat collected by MDH for study No stong epi associations to a specific brand 22 V. parahaemolyticus isolates (obtained by BAM method) 7 representative isolates forwarded for PFGE and WGS 12 # of clinical isolates Source PFGE Pattern PCR Toxin Results MLST SfiI NotI Tdh Trh Tlh 16S 10 Feces K16S K16N ST Feces K16S K16N ST Feces K16S K16N ST Wound K16S K16N ST Feces K16S K16N ST Feces K16S K16N ST Feces K16S K16N ST

13 Environmental V. Parahaemolyticus results Initial survey: All 7 isolates had unique PFGE/novel MLST patterns all environmental non-toxigenic All isolates were tdh negative and did not correlate to outbreak patterns Only two isolates shared a common PFGE and MLST pattern, and were from the same crab meat source PFGE Pattern PCR Toxin Results Follow-up survey: analyzing starting material homogenate Internal # Source Species SfiI NotI MLST Tdh Trh Tlh 16S PCR screen: tdh tdh detected in 2 crab sources (high Ct value; low abundance) 23 more isolates prepared from these crab sources C 2 Isolate from crab V. para by MALDI-TOF and PCR K16S K16N novel a Isolate from crab V. para by MALDI-TOF and PCR K16S K16N novel c Isolate from crab V. para by MALDI-TOF and PCR K16S K16N novel x Isolate from crab V. para by MALDI-TOF and PCR K16S K16N novel b Isolate from crab V. para by MALDI-TOF and PCR K16S K16N novel b Isolate from crab V. para by MALDI-TOF and PCR K16S K16N novel Results: tdh not detected in any isolates from new preparation 67-1b Isolate from crab V. para by MALDI-TOF and PCR K16S K16N novel out of 49 total isolates discontinued pursuing tdh + environmental isolate due to screening limitations and high non-toxigenic representation (low abundance as determined by qpcr) 13

14 Foodborne V. Parahaemolyticus investigation: take-away Environmental V. parahaemolyticus organisms are highly represented in seafood and highly diverse Culture-dependent methods can make it difficult to find the needle in the haystack isolates from food/environmental samples 14 Future directions: Colony lift assay (BAM method) for screening colonies with tdh/trh DNA probes Metagenomics implementation for pathogen fishing from difficult environmental sources PCR-screen environmental/food source homogenate Deep sequencing of positive hits

15 WGS Considerations for New and Unusual Organisms WGS Considerations for New and Unusual Organisms 1. Can it be sequenced/cultured? (e.g., Cyclospora is particularly difficult from food matrices; viruses are difficult). Does it have a large genome few samples on a single run thus increasing costs (e.g., Cyclospora is 42 Mbp or ~10X larger than Salmonella) Vibrio parahaemolyticus: Two circular chromosomes of 3.3 Mbp and 1.9 Mbp is that an issue for analysis pipelines? 15

16 WGS Considerations for New and Unusual Organisms WGS Considerations for New and Unusual Organisms 1. Can it be sequenced/cultured? (e.g., Cyclospora is particularly difficult from food matrices; viruses are difficult). Does it have a large genome few samples on a single run thus increasing costs (e.g., Cyclospora is 42 Mbp or ~10X larger than Salmonella) Vibrio parahaemolyticus: Two circular chromosomes of 3.3 Mbp and 1.9 Mbp is that an issue for analysis pipelines? 2. Is there a database against which isolates can be compared but have to start somewhere. 16

17 WGS Considerations for New and Unusual Organisms WGS Considerations for New and Unusual Organisms 1. Can it be sequenced/cultured? (e.g., Cyclospora is particularly difficult from food matrices; viruses are difficult). Does it have a large genome few samples on a single run thus increasing costs (e.g., Cyclospora is 42 Mbp or ~10X larger than Salmonella) Vibrio parahaemolyticus: Two circular chromosomes of 3.3 Mbp and 1.9 Mbp is that an issue for analysis pipelines? 2. Is there a database against which isolates can be compared but have to start somewhere. 3. Are there sufficient computing resources storage, computational (HPC), and analytical? 17

18 WGS Considerations for New and Unusual Organisms 1. Can it be sequenced/cultured? (e.g., Cyclospora is particularly difficult from food matrices; viruses are difficult). Does it have a large genome few samples on a single run thus increasing costs (e.g., Cyclospora is 42 Mbp or ~10X larger than Salmonella) Vibrio parahaemolyticus: Two circular chromosomes of 3.3 Mbp and 1.9 Mbp is that an issue for analysis pipelines? 2. Is there a database against which isolates can be compared but have to start somewhere. 3. Are there sufficient computing resources storage, computational (HPC), and analytical? 4. Participation from labs and different agencies (CDC/FDA/USDA/etc) 18

19 Sufficient database to compare samples

20 CFSAN s open-access peer reviewed methods for analyzing and differentiating among samples based on WGS data.

21 WGS for Vibrio clinical and product isolates

22 Development of taxon specific SNP distance guidelines 22

23 Development of taxon specific SNP distance guidelines 23

24 WGS Considerations for New and Unusual Organisms WGS Considerations for New and Unusual Organisms 1. Can it be sequenced/cultured? 2. Is it a large genome (>100 Mbp genome = singleplex)? 3. Is there a database against which isolates can be compared but have to start somewhere. 4. Are there sufficient computing resources storage, computational (HPC), and analytical? 5. Participation from labs and different agencies (CDC/FDA/USDA/etc) 24

25 Acknowledgements FDA Center for Food Safety and Applied Nutrition Center for Veterinary Medicine Office of Regulatory Affairs GenomeTrakr CDC Enteric Diseases Laboratory INEI-ANLIS Carolos Malbran Institute, Argentina National Institutes of Health National Center for Biotechnology Information USDA/FSIS Eastern Laboratory State Health and University Labs Alaska, Arizona, California, Florida, Hawaii, Maryland, Minnesota, New Mexico, New York, South Dakota, Texas, Virginia, Washington Centre for Food Safety, University College Dublin, Ireland Food Environmental Research Agency, UK Public Health England, UK WHO Other independent collaborators 25

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