How bacteria build biofilms

Princeton researchers have for the first time revealed the mechanics of how bacteria build up slimy masses, called biofilms, cell by cell. When encased in biofilms in the human body, bacteria are a thousand times less susceptible to antibiotics, making certain infections, such as pneumonia, difficult to treat and potentially lethal. 

biofilm-illustration_1150In a study published Sept. 6 in the Proceedings of the National Academy of Sciences, a team at Princeton tracked a single bacterial cell as it grew into a mature biofilm of 10,000 cells with an ordered architecture. The findings should help scientists learn more about bacterial behavior and open up new ways of attacking biofilms with drugs.

“No one’s ever peered inside a living biofilm and watched it develop cell by cell,” said Bonnie Bassler, a senior author of the paper and the Squibb Professor in Molecular Biology at Princeton, as well as a Howard Hughes Medical Institute Investigator. “With this paper, we can now understand for the first time how communities of bacteria form a biofilm.”

The discovery became possible thanks to a special microscopy method pioneered at Princeton by a former postdoctoral research associate, Knut Drescher, which allowed the imaging of single cells, letting researchers follow a budding biofilm in real time.

“We have used a state-of-the-art technique to see into the core of a living, growing biofilm,” said postdoctoral research associate Jing Yan, lead author of the new study. Along with membership in Bassler’s lab, Yan belongs to the Complex Fluids Group led by paper senior co-author Howard Stone, the Donald R. Dixon ’69 and Elizabeth W. Dixon Professor of Mechanical and Aerospace Engineering at Princeton. Yan is further advised by paper senior author Ned Wingreen, the Howard A. Prior Professor of the Life Sciences and acting director of the Lewis-Sigler Institute for Integrative Genomics at Princeton.

“The research that produced this paper sits at the frontier between materials science, engineering, physics and biology and represents a fantastic collaboration across Princeton University,” said Bassler.

Along with Yan, Bassler, Stone and Wingreen, a fifth co-author of the paper is Andrew Sharo, a former undergraduate in Princeton’s Department of Physics and now at the University of California-Berkeley.

The researchers chose Vibrio cholerae for their model biofilm organism because of its long history of study and threat to human health, causing the diarrheal disease cholera. A curved, rod-shaped bacterium, V. cholerae lives as a free-swimming cell in brackish water or saltwater. When V. cholerae makes contact with a food particle, perhaps on the shell of a crab or a shrimp, or a human intestinal cell during disease, the bacterium attaches itself and begins to reproduce. The expanding colony’s members secrete a glue-like substance to keep from getting washed away and to protect themselves from competing bacteria.

Previous efforts to delve into how the cells in a burgeoning biofilm interact had failed because of insufficient optical resolution; basically, what one cell was doing in the opaque mass could not be distinguished from its neighbors. 

The Princeton researchers overcame this problem in several ways. First, they genetically modified the bacterial strain so the cells produced proteins that glow brightly when illuminated by specific colors of light. The proteins selected offer the brightest available fluorescence, making each cell easier to pick out, while reducing the intensity of potentially cell-damaging light required for the experiment.

The team then used a confocal microscope, a device that focuses on a single portion of a specimen from a certain distance. By making hundreds of such observations, images can be stacked together to create a three-dimensional image of the entire specimen. “It’s like looking deep into the interior of a biofilm without having to slice it open,” said Yan.

Another boost for the research team came from computer algorithms originally developed for fields like materials science. The algorithms differentiated closely clustered sources of light, in this case the many bunched-up V. cholerae cells in a thickening biofilm. 

What the Princeton team saw was remarkable. At first, the bacterial colony expanded horizontally on the given surface in the experiment. As each cell split, the resulting daughter cells firmly attached to the surface alongside their parent cells. Squeezed by increasing numbers of offspring bacteria, however, the cells at the heart of expanding colony were forced to detach from the surface and point vertically. The bacterial colony thus went from a flat, two-dimensional mass to an expanding, three-dimensional blob, all held together by gunk in the developing biofilm.

The Princeton team dug a bit deeper into the genetics behind this cellular behavior. A single gene, dubbed RbmA, is key to behavior in which new cells connect in such a way to develop a three-dimensional biofilm. When the researchers deactivated the gene, a big, diffuse and floppy biofilm formed. When RbmA performed as normal, though, a denser, stronger biofilm resulted as the cells stayed linked to each other. Thus, RbmA provides the biofilm its resilience, providing insight into a potential Achilles heel that could be targeted for therapeutic intervention.

Ongoing work is now measuring the physical forces experienced by cells uplifting at the biofilm’s center so the overall mechanics can be precisely worked out. “We are currently trying to develop a mathematical model for how the bacterial colony grows in time and how the spatial features are linked to typical mechanical features of the biofilm,” said Stone. 

Crabs, marinade and pathogens

Knowing the survival characteristics of foodborne pathogens in raw ready-to-eat (RTE) seafood is the key to predicting whether they pose a microbiological hazard. The present study examined the survival of Escherichia coli O157:H7, Salmonella Typhimurium, Vibrio parahaemoliticus, Listeria monocytogenes, and Staphylococcus aure- us in raw RTE crab marinated in soy sauce.

cartooncrab_1_jpg103a1464-d351-465e-90ed-f6ab47e3c8bboriginalInoculated crabs (initial bacterial population = 4.1–4.4 log CFU/g) were immersed in soy sauce and then stored at refrigeration (5 °C) or room temperature (22 °C) for up to 28 days. At 5 °C, all bacteria (except V. parahaemolyticus) survived in crab samples until Day 28 (counts of 1.4, 1.6, 3.1, 3.2 log CFU/g for E. coli O157:H7, S. Typhimurium, L. monocytogenes, and S. aureus, respectively). Howev- er, at 22 °C, all tested bacteria were more susceptible to the antimicrobial effects of marination. Regardless of tem- perature, foodborne pathogens attached to crab samples were more resistant to marination than those suspended in soy sauce samples; however, the survival pattern for each species was different. Gram-positive bac- teria were most resistant to marination conditions (high salinity, low pH), whereas V. parahaemolyticus was ex- tremely susceptible.

Marination is the only antibacterial step in the manufacturing processes; however, the results presented herein reveal that this is not sufficient to inactivate foodborne pathogens. In particular, the survival of pathogens on crabs at refrigeration temperature may pose a major hazard for the consumption of raw RTE seafood. Thus, appropriate decontamination methods and implementation of safety management practices are needed.

This study provides predictive microbiological information of foodborne pathogens in raw RTE seafood with margination.

Survival of foodborne pathogens (Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus) in raw ready-to-eat crab marinated in soy sauce

International Journal of Food Microbiology 238 (2016) 50–55, DOI: http://dx.doi.org/10.1016/j.ijfoodmicro.2016.08.041

TJ Cho, NH Kim, SA Kim, JH Song

Minneapolis sees rise in foodborne illness from nororvirus, Vibrio in oysters

When I think Minnesota, I think raw oysters.

No, I never think that about anywhere.

raw.oysters.minnJeremy Olson of the Star Tribune reports that city health inspectors in Minneapolis are investigating a summer increase in foodborne illnesses related to norovirus and Vibrio, a bacteria found in raw oysters.

The increases were highlighted in the city’s “food establishment” newsletter, released Thursday.

“The reason for the spike in norovirus outbreaks is not known,” the advisory stated. “The Vibrio outbreaks are due to higher concentrations of bacteria in some oyster beds during the summer.”

Cases of norovirus, a highly contagious bug that causes severe vomiting and diarrhea, are not required to be reported to the Minnesota Department of Health, but the state agency has received reports of a slight uptick that is unusual for this time of year.

Raw is risky: 2 Tampa Bay residents die from shellfish

Jacqueline Ingles of WFTS Tampa Bay reports, take a ride on Captain Nick Warhurst’s boat and there is just one rule: do not eat raw shellfish.

seven.fish.bbq.dec.11“I’d rather you not eat anything raw on my boat,” said Warhurst. “If you want to eat them raw you wait till you get to the dock and you’re on your own.”

Married to a nurse, Warhurst says he knows the dangers of eating raw or undercooked shellfish.

“Some people die from this stuff,” he explained.

According to the Florida Department of Health, two Bay area residents did get infected with Vibrio Vulnificus and died this year. One resident was from Citrus County, the other resided in Sarasota County.

Vibrio is a bacteria that occurs naturally in Gulf Coast waters.

You can also get infected if you go into water with an open cut or sore.

So far this year, 23 people have been infected by the bacteria across the states. A total of five people have died from the infections.

However, contracting it is rare.

“It is really, really, really rare, but why take the chance,” asked Terry Natwick, the director of sales and marketing at the Plantation Inn in Crystal River.

The inn, which is a hotspot for tourists who’ve come to scallop stay, offers a catch and cook program.

“Not only do we have somebody who will professionally shuck the scallops for you and keep it on ice and then put it in a Ziplock and then you bring it right to our kitchen where we refrigerate it at the proper temperature and cook if for you either that day at lunch or that night for dinner,” Natwick said.

First time scalloper Nick Tulse is taking the Inn up on it’s offer.

“Oh no no, you cook ’em,” said Tulse, who drove up from Bradenton.

Raw is risky: Study links global warming to rise in waterborne illnesses

Rising global temperatures are clearly linked to increasing waterborne food poisoning, particularly from eating raw oysters, along with other nasty infections, a new study shows.

Oyster-Vancouver, B.C.- 07/05/07- Joe Fortes Oyster Specialist Oyster Bob Skinner samples a Fanny Bay oyster at the restuarant. Vancouver Coastal Health now requires restaurants to inform their patrons of the dangers of eating raw shellfish. (Richard Lam/Vancouver Sun) [PNG Merlin Archive]

About a dozen species of vibrio bacteria make people sick from eating raw or undercooked seafood or drinking or swimming in tainted water. It also causes cholera, although that was not the focus of the research.

Lab-confirmed vibrio infections in the United States have increased from an average of about 390 a year from the late 1990s to an average of 1,030 in recent years, according to the Centers for Disease Control and Prevention. But most cases aren’t confirmed by tests and reported.

“It’s a remarkable increase on an annual basis,” said study lead author Rita Colwell of the University of Maryland, a top microbiologist who used to head the National Science Foundation.

The study examined Europe and North America, but the most consistent tracking of vibrio illnesses were in the United States. The CDC blames about 100 deaths a year on vibrio on average.

Even Alaska, where such outbreaks used to be unheard of because the bacteria needs warm water, is getting cases from people eating vibrio-infected oysters, Colwell said. Her study, published in Monday’s Proceedings of the National Academy of Sciences , highlights an unprecedented wave of vibrio illnesses from swimming in northern Europe during heat waves in 1994, 1997, 2003, 2006 and 2010.

Climate influence on Vibrio and associated human diseases during the past half-century in the coastal North Atlantic

22.jun.16

DOI: 10.1073/pnas.1609157113

Vezzulli, C. Grande, P.C. Reid, P. Hélaouët, M. Edwards, M.G. Höfle, I. Brettar, R.R. Colwell, C. Pruzzo

http://www.pnas.org/content/early/2016/08/02/1609157113.abstract

Climate change is having a dramatic impact on marine animal and plant communities but little is known of its influence on marine prokaryotes, which represent the largest living biomass in the world oceans and play a fundamental role in maintaining life on our planet. In this study, for the first time to our knowledge, experimental evidence is provided on the link between multidecadal climatic variability in the temperate North Atlantic and the presence and spread of an important group of marine prokaryotes, the vibrios, which are responsible for several infections in both humans and animals. Using archived formalin-preserved plankton samples collected by the Continuous Plankton Recorder survey over the past half-century (1958–2011), we assessed retrospectively the relative abundance of vibrios, including human pathogens, in nine areas of the North Atlantic and North Sea and showed correlation with climate and plankton changes. Generalized additive models revealed that long-term increase in Vibrio abundance is promoted by increasing sea surface temperatures (up to ∼1.5 °C over the past 54 y) and is positively correlated with the Northern Hemisphere Temperature (NHT) and Atlantic Multidecadal Oscillation (AMO) climatic indices (P < 0.001). Such increases are associated with an unprecedented occurrence of environmentally acquired Vibrio infections in the human population of Northern Europe and the Atlantic coast of the United States in recent years.

Raw is risky: Canada reports 1st case this year of illness linked to eating raw oysters

CBC News reports British Columbia has recorded its first case this year of someone being sickened by eating raw oysters contaminated with Vibrio bacteria.

oysters.grillThe B.C. Centre for Disease Control (BCCDC) said the illness was reported June 30 in the Vancouver area.

Vibrio parahaemolyticus bacteria grow in seawater and can end up in shellfish like oysters and clams. When water temperatures rise in the summer, the accumulations of the naturally occurring bacteria increase to the point that eating undercooked shellfish can give people nausea, fever and diarrhea.

Last year’s outbreak of the Vibrio-caused illness was the biggest in Canadian history and sickened at least 73 British Columbians. Sixty of the illnesses were due to eating contaminated raw or undercooked B.C. oysters in restaurants. The other 13 illnesses were traced to exposure to seawater with high levels of the bacteria.

At the height of the outbreak last summer, Vancouver Coastal Health ordered restaurants not to serve raw oysters harvested from B.C. waters and the Canadian Food Inspection Agency issued a food recall for B.C. oysters. 

“Eating raw shellfish increases your risk of Vibrio and other infections,” said Dr. Eleni Galanis, epidemiologist at the BCCDC, in a release.  

“It’s best to eat them cooked, but if you choose to eat raw shellfish like oysters, then understand the risks and take steps to reduce your likelihood of illness.”

Meanwhile, Florida health officials have reported 13 Vibrio vulnificus cases as of July 5, including four fatalities thus far in 2016.

Last year, Florida saw 45 cases and 14 deaths, the most since 2003.

Healthy individuals typically develop a mild disease; however, Vibrio vulnificus infections can be a serious concern for people who have weakened immune systems, particularly those with chronic liver disease.

Oyster-Vancouver, B.C.- 07/05/07- Joe Fortes Oyster Specialist Oyster Bob Skinner samples a Fanny Bay oyster at the restuarant. Vancouver Coastal Health now requires restaurants to inform their patrons of the dangers of eating raw shellfish.  (Richard Lam/Vancouver Sun)   [PNG Merlin Archive]

So don’t be a drunk and eat raw.

I BBQ them, and prefer scallops on the half-shell.

In other Virbrio news, UT Southwestern Medical Center researchers have uncovered a mechanism that a type of pathogenic bacteria found in shellfish use to sense when they are in the human gut, where they release toxins that cause food poisoning.

The researchers studied Vibrio parahaemolyticus, a globally spread, Gram-negative bacterium that contaminates shellfish in warm saltwater during the summer. The bacterium thrives in coastal waters and is the world’s leading cause of acute gastroenteritis.

“During recent years, rising temperatures in the ocean have contributed to this pathogen’s worldwide dissemination,” said Dr. Kim Orth, Professor of Molecular Biology and Biochemistry at UT Southwestern and senior author of the study, published today in the online journal eLife.

About a dozen Vibrio species cause infection in humans, according to the Centers for Disease Control and Prevention, and Vibrio parahaemolyticus is one of the three most common culprits. Vibrio infections cause an estimated 80,000 illnesses and 100 deaths in the United States every year.

The study found that two proteins made by Vibrio parahaemolyticus work together to detect and capture bile salts in the intestines of people who eat raw or undercooked seafood containing the bacteria.

“When a person eats, acids in the stomach help break down the meal, and bile salts in the intestine aid in the solubilization of fatty food. When humans eat raw or undercooked shellfish contaminated with Vibrio parahaemolyticus, the bacteria use those same bile salts as a signal to release toxins,” said Dr. Orth, also an Investigator with the Howard Hughes Medical Institute (HHMI), holder of the Earl A. Forsythe Chair in Biomedical Science, and a W.W. Caruth, Jr. Scholar in Biomedical Research. Dr. Orth studies the strategies that bacterial pathogens use to outsmart their host cells.

Evidence is increasing that several bacterial pathogens that cause gastrointestinal illness, including the extremely toxic Vibrio cholerae, sense bile salts. But until now, the mechanism that those pathogens use for doing this has remained unknown, Dr. Orth said. In previous studies, only one bacterial gene had been implicated in receiving and transmitting the gut-sensing signal, Dr. Orth said.

“We discovered that not one, but two genes are required for Vibrio to receive the bile salt signal. These genes encode two proteins that form a complex on the surface of the bacterial membrane. Using X-ray crystallography, we found that these proteins create a barrel-like structure that binds bile salts and receives the signal to tell the bacterial cell to start making toxins,” she said.

Future experiments will aim to understand how binding of bile salt by this protein complex induces the release of toxins.

“Ultimately, we want to understand how other pathogenic bacteria sense environmental cues to produce toxins. With this knowledge, we might be able to design pharmaceuticals that could prevent toxin production, and ultimately avoid the damaging effects of infections,” she said.

The receptor pair could possibly act as a model to discover sensors in other bacteria where pharmaceuticals might be more applicable, Dr. Orth said, adding “we are in the early stages of this research.”

Co-lead authors were graduate student Peng Li and research scientist Dr. Giomar Rivera-Cancel, both in Molecular Biology. Other contributing authors included Dr. Lisa Kinch, an HHMI bioinformatics specialist; Dr. Dor Salomon, postdoctoral researcher; Dr. Diana Tomchick, Professor of Biophysics and Biochemistry and Director of the Structural Biology Core Facility; and Dr. Nick Grishin, Professor of Biophysics and Biochemistry, an HHMI Investigator, and a Virginia Murchison Linthicum Scholar in Biomedical Research.

The research was supported by the National Institutes of Health, the Welch Foundation, the Department of Energy, and the HHMI.

And finally, bacterial infections from various organisms including Vibrio sp. pose a serious hazard to humans in many forms from clinical infection to affecting the yield of agriculture and aquaculture via infection of livestock. Vibrio sp. is one of the main foodborne pathogens causing human infection and is also a common cause of losses in the aquaculture industry. Prophylactic and therapeutic usage of antibiotics has become the mainstay of managing this problem, however this in turn led to the emergence of multidrug resistant strains of bacteria in the environment; which has raised awareness of the critical need for alternative non antibiotic based methods of preventing and treating bacterial infections. Bacteriophages – viruses that infect and result in the death of bacteria – are currently of great interest as a highly viable alternative to antibiotics. This article provides an insight into bacteriophage application in controlling Vibrio species as well underlining the advantages and drawbacks of phage therapy.

Insights into bacteriophage application in controlling Vibrio species

Front. Microbiol. | doi: 10.3389/fmicb.2016.01114

http://journal.frontiersin.org/article/10.3389/fmicb.2016.01114/abstract

Vengadesh Letchumanan,  Kok Gan Chan,  Priyia Pusparajah,  Surasak Saokaew,  Acharaporn Duangjai,  Bey Hing Goh,  Nurul-Syakima Ab Mutalib and  Learn-Han Lee

I’m not your guy, pal: Raw oysters risky for wine drinkers

When Canada’s food safety agency announced a recall of B.C. oysters last August, it meant producers like Steve Pocock had to ensure every last oyster they had shipped after a certain date was accounted for.

Oyster-Vancouver, B.C.- 07/05/07- Joe Fortes Oyster Specialist Oyster Bob Skinner samples a Fanny Bay oyster at the restuarant. Vancouver Coastal Health now requires restaurants to inform their patrons of the dangers of eating raw shellfish.  (Richard Lam/Vancouver Sun)   [PNG Merlin Archive]

Along with a recall – issued by the Canadian Food Inspection Agency (CFIA) after dozens of people got sick as a result of eating raw oysters contaminated with Vibrio parahaemolyticus – there was a ban on restaurants serving raw oysters from British Columbia.

The inconvenience and forgone sales added up to a big hit for Mr. Pocock and other producers in British Columbia’s oyster sector.

Over the past few months, they have been working to prevent a repeat scenario.

“The recall had a very serious impact on our industry – and it should be taken very seriously,” Mr. Pocock said in a recent interview. He owns and operates Sawmill Bay Shellfish and is also president of the BC Shellfish Grower’s Association.

“And I’m not just talking about the farmers; I’m talking about everyone right through to the server in the restaurant,” he added.

A workshop last November spawned a national working group focused on Vibrio with representatives from the Canadian Food Inspection Agency, Fisheries and Oceans Canada, Health Canada, the BC Centre for Disease Control (BCCDC) and provincial health authorities.

That group developed a prevention program for Vibrio, focusing on education, enhanced testing and improved communication between producers and government agencies.

On the education front, workshops for producers emphasized measures to control Vibrio, such as proper refrigeration during transport.

Oysters represent a relatively small chunk of British Columbia’s aquaculture sales – $13-million, compared with $380.4-million for salmon, according to a 2015 report by British Columbia’s Ministry of Agriculture – but are prized for their taste and local appeal.

“Shellfish are an important part of our business, and especially in the summertime, when patios are open, [oysters] go great with wine and it was disappointing we were unable to offer B.C. product for raw consumption,” said Guy Dean, vice-president of seafood distributor Albion Fisheries.

Yeah, especially since Vibrio produces a toxin that attacks the weak livers of persistent wine drinkers.

Raw is risky.

And this Guy ain’t your buddy. Or friend.

Canadians say: Update to the Vibrio parahamolyticus guideline

This notice provides an update to the information published on October 20, 2015 regarding the management of the risks of Vibrio parahaemolyticus (Vp) in raw oysters. Effective immediately, the Canadian Food Inspection Agency (CFIA) is expanding the scope of application of the bacteriological guideline for Vp in live oysters.

Oyster-Vancouver, B.C.- 07/05/07- Joe Fortes Oyster Specialist Oyster Bob Skinner samples a Fanny Bay oyster at the restuarant. Vancouver Coastal Health now requires restaurants to inform their patrons of the dangers of eating raw shellfish. (Richard Lam/Vancouver Sun) [PNG Merlin Archive]

The interim bacteriological guideline for Vp, found in the CFIA’s Fish Products Standards and Methods Manual will now apply to all live oysters (end product), whether domestically produced or imported. This means that no sample can exceed 100 MPN per gram in each of five subsamples.

Importers, domestic processors and exporters are responsible for ensuring that fish and seafood products meet all applicable regulatory requirements, including the regulations made under the authority of the Fish Inspection Act.

Quality Management Program Importers (QMPI) and fish processing establishments must outline and implement controls to ensure that any significant health and safety hazards identified are controlled for fish imported into Canada, or processed in Canada.

Federally registered establishments must review their Hazard Analysis Critical Control Point (HACCP) plan to ensure that the measures, as a whole, to eliminate or reduce Vp to an acceptable level are effective in ensuring the live oysters meet the updated Vp guideline. Until the review of the HACCP plan is completed and the measures are determined to be effective, additional interim measures (e.g. lot by lot testing of the oysters) are necessary to ensure compliance. Interim measures must be initiated when conditions are favourable for Vp (identified as water or oyster meat temperature at point of harvest equal to or greater than 15°C or testing of Vp in oysters at the harvest area showing persistent levels at or near 100 Vp MPN/g).

Importers must ensure that the live oysters they import meet Canadian regulatory requirements, including the updated Vp guideline. They must also verify that the oysters have been harvested, handled, stored and conveyed in a manner which adequately manages the risk of Vp.

QMPI licence holders that import live oysters must review and amend their QMPI plan. This will ensure that effective controls are in place so that the oysters comply with the updated Vp guideline.

The CFIA will continue to verify that appropriate controls for Vp in oysters have been implemented; through its regular activities at federally registered processing establishments and with importers using inspections, audits and sampling and testing.

Health Canada is reviewing available Vp data, which may result in further revision to the interim guideline. Until this review is completed, the CFIA will apply the interim guideline to all live oysters sold in Canada, in order to continue to protect consumers. 

The CFIA is preparing additional guidance to assist industry in understanding and managing the Vp hazard.  This information will be available on CFIA’s website.  Industry is encouraged to subscribe to the e-mail notification service to be notified when CFIA manuals are updated.

I don’t eat raw oysters: Gross and may have Vibrio

Following up a scientific report, Timothy B. Wheeler of the Bay Journal reports a 6-year-old outbreak of food poisoning linked to eating raw Chesapeake Bay oysters has left behind a lingering mystery. Scientists seeking to identify the water-borne pathogen that sickened a pair of Baltimore restaurant patrons have tracked the culprit to Asia.

Raw oystersHow a potent strain of Vibrio bacteria seemingly from so far away wound up in the Bay continues to puzzle Maryland health officials, who worked with researchers at the U.S. Food and Drug Administration to investigate the 2010 cases.

The microorganism could have gotten here in the ballast water of the many oceangoing ships that ply the Chesapeake every year, state and federal scientists suggested in a recently published journal article. Or, they added, perhaps it came via the introduction of non-native oysters or some exotic fish.

“It really is speculation,” acknowledged Dr. Clifford Mitchell, environmental health bureau director for the Maryland Department of Health and Mental Hygiene. “We didn’t sample ballast water. We didn’t take specimens that would lead us to know that we had fish coming over, or migration.”

But the case, published in the June issue of Applied and Environmental Microbiology, illustrates how disease-carrying organisms may travel around the world, researchers said. And while steps have been taken since 2010 to prevent the unintentional transport of pathogens, parasites and other potentially harmful organisms via ships’ ballast water, those safeguards still have significant gaps in them.

The bacterium involved in the 2010 food poisoning outbreak was Vibrio parahaemolyticus, strains of which are commonly found in coastal waters worldwide — including the Bay — though only some have been found to cause illness. When those are ingested, they can cause acute gastrointestinal distress, including diarrhea, stomach pain, nausea, fever and chills. It usually passes within a few days, but in rare cases can be more severe, especially in people with weakened immune systems.

There were 45 cases of Vibrio infections reported in Maryland in 2010, but it’s not that often, state health officials said, that they’re able to pinpoint the source of the bacteria that may have sickened a particular person. By the time laboratory tests identify Vibrio as the cause of someone’s GI distress and the information gets reported to the state, days or even weeks may have passed, and the food that person had eaten is long gone.

In this case, though, state health investigators got a lucky break. Two individuals who got sick said that shortly before they became ill that summer, they had eaten raw oysters at different Baltimore restaurants. They hadn’t traveled out of state or done anything else that likely could have exposed them.

When investigators visited the restaurants, they found the half-shells eaten by the two victims were from the Bay. And when they visited the Maryland aquaculture operation that supplied both eateries, investigators pulled some oysters from the water and discovered that they had Vibrio in them as well — 11 different potentially disease-causing strains, in fact. One of those appeared to match the Asian strain found in the two food poisoning victims.

Coos Bay Oyster Co.The investigation ended there, for the time being. Even though the Vibrio involved were similar, researchers couldn’t positively identify them as the same, using the analytic techniques they had at the time. “The chromosome patterns matched, but we weren’t sure how common that pattern was in the environment,” explained Robert Myers, director of the state health department’s laboratory administration. “We hadn’t seen it before.”

A few years later, though, “whole genome sequencing” technology became available, Myers said, giving researchers the ability to draw a more detailed map of an organism’s genetic makeup.

With that new, more powerful analytical tool, FDA researchers re-examined the Vibrio strains involved in the 2010 outbreak and those from the oysters that state health investigators had sampled. They identified them as belonging to a family of strains known as “sequence type 8.” 

When researchers consulted a worldwide Vibrio database, they found that the Maryland strains were unlike any seen to date in the United States. Instead, they were closely related to strains reported only in Asia, most recently in Hong Kong about four years before the outbreak.

Changes were made to shellfish safety protocols after a larger outbreak in 2013of Vibrio parahaemolyticus illnesses associated with eating raw oysters harvested along the Atlantic Coast. More than 100 people in 13 states, including Maryland and Virginia, became ill.  According to a spokesman for the Maryland Department of the Environment, which regulates shellfish harvest waters in the state, the Interstate Shellfish Sanitation Conference, a joint state-federal body, tightened its requirements for investigating such cases, closing implicated harvest areas and ordering a product recall when more than 10 cases are traced to a given area. 

But officials caution that the protocols are not foolproof, and cases like this are a reminder of the risk people run in consuming raw seafood, Mitchell said, especially if they have underlying health conditions.

The number of reported Vibrio infections in the state varies from year to year, but has been trending upward since 2005, according to state health data. Concentrations of the bacteria increase in warmer weather, and climate change could be a factor as Bay water temperatures tick upward. But Mitchell cautioned that the bacteria are present year-round.

“Given the number of people who eat oysters, certainly it’s a relatively small number of infections, but it can be a very significant one,” Mitchell said.

WGS, Vibrio and traceback in oysters

In the summer of 2010, Vibrio parahaemolyticus caused an outbreak in Maryland linked to the consumption of oysters. Strains isolated from both stool and oyster samples were indistinguishable by pulsed-field gel electrophoresis (PFGE). However, the oysters contained other potentially pathogenic V. parahaemolyticusstrains exhibiting different PFGE patterns.

Oyster-Vancouver, B.C.- 07/05/07- Joe Fortes Oyster Specialist Oyster Bob Skinner samples a Fanny Bay oyster at the restuarant. Vancouver Coastal Health now requires restaurants to inform their patrons of the dangers of eating raw shellfish.  (Richard Lam/Vancouver Sun)   [PNG Merlin Archive]

In order to assess the identity, genetic makeup, relatedness, and potential pathogenicity of the V. parahaemolyticusstrains, we sequenced 11 such strains (2 clinical strains and 9 oyster strains). We analyzed these genomes by in silico multilocus sequence typing (MLST) and determined their phylogeny using a whole-genome MLST (wgMLST) analysis. Ourin silico MLST analysis identified six different sequence types (STs) (ST8, ST676, ST810, ST811, ST34, and ST768), with both of the clinical and four of the oyster strains being identified as belonging to ST8.

Using wgMLST, we showed that the ST8 strains from clinical and oyster samples were nearly indistinguishable and belonged to the same outbreak, confirming that local oysters were the source of the infections. The remaining oyster strains were genetically diverse, differing in >3,000 loci from the Maryland ST8 strains. eBURST analysis comparing these strains with strains of other STs available at the V. parahaemolyticus MLST website showed that the Maryland ST8 strains belonged to a clonal complex endemic to Asia. This indicates that the ST8 isolates from clinical and oyster sources were likely not endemic to Maryland. Finally, this study demonstrates the utility of whole-genome sequencing (WGS) and associated analyses for source-tracking investigations.

A nonautochthonous U.S. strain of Vibrio parahaemolyticus isolated from Chesapeake Bay oysters caused the outbreak in Maryland in 2010

Appl. Environ. Microbiol. June 2016 vol. 82 no. 11 3208-3216

Julie Haendiges, Jessica Jones, Robert A. Myers, Clifford S. Mitchell, Erin Butler, Magaly Toro and Narjol Gonzalez-Escalona

http://aem.asm.org/content/82/11/3208.abstract?etoc