text:
Remember when barcode label printers could only print one color at a time? Now you can rint any label in two colors, with the Plexo! two color label printer family. Print labels with two-color graphics, barcodes, and variable fields that pull information directly from a database or production system.
The Plexo! family is especially ideal for printing GHS labels, where red and black pictograms, chemical names, and precautionary statements must be printed in color on hazardous chemical labels.
Each Plexo! comes with a one-year warranty and optional Customer Support Agreement with QuickSwap™ Repair and Replacement Program.
- Overview
- Price
- Specifications
- Labels & Inks
- Accessories
Two Colors Are Better Than One:
QuickLabel Systems is known for its color label printers and barcode label printers. Now you can have the best of both worlds. The Plexo! family is a line of two color label printers that operate just like barcode label printers. If you're familiar with printing labels from a barcode printer, you'll find it comfortable and easy to print two color labels with a Plexo! color label printer.
Two Color Label Printer Models:
- The Plexo! 453 is a two color label printer that makes labels up to 4 inches wide and 79 inches long. The 4 inch wide model is used to print a wide variety of labels and tags, including GHS chemical labels, asset tracking labels, food labels, and other product labels.
- The Plexo! 653 is a two color label printer that makes labels up to 6 inches wide and 59 inches long. The 6 inch wide model is popular among chemical producers who require a 6 inch wide label for use in drum labeling and barrel labeling, and especially for printing large GHS labels.
GHS Label Printing in Two Colors:
When the EC declared December 2010 as the date by which all chemical manufacturers must comply with GHS labeling regulations, it did not describe a way to put its regulations into effect. How can chemical processors who have dozens or hundreds of different products suddenly begin to make GHS chemical labels in two colors, with a unique black and red pictogram symbol, signal word, hazard statement, and precautionary statement for each chemical? The costs of printing these labels and storing them in inventory could be astronomical, and could easily result in labeling errors when operators retrieve stock to apply labels to each product.
That's why QuickLabel offers the Plexo Family, capable of printing GHS labels in two colors. The Plexo! printers allow you to print any label content on-the-fly, including barcodes and unique, variable information. QuickLabel also offers the Vivo! Touch for printing GHS labels in 4 colors through CMYK process color label printing.
Low Cost per Label with Ribbon Saver:
The Plexo! printers use standard thermal transfer labels and thermal transfer ribbons, allowing you to select the most cost-effective media for your label printing project. The Plexo! printers also have a hardware advantage: a built in ribbon saving mechanism. When you print labels in two colors, you may find that one color is used in a small area, as is the case with GHS labels, where the red portion of the GHS pictogram is in a small spot on the label. In that situation, your Plexo! will conserve the use of thermal transfer ribbon, saving you money.
Prices of the Plexo! Two Color Label Printer Family:
Please contact us for price information in your local area, since prices vary around the world
Plexo! 453:
- Two color label printer
- 4 inch print width
Plexo! 653:
- Two color label printer
- 6 inch print width
- Print Technology: Multi-color thermal transfer label printing
- Print Resolution: 300 dpi
- Print Speeds: 1, 1.5, 2, 3, 4, 5 ips (30-125mm/s). User selectable.
- Print Width:
- Print Length:
- On-board Processor: 266 MHz processor
- Memory and Storage: 64 MB RAM, 8 MB Flash Memory
- User Interface: Backlit LCD control panel
- Optional Accessories: Automatic Label Rewinder, Label & Tag Cutter-Stacker, Label & Tag Cutter, Large Roll Unwinder (to accommodate supply roll larger than 12" OD (300mm OD), QuickDispense™ Label Dispenser (for faster hand apply labeling)
Plexo! 453: 0.78" to 4.1" wide (20mm to 106mm)
Plexo! 653: 2" to 6.4" wide (50mm to 163mm)
Plexo! 453: 1.18" to 79" long (30 mm – 2000mm)
Plexo! 653: 1.18" to 59" long (30mm- 1500mm)
- Two built in USB 2.0 ports; Built-in 10/100Base-T Ethernet port with TCP/IP; available slot for Wireless LAN card; available slot for Compact Flash Type 1 Memory card
Barcodes, Graphics, and Fonts Supported:
- Fonts: Any TrueType® font can be downloaded; 5 bitmap and 3 vector fonts on-board
- Graphics: .BMP, .GIF, .JPG, .PCX, .PNG, .TIF
- Linear Barcodes: Code 39, Code 39 Full ASCII Indent and lead (extended Code 3 of 9), Code 93, Code 128 A, B, C, Interleaved 2 of 5, Codabar, EAN 8, EAN 13, EAN/UPC Appendix 2, EAN/UPC Appendix 5, FIM, RSS 14, GS1-128 (formerly known as UCC 128), HIBC, 13 JAN 8, Identcode (German Post Code), MSI, Plessey, Postnet, RSS 14, UPC A, UPC E, E0
- 2D Barcodes: Aztec, Codablock F, EAN-Datamatrix, Data Matrix, GS1 DataBar, IUID/UID, Micro PDF 417, PDF 417, QR-Code, RSS 14 truncated, limited, stacked and stacked omnidirectional, UPS Maxicode.
Label & Ink Supplies:
- Ink: Thermal transfer ribbon (ink): two rolls in any color(s)
- Ribbon Saver: built-in.
- Labels: Rollstock or fanfold paper and synthetic labels and tags
- Maximum Label Roll Outside Diameter: 12" OD (300mm OD) wide
- Dimensions:
- Weight:
- Environmental Conditions: 41◦F - -40◦ F (5◦ C- -40◦ C), 10-85% Relative Humidity
- Safety Standards: UL, CE, CCC, FCC Part 15 Class A, CB, ROHS-compliant
- Power Requirements: 100 - 240 VAC, 50/60 Hz
Plexo! 453: 15.5" H x 21.8" D x 9.8" W (395 mm H x 554 mm D x 248 mm W)
Plexo! 653: 15.5" H x 21.8" D x 14" W (395 mm H x 554 mm D x 358 mm W)
Plexo! 453: 48.5 lbs. (22 kg)
Plexo! 653: 53 lbs. (24 kg)
Labels and Thermal Transfer Ribbons for the Plexo! Family:
The Plexo! 453 & 653 offer some label printing advantages due to its unique thermal transfer printer technology: the ability to print long-lasting, durable labels and the ability print labels in the color white, and in special, dedicated colors including metallic gold and silver. This is possible because, instead of using inkjet ink or laser toner, the Plexo! 453 & 653 use color thermal transfer ribbons as printing ink. With the largest inventory of colored thermal transfer ribbons in the world, QuickLabel sells over 70 different formulations of thermal transfer ribbons in a rainbow of colors. Request a quote on thermal transfer ribbon.
Labels printed by the Plexo! 453 & 653 are finished, dry, and need no additional finishing. Users can printed directly on pre-die cut label materials, producing a range of label shapes and sizes. Request a quote on labels to supply a Plexo! Family label printers.
Compatibility with a wide range of thermal transfer labels and tag materials, including both pressure sensitive paper label materials and synthetic label materials that are not compatible with other printing technologies, is another advantage of color thermal transfer printing. Many rollstock packaging films are compatible with the Plexo! 453 & 653 label printers. If you'd like our Media Test Lab to evaluate your material for compatibility with the Plexo! 453 & 653, Contact a Media Sales Specialist.
Software & Accessories for the Plexo! Family:
One of the best features of Plexo! family is that the functionality of each printer is designed to be extended by accessories, allowing you to create a true tabletop in-house labeling center. An integrated label cutter and cutter-stacker can be connected via a USB port located on the front panel. Off-line accessories such as an external label rewinder can also be easily mated to the Plexo! printers.
QuickLabel offers a full line of accessory devices for use with the Plexo! two color label printers:
Labeling Software (Optional):
NiceLabel® is a professional labeling software that offers a modular solution that fits into your existing system seamlessly for bar code and RFID label design and printing.
External Label Rewinder:
Compact device automatically winds labels onto a roll as they exit the printer. Finished rolls are ready to be loaded into an automatic label applicator, label dispenser, or stored for later use.
Label & Tag Cutter:
This attachment integrates the cutting of pressure-sensitive labels and non-adhesive tags with the Plexo! printers so that users can print and accurately cut tags in a single process. Labels can be cut individually, or a strip of labels can be cut at the end of a batch. Available with attachable tray to collect cut labels and tags. Minimum cut length: 0.47" (12 mm). Maximum label/tag thickness: 12 Mil (0.012", 0.30 mm) on tag stock.
Label & Tag Cutter-Stacker:
This integrated accessory attaches to the front of the Plexo! 453 two color label printer, so that labels are cut and stacked as they are printed. Speed is up to 120 cuts per minute. Printing and cutting will stop automatically when the maximum height of the stack is reached (5.5" high, 140mm). Printer front panel will display Cutter-Stacker status messages, including maximum stack height and label jam error. To initiate cutting, select "Cutter" from the Plexo! Windows® driver.
QuickDispense™ Label Dispenser:
Using a roll of labels, the QuickDispense
automatically dispenses one label at a time, reducing time and effort in hand-applying labels. Typically used after labels have been printed and wound onto a core by a label rewinder. Accepts label sizes between 0.5" – 6.55" wide and between 0.25" – 8" long (13mm – 156mm wide x 6mm – 203mm long).
Compact Flash Card:
2 Gb card stores label files, images, and fonts where they can be rapidly retrieved during printing, reducing processing time for the host computer. In addition, the Compact Flash card can store custom software programs written to interpret data coming from another system into a language the Plexo! can print.


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Prompt sales by adding the BuyNow button to your mailings.
More in this category: Visual Website Optimizer Google AdWords Facebook Pixel MonkeyData Survicate OptiMonk Integration Privy Amazon Payments Integration Google Checkout Integration
Author: PayPal integrator
Latest update: October 12, 2011
PayPal Integration
Integrate your GetResponse account with your PayPal services account and maximize your conversion rate.
Extend the reach of your newsletters and start building new relationships right away by turning your PayPal customers into email subscribers the moment they complete a sale.
Add a GetResponse notification URL into the Instant Payment Notifications settings. Then create a BUY NOW button and add your campaign as a custom value in the third step during the button creation. And that's it! Now, whenever the generated button or URL is clicked it will launch the PayPal transaction and in the same time add the contact into your GetResponse campaign, significantly minimizing the time and resources necessary for a subscriber to convert.
For more information, see How do I add contacts after they use a PayPal "Buy now" button on my website?

subset: commoncrawl


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|Home Decor
|Kitchen Accessory
Currently a stay at home mom to two little ones under 2. We also share our house with a dog and a cat.View all patterns by designer (40) »
My toddler likes to pull things, get into things, put things where they don't belong, etc. You moms of toddlers know what I'm talking about! I didn't want to keep picking the kitchen towels up from the floor, so I came up with this quick and easy pattern. My toddler has yet to pull the towels down!!
Includes patterns for two types of towel holders.


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on January 15, 2014 - 12:01 AM
State Comptroller Thomas P. DiNapoli's positive report on the City of Buffalo's finances results from the combination of good work by the mayor, oversight by the control board and a healthy dose of state aid.
The Brown administration gets credit for returning the city from the fiscal wilderness. Byron W. Brown moved in to City Hall just as the state control board took over while the city was near its constitutional taxing limit.
Helped by a control board that froze wages, Brown and his staff were able to institute their own progressive fiscal policies. The result is that Buffalo has fared better than other comparable cities, and has weathered the worst recession since the Great Depression.
The city's credit ratings from all three rating agencies – Standard & Poor's, Fitch and Moody's – reached high levels. At the same time, Buffalo is benefiting from more than $2.2 billion in construction, including the $172 million privately financed HarborCenter and the $375 million University at Buffalo School of Medicine. Over the past decade, Buffalo's revenues have grown 20 percent to $447 million in 2012.
As DiNapoli said, "Buffalo's finances are trending in the right direction." The comptroller praised the mayor and control board, which under Brown's administration is down to advisory status, but not all is rosy. DiNapoli raised a number of red flags, cautionary notes that echo concerns raised by the control board.
While the Brown administration was able to negotiate a new firefighter contract, it still has not reached an agreement with the police union. That fiscal uncertainty could bring new pressure to the city budget.
Brown's administration remains weighed down by a dependent city school district that is facing a $35 million budget gap this year. Even though the district gets part of its funding from the city, it unfortunately was not part of the comptroller's report.
DiNapoli also noted the demographic challenges facing one of the poorest big cities in the nation. Property values grew at less than half the rate of other cities in the state between 2002 and 2013, the population continues to decline, the city has relatively low housing values and childhood poverty is pervasive.
Improving education to better prepare residents for jobs should improve the city's outlook. Brown can play a role in helping to create better schools, and lately he seems to be embracing the opportunity.
The city has risen far since the depths of its fiscal crisis. While there's still a long way to go, the progress so far is worth celebrating.


text:
World Snooker Championship: Barry Hawkins thumps the table after victory
- From the section Snooker
Watch Barry Hawkins thump the table in celebration after securing his semi-final place with a dramatic win over Dominic Dale at the World Snooker Championship.
Hawkins could not hide his emotions after making the decisive pot in the final frame, having withstood a spirited fightback from Dale.
Dale won seven frames in a row from 11-5 down, but Hawkins claimed the final two frames to clinch victory.
Available to UK users only.
World Snooker Championship
- Venue:
- The Crucible, Sheffield
- Date:
- 19 April - 5 May
- Coverage:
- Watch live on BBC TV, Red Button, online, connected TVs, mobiles and tablets. Updates on BBC Radio 5 live


text:
Preview of KDE 4.0
The first KDE 4 beta release gives users a sneak preview of the many innovations the next K Desktop generation will introduce.
Right from the start of development, the KDE project raised expectations – the developers' blogs boasted that the desktop would be more attractive and work differently and the new release would also be much faster than its predecessor. Just a few months before the release, and shortly after the first beta version became available, we decided to find out how the KDE project is faring.
Buy this article as PDF
(incl. VAT)
Buy Linux Magazine
Visit Our Shop
Direct Download
Read full article as PDF:KDE_4.0_Review.pdf (512.60 kB)
Mirantis to Refactor OpenStack Fuel for Kubernetes
The company is collaborating with Google and Intel to use Kubernetes as an engine for Fuel
Canonical Joins Document Foundation Advisory Board
The upcoming release of LibreOffice will be available as a snap package
SUSE Joins the Microsoft Test Drive Program
Customers can take a free test drive of SLES for HPC on the Azure Cloud
SiFive Launches a Line of Open Source Chips
San Francisco-based chip company announces their first fully open source chip platform.
Fedora 24 Released
The whole distro gets rebuilt on glibc 2.3
Canonical Announces Cross-Distro Support for Snaps
Ubuntu Vendor tries to solve app packaging and distribution problem across distributions.
ownCloud Forked
Founder of ownCloud launches the Nextcloud project.
HP Enterprise Unveils a Futuristic Computer
Will The Machine change the way future programmers think about memory?
CoreOS Announces Distributed Storage System
The new Torus distributed storage system is available under an open source license on GitHub
Google Beats Oracle in the Android Lawsuit
Juries decides Google's use of Java APIs Was Fair Use


text:
Based on a 2,000 calorie dietSee full nutrition
Ready In
I was skeptical, but these were very, very good!! I'm not sure what asian chili paste is, and couldn't find it at my grocery store, so I subbed for sriracha sauce. Very spicy, but soo good!! I ...
the sauce is very tasty.. i had to sub 1/8 tsp ground ginger for the fresh and i added about 1/4-1/2 tsp sesame oil for extra pop.. im sure this would make a great marinade.. ty for the recipe


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Florida Cracker houses, often referred to as "vernacular" home design, have been around for a long time and are probably still the most energy efficient homes for our area.
South Anastasia Communities Association will hear from University of Florida professor of architecture Ron Haase at its March 2 meeting. The meeting is open to the public and will begin after a brief association business meeting starting at 7 p.m. at Marineland's Whitney Lab.
Haase will discuss "Cracker Houses of Florida -- Living with the Environment." His talk will focus on Florida's wood-frame homes that are designed to fit with the climate, culture and rural lifestyles of Florida's small towns. Haase is author of a book, "Classic Cracker, Florida's Wood-Frame Vernacular Architecture." He won first place in an energy efficient Cracker home design contest sponsored by Florida Solar Energy Center and Florida's State Energy Office.For information on the community association and the program, call Evelyn Kahrs at 471-6735.
Indians of the Estuary Kayak Tours will be conducted between Feb. 27 and March 27 from Washington Oaks Gardens State Park. Kayaking experience is not required. Basic paddling instruction will be given before the tour proceeds to explore the Matanzas Estuary. The naturalist-led tour will include stories about the Timucuan indians and the important role the estuary played in their everyday lives. Cost for the two-hour program is $40 per person. The fee includes kayaking equipment, instruction, park entrance fee and bottled water. Reservations are recommended. Call Coastal Outdoor Center at (904) 471-4144 for tour departure dates and times, or go to Web site at www.coastaloutdoorcenter.com .Washington Oaks Gardens State Park is located at 6400 N. Oceanshore Blvd., off State Road A1A South near Palm Coast. The park phone number is (386) 446-6783.
The 11th Annual Arts and Crafts Show will take place at the St. Anastasia Catholic Church community center from 9 a.m. to 3 p.m. March 5. Some 65 artisans will be on hand with fine art, pottery, woodcraft, jewelry and more. There will also be a bake sale. Hot dogs and beverages will be available for purchase.
Proceeds of the sale will benefit the church's outreach program to the Missionaries of the Poor's Haitian site.
St. Anastasia Catholic Church is located at 5205 S.R. A1A S. on Anastasia Island.
Local artists and crafts persons who are interested in reserving a table may call Terry McKay at 471-3948
The faculty and staff of R.B. Hunt Elementary School have selected Judy Johnson as the school's Teacher of the Year for the 2005-06 school year. Hunt school is located on Anastasia Boulevard.
Johnson, a second grade teacher since 1987, has been instrumental in developing the school's reading resource room and has provided many literacy initiatives for the school. Johnson will be recognized at the St. Johns County Education Foundation's Teacher of the Year Horizon Banquet on March 22.
Got news? If it takes place on Anastasia Island or the adjoining areas you can send your information about people and events to email@example.com, or call 471-4851.
Read more Neighbors: Beaches online
© 2016. All Rights Reserved. Contact Us


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looking through our emergency bags during our practice drill
nurse Cassidy pretending to care for our "sick" boys
my 3-course-meal-in-1-hour challenge... I need a fancier veggie platter :p
he's a cutie!
at Daddy's feet
Galen has perpetual bruises on his head...
Galen hanging out with Daddy
forgot about the hamburgers...oops
well, we can get him to eat the tags...
Look at those eyes! And that hair!
I know I'm cute
Look ma, it's Windows 7!
are we done yet?


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- WordPress: Current
- NextGEN: Current
Currently running this all via wamp server not yet got a host.
I've been working on my site for a while now I'm using nextgen. Each image has and extra field added using the Nextgen custom fields plug-in. In this field it contains the following code/trigger text (Currently Test code) below which is used in conjunction with WP Ultra simple Paypal Cart.
wp_cart:Sunrise over Pen Ponds :price:[SIZE|6x4 - £10,10|7x5 - £20,20|8x6 - £30,30|9x5 - £40,40|12x8 - £50,50]:end]
When you click on an image from gallery it loads up in imagebrowser page Title at the top followed by the image then navigation description and then extra field. Via this code added to imagebrowser.php

<?php echo $image->ngg_custom_fields["Product-Item"]; ?>

This works absolutely fine until I enable shutter ajax now the page is not reloaded each image which is much smoother but I have a new problem.
Now on the very first time imagebrowser page opens it's fine as soon as you move forward or back. The code reverts to what's shown above rather than being a drop-down box and a purchase button.
Does anyone have any ideas how I get this to be interpreted properly as it would without the ajax being enable or a way I can stop the page being refreshed with out the need to enable the ajax.


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I don't know when it started lurking around my blog but now I see a category named "Plastic Surgery" (which I have not defined) in the Write > Post page. I checked on Manage > Categories and indeed it was not there.
How could this be possible? Is it spam or something like a side effect of a hack attempt?


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Despite protestations that it was "unfilmable," the upcoming movie "Cloud Atlas" has answered with a first look at the film, in the form of a jaw-dropping six minute trailer.
Adapted by sci-fi vets Andy and Lana Wachowski ("The Matrix"), along with Tom Tykwer ("Run Lola Run"), the movie is based on David Mitchell's ambitious, century-spanning novel.
Here, the cast — Tom Hanks (at times wearing a rather unfortunate blond wig), Halle Berry, Hugh Grant, Jim Sturgess, and Susan Sarandon — all do double duty (hexa-duty?) as they play out six story lines, which span time and space.
The film delves into the impressions that one leaves on future generations. Or, more poetically, as the trailer explains: "We cross and re-cross our old tracks like figure skaters."
For any disbelievers out there, take a look at the stunner above.
"Cloud Atlas" hits theaters October 26.
You can check out the trailer over on Apple.
[via Apple]
Earlier on Moviefone:
« Best Dance Movies: The Evolution Of The Genre, From 'Dirty Dancing' To 'Step Up' Guardians of the Galaxy Team Up with The Avengers in Fan-Made Banner »


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text:
From Booklist
"Zippy! . . . These tales are about plot and economy and speed [and] make great bedtime read-alouds for children who can handle a little gore. . . . The original tales weren't for children, of course; they were for everyone. So is this book. . . . Pullman is both erudite and funny. . . . He has fun with dialogue, and is particularly snappy with dwarfs. . . . You know what? He crushes it, as the youth of today are wont to say. . . . His translations are perfection." —The New York Times Book Review
"It is such a pleasure to read these tales again, to experience their strangeness and richness, their violence and beauty, their sheer nonsense. . . . Reading Pullman's version it is impossible not to hear Pullman's own gentle voice; he is present on every page. . . . [His] interventions work brilliantly." —The Boston Globe
"These wonder tales . . . compel belief as powerfully as any adventure or thriller." —Marina Warner, The Times Literary Supplement
"Excellent . . . His beginnings are like invitations that cannot be refused. . . . Pullman shows how completely he understands the Grimms. . . . [He] pays homage to the Brothers' pioneer work and simultaneously breathes new life into a great, venerable tradition of magical storytelling." —Jack Zipes, Los Angeles Review of Books
"A real pleasure to read . . . This is the kind of writing that stands up to years of bedtime repetition. . . . The author's best appearances are in the notes, which are often as entertaining as the stories themselves. . . . Swiftness and clarity, he says, were his guiding principles; to which he has added wit and invention. . . . Beautiful or grotesque, the mad poetry of these tales is often delightfully funny too." —The Economist
"A fresh, sparkling collection of the finest stories from the Brothers Grimm, hand-picked by an author perfectly suited to the task. This volume is a must-have for any lover of fairy tales. . . . In the hands of a master storyteller such as Pullman, the Grimms's tales take on a whole new life." —Library Journal (starred review)
"A wonderfully rich reading experience . . . Stylish in its simplicity [and with] a salutary clarity and directness . . . In addition to his elegant introduction, [Pullman] concludes each tale with his own always interesting commentary. . . . There are, of course, any number of English-language versions and editions of Grimm, but few are as felicitous in their telling as Pullman's. His book surely belongs on the same shelf as the very best of those that appeal to general readers of all ages." —Booklist (starred review)
"Readers will enjoy not only returning to European fantasy's roots but seeing how the tree still blooms. . . . Smooth narration makes every tale accessible while keeping the mystical and lyrical qualities that make fairy tales so beloved." —Publishers Weekly (starred review)
"You didn't know you needed to reread Grimm. You do. This is a grand and a great book. With confidence and modesty alike, Pullman adds just enough Pullman to remind us that the oldest stories are always best told by someone who knows how to do the job of storytelling. No grandstanding here, no posturing or poesy-making. Pullman selects familiars and exotics, and gives us the goods anew—the ashes never grittier, the golden shoes never more lively, and the teller's notes concise, witty, scholarly even. Older Grimms—put them on the top of the bookcase. This one needs to be closer to hand. I read it ravenously, rapturously."—Gregory Maguire, author of Wicked and Making Mischief: A Maurice Sendak Appreciation
"I could not imagine a better emissary for the Brothers Grimm than Philip Pullman. His translations have the timeless quality of a voice speaking in a quiet room, at once ancient and immediate to the senses. What a pleasure it is to be reacquainted with these stories in all their swiftness, wonder, horror, and charm." —Kevin Brockmeier, author of The Brief History of the Dead
"Philip Pullman's Grimm is quite eloquent, and his commentary is witty and historically accurate. There is no doubt in my mind that the Grimms would have been delighted with what he has accomplished." —Jack Zipes, Professor Emeritus, University of Minnesota
"In this pitch-perfect retelling of the Grimms' fairy tales, Philip Pullman reminds us that the stories have lost none of their relevance or racing energy, even two hundred years after they were written down. As storyteller and sage, he preserves the flavors and aromas of fine, old wines from times past and delivers them to us in sparkling new bottles." —Maria Tatar, Harvard University; author of The Classic Fairy Tales
"I've admired Philip Pullman since his early fantasy Galatea on through the splendid trilogy His Dark Materials. All of his gifts, including his prose eloquence, and his endless high Romantic imagination, are manifested in this marvelous retelling of Grimm." —Harold Bloom


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Mildred Ziegeldorf, 79, of Carrington, N.D., died Thursday, May 16, 2013 at Carrington Health, Carrington.
Her memorial service will be held on Tuesday, May 21, 2013, at 2 p.m. at Grace Lutheran Church, Carrington. Visitation will be held on Tuesday from 9 a.m. until 12 Noon at Evans Funeral Home, Carrington. Burial will be at Carrington Cemetery.
Evans Funeral Home, Carrington, is in charge of arrangements.
Location, ST | website.com


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Cammy's Comic Corner – Geeky Talky – Episode 106
Posted on January 11th, 2012 by Cameron
In this episode I'm joined by Haley Manbat as we discuss Hunter S. Thompson in cinema. We cover Where The Buffalo Roam, Fear And Loathing In Las Vegas, and The Rum Diary. Spoilers!
Filed under: Cammy's Comic Corner
Filed under: Cammy's Comic Corner
Tags: Aaron Eckhart, benicio del toro, bill murray, fear and loathing in las vegas, hunter s thompson, Johnny Depp, Peter Boyle, Terry Gilliam, the rum diary, where the buffalo roam


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Two million people are using Slack to chat, send emojis and encourage creativity at work. Nasa and Harvard are on board – will your company be next?
British mapping agency Ordnance Survey creates easy-to-read map using Nasa open data, which could be used while walking on the surface of Mars
Google Photos labelled a picture of two black people as 'gorillas' while Google Maps and Flickr have also suffered from race-related problems
Auto-tagging system slaps 'animal' and 'ape' labels on images of black people, and tags concentration camps with 'jungle gym' and 'sport'
The chat app for business has a new, sky-high valuation. But is it really that groundbreaking, and why is Silicon Valley so excited? By Alex Hern
Ruby Tandoh, Flickr, Angry Birds Transformers, Best Fiends, Inbox by Gmail, Toca Boo, Skylanders and more. By Stuart Dredge
Know your lolz from your lulzsec, and your belfies from your selfies? Hannah Jane Parkinson is here to help with an almost definitive list of digital geekery
Barbara Speed: Llamas and coffee cups also get you clicks, say researchers, who analysed pictures to see what made them popular on Flickr
Privacy flaw on photo sharing website initially dismissed as 'working as designed' and not making sensitive data available. By Samuel Gibbs
Here's our roundup of great urban content we've seen online lately – featuring public art, a diaper store and two years' worth of underground trips
City 'selfies', overcrowding misconceptions and how weather affects New Yorkers' generosity feature among the best city content we've seen online this week
Following a spate of colourful winter sunsets across Britain, Jonny Weeks explores our fascination with the photographs some people love – and others love to hate
Jonathan Jones: The British Library has made a million of its images available online – but not its masterpieces. Isn't it time to go where Nasa has boldly gone and make all pictures free?
Editors call for boycott of officially released mages
In a world where we can 'curate' our Whole Foods basket, asks Daniel Blight, what cultural currency does curation now hold?
Roy Greenslade presents a fifth extract from the updated second edition of the book What do we mean by local? It's by Marc Astley
From the whiff of your first pet to the smell of that seaside holiday, the Madeleine captures the scent of your memories
Yahoo hopes its rebooted photography service is 'not only beautiful, but bigger and better than anything else'. By Stuart Dredge
Messages apparently from Anonymous activists appear on North Korean social media accounts
Companies discussed how information was shared online during the storm at a Social Media Week event in New York
Instagram announces an unpopular change to its terms of service less than a week after Flickr relaunches its i0S app
As Bruce Willis considers a legal bid to bequeath his iTunes library, we look at who actually owns your digital content – from music and books to film – and what your rights are
We say goodbye for this year to the Edinburgh festival with a selection of our favourite reader photographs from the last week
Dan Gillmor: Its new CEO inherits a big challenge to turn around the sleeping tech giant, but Yahoo still has major assets to build on
Hannah Waldram: As we reach the halfway mark of 2012, we take a look at the 52 weeks Flickr photo project to document the year in pictures
Images from the weekend celebrating the Queen's 60-year reign
Roy Greenslade presents, after a short break, a further extract from What do we mean by local?. It's taken from chapter by David Baines
Instagram gives Facebook the hottest app in mobile, now the cutting edge of tech
Datablog Open data journalism at the Guardian: who's who on the Datastore and Datablog, and how you can get involved
Open journalism at the Guardian means open data journalism. Every day, on our Datablog and Datastore, we publish the raw data behind the news for you to explore, visualise and debate. Find out who puts the Datablog together - and how you can take part


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When Donald Trump is president there's going to be hell toupee.
Type: Posts; User: Keef Riffers
When Donald Trump is president there's going to be hell toupee.
I don't mean to be an alarmist, but if this guy gets elected we are fucked
Well there's this
Awesome. Thank you
Hey AC. I had some friends a few years ago that lived on the ridge where Red lives and I remember the Aslan Foundation bought that property. What is the Aslan Foundation? At the time none of the...
A Republican controlled state government?
What say you Amercia?
Is Obama Ra's Al Ghul?
Missing? I said I was going to Muncie for a while. No wonder youns couldn't find me I was done acrost the plattoo.
And damnit Earl I told you not to show Crow where them "Eleusian strawberries"...
I lost your number. Got my tele tuned up at the nashville guitar center and when I told them I was from Knoxville they asked me ifn I knew you. Mark valentine maybe? Anyways he as a nice guy and I...
Who cares about the happiness of the body politic at a moment like that? What about the implications for the Constitution? This would be unprecedented I think. The constitution becomes invalid...
They will grow bored of balancing the Rothchild ledger pretty quick I think.
From there they will fall in love with the essence of Alan Watts, extract him from his audio lecture archives and...
Facebleck participates in DARPA/DoD "narrative analysis" ""experiment""
best "my opponent is not a real person" ever
I contacted the NSA to see if we can get a copy but have not yet heard back. Will keep y'all posted.
You gonna eat your jello?
Gah people. Just take the Internet to work
Page 3 and finally somebody have something intelligent to say.
Also contact Eric at tone revival
Those monoliths are GINORMICAL
Expect some awesome commentary from the rune soup guy pretty soon.
Didn't the Westbrook baptist guy die?
Just now read this thread. Damn. What an amazing guy! I always had so much fun whenever I ran into him. A shining example of how to live and die.
like nobody saw THAT coming...
Awesome! Good to hear your doing better.
Alright! We can finally get the blab going again!
That's sayIng something. If Rush will go to bat for Africa's "Lords Resistance Army" but not Christie then Christie must have a graveyard closet we are still unaware of.
Besides, Putin is an IMF/UN Trojan horse.


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User/Comment Login
OAKMONT, Pa. — Oakmont Country Club's first family of caddying spans three generations, boasts nearly 100 years of combined experience and has only so much sympathy for the players trying to tame their notoriously tough home course at the U.S. Open.
"Personally, I like seeing them suffer," Bob Bugna chuckled softly, taking a break Saturday afternoon from watching golf to run some errands. "The fairways and greens are pretty much the same as what the members run into, but man, that rough is something."
CHICAGO — Manny Ramirez's latest gig with the Cubs is proof that if you hang around the game long enough, you'll see it all.
Among the greatest right-handed hitters of his generation and definitely one of the least coachable, he's back in baseball as a coach. Exactly what kind of coach, well, no one in the organization seems to be certain.
LAKE FOREST, Ill. — Jason Day has plenty of rivals at the moment, but very few peers.
Day's runaway victory Sunday in the BMW Championship vaulted him to No. 1 in the world ranking, backing up a boast the then little-known Australian made during a conference call with reporters in 2007 — and for which he was roasted on more than one occasion since.
"I remember sitting on my mom's bed and thinking that (predicting he'd be No. 1 someday) might not go over too well," laughed Day, who turns 28 next month.
It didn't.
LAKE FOREST, Ill. — Instead of running away from the field one more time, Jason Day spent most of the third round at the BMW Championship running in place.
He was hardly the only player struggling to cope with the suddenly cool, wet and windy conditions at Conway Farms left behind by a wave of overnight storms. After opening rounds of 61 and 63, the Aussie made his first bogey in 20 holes at No. 6, then piled on three more to offset a half-dozen birdies Saturday en route to a 2-under 69.
"Obviously, the most difficult round that we've had this week by far," Day said.
LAKE FOREST, Ill. — As Jason Day's long eagle putt at the last hole rolled down the hill and toward the hole, every revolution of the ball only added to the sense of inevitability.
When it disappeared into the cup for an 8-under 63, a PGA Tour record-tying 36-hole score of 18 under and a five-shot lead at the BMW Championship, Day pumped his fist as playing partners Jordan Spieth and Rickie Fowler broke into widening grins and then applause.
Their expressions said it all about golf's man of the moment: Day is making everything that matters.
SHEBOYGAN, Wis. — The family business, at least on the athletic side of the Finau family, was pro football or basketball.
Tony Finau, all 6-foot-4 and 200 pounds of him, was determined to play golf.
So if nothing else, his play Saturday was further validation of a story that begins with two poor kids learning to play the game by banging balls against a mattress in their garage, and one of them winding up with a shot at a major title on the final day of the PGA Championship.
CHICAGO — Don't bother asking whether this is the year.
Theo Epstein's answer to the question that's haunted Cubs fans for a century-and-counting hasn't changed. It's the same one he gave four years ago, when Epstein was introduced as the franchise's latest savior to great fanfare and pleaded for patience: He has no idea. Neither does anyone else.
When the club broke training camp back in spring, 2016 seemed like a reasonable guess, or maybe '17. But if Epstein's suddenly fast-filling schedule is any indication, this just might be the one.
CHICAGO — The owner made a beeline through the Cubs' clubhouse looking for his rookie slugger.
"Hey, take it easy," Tom Ricketts told Kris Bryant, trying not to laugh. "That thing costs a lot of money."
"Gotcha," Bryant smiled, trying not to blush.
"That thing" is a brand-new, 4,000-square-foot Jumbotron atop the bleachers and the ivy-covered outfield wall in left, an expensive piece of furniture in Wrigley Field's $500 million makeover.
CHICAGO — The NFL draft is back in the City of Big Shoulder Pads for a seventh time, expecting a much warmer reception than it got nearly 80 years ago from the very first pick in its very first draft.
The man who stiff-armed the NFL back then at the draft in Philadelphia was University of Chicago running back and Heisman Trophy winner Jay Berwanger.
INDIANAPOLIS — Prospective NFL draftees don't flinch at benching 225 pounds until exhaustion or trying to beat their personal best at the 40-yard dash. They do dread one sentence: "The doctor will see you now."
New England at Green Bay features the two best teams with the most potent offenses in the NFL at the moment.
It's being billed in some quarters as a Super Bowl preview and, if so, watch how Pats coach Bill Belichick and Packers counterpart Mike McCarthy react to each other's moves. If nothing else, this meeting figures to be an important test of their wills.
St. Augustine Record ©2016. All Rights Reserved.


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Rosacea Center - Enid, OK
- WebMD Physician Directory of Enid Dermatologist Doctors
- Related Rosacea Articles
- Enid Emergency Contacts
- Nearby Enid Hospitals
- Find a Local Pharmacy on RxList - Enid
Enid Dermatologist Doctors for Rosacea
Type of Physician: Dermatologist
What is a Dermatologist? A certification by the Board of Dermatology; practitioners treat pediatric and adult patients with disorders of the skin, mouth, hair and nails as well as a number of sexually transmitted diseases. They also have expertise in the care of normal skin, the prevention of skin diseases and cancers, and in the management of cosmetic disorders of the skin such as hair loss and scars.
Specialty: Dermatology
Common Name: Skin Doctor
Dermatologist Doctors in Enid * results
- 2
David L. Graham
Silverleaf Dermatology Center
609 S Kelly Ave Ste E2
Edmond, OK 73003
(405) 216-0100
- 38 years experience
- 2 office location(s)
- 3
Sara K. Metcalf
Metcalf Dermatology
1329 S Sangre Rd
Stillwater, OK 74074
(405) 372-7575
- 11 years experience
- 1 office location(s)
- 4
Kenda G. Baker
Metcalf Dermatology
1329 S Sangre Rd
Stillwater, OK 74074
(405) 372-7575
- 1 office location(s)
- 5
Robert A. Breedlove
Stillwater Skin & Cancer Medical Clinic
1604 W 8th Ave
Stillwater, OK 74074
(405) 624-1077
- 1 office location(s)
- 6
Thomas C. Hall
Stillwater Dermatology Clinic
1329 S Sangre Rd
Stillwater, OK 74074
(405) 533-3376
- 1 office location(s)
Don't see your city? Use the WebMD Physician Directory to expand your search.
Rosacea facts
- Rosacea is a common, chronic, incurable, adult acne-like skin condition that is easily controllable and medically manageable.
- Rosacea commonly affects the central third of the face, especially the nose, and has...
Other Related Rosacea Articles
Emergency Contact for Enid
- In case of Emergency, call 911
Nearby Enid Hospitals results
- 1Integris Bass Health Pavilion
401 S 3rd St
Enid, Oklahoma 73701
(580) 233-230
- 2St Mary's Regional Medical Ctr
305 S 5th St
Enid, Oklahoma 73701
(580) 233-610
- 3Integris Bass Baptist Hlth Ctr
600 S Monroe St
Enid, Oklahoma 73701
(580) 233-230
- 4Integris Behavioral Health
2216 S Van Buren St
Enid, Oklahoma 73703
(580) 233-230
- 5Okeene Municipal Hospital
207 E F St
Enid, Oklahoma 73763
(580) 822-441
- 6Perry Memorial Hospital
501 N 14th St
Enid, Oklahoma 73077
(580) 336-354
- 7Fairview Regional Medical Ctr
523 E State Rd
Enid, Oklahoma 73737
(580) 227-372
* Provider Directory Terms of Use:
The WebMD 'Provider Directory' is provided by WebMD for use by the general public as a quick reference of information about Providers. The Provider Directory is not intended as a tool for verifying the credentials, qualifications, or abilities of any Provider contained therein. Inclusion in the Provider Directory does not imply recommendation or endorsement nor does omission in the Provider Directory imply WebMD disapproval.
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By using the WebMD Provider Directory, you agree to these Terms and Conditions.


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This plugin only notifies me of my published posts, not of the published posts of my authors. Is that how its supposed to work? I want to know when an author has published a post.
I want to be the only one getting notification so the others are excluded in the post notification settings. Since the other authors are checked-off, is that also excluding me from getting a notification when they publish?


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Harmans Water
|Harmans Water
St Paul's Church
Harmans Water shown within Berkshire
|OS grid reference
|Metropolitan borough
|Bracknell Forest
|Metropolitan county
|South East
|Sovereign state
|United Kingdom
|Post town
|Postcode district
|Dialling code
|Thames Valley
|Royal Berkshire
|South Central
|EU Parliament
|South East England
|UK Parliament
Coordinates: 51°24′13″N 0°44′20″W / 51.403473°N 0.738849°W
Harmans Water is a suburb of Bracknell in Berkshire, England. It takes its name from Harman's Water Lake, long gone.[1] Building of the estate began around 1960 and was the fourth and last estate to be built as part of the original plan for the new town.
The estate lies approximately 1 mile (1.6 km) south-east of the town centre, to the east of the A322 road and south of the A329 road. It is in Harmans Water ward, which following boundary changes now includes parts of Bullbrook, Martins Heron and The Parks.[2]
Facilities include a shopping centre, a library, several public houses and Harmans Water Primary School [1]. St. Pauls Church has shared Church of England and United Reformed Church services and is situated adjacent to the shopping centre. There are a few office buildings in Broad Lane but otherwise the estate is largely residential.
The Parks[edit]
The Parks is a recent development and is on the site of the former RAF Staff College which closed in 2004. The first phase of the development, of 220 homes was developed by English Partnerships and completed in 2008.[3] As of 2012 development continues.
Ramslade House, the former headquarters building of the staff college, was originally planned to become a community centre but its future currently remains uncertain.[4]
- ^ "History of the Hamlets of Winkfield, Berkshire". David Nash Ford. Retrieved 28 September 2012.
- ^ "Ward Map for Harmans Water" (PDF). Bracknell Forest Council. Retrieved 28 September 2012.
- ^ "The Parks, Bracknell". English Heritage. Retrieved 4 October 2012.
- ^ "Uncertain future for Historic Ramslade House". Bracknell Forest Standard. Retrieved 4 October 2012.


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Vibrio parahaemolyticus is a foodborne pathogen that has become a public health concern at the global scale. The epidemiological significance of V. parahaemolyticus infections in Latin America received little attention until the winter of 1997 when cases related to the pandemic clone were detected in the region, changing the epidemic dynamics of this pathogen in Peru. With the aim to assess the impact of the arrival of the pandemic clone on local populations of pathogenic V. parahaemolyticus in Peru, we investigated the population genetics and genomic variation in a complete collection of non-pandemic strains recovered from clinical sources in Peru during the pre- and post-emergence periods of the pandemic clone. A total of 56 clinical strains isolated in Peru during the period 1994 to 2007, 13 strains from Chile and 20 strains from Asia were characterized by Multilocus Sequence Typing (MLST) and checked for the presence of Variable Genomic Regions (VGRs). The emergence of O3:K6 cases in Peru implied a drastic disruption of the seasonal dynamics of infections and a shift in the serotype dominance of pathogenic V. parahaemolyticus. After the arrival of the pandemic clone, a great diversity of serovars not previously reported was detected in the country, which supports the introduction of additional populations cohabitating with the pandemic group. Moreover, the presence of genomic regions characteristic of the pandemic clone in other non-pandemic strains may represent early evidence of genetic transfer from the introduced population to the local communities. Finally, the results of this study stress the importance of population admixture, horizontal genetic transfer and homologous recombination as major events shaping the structure and diversity of pathogenic V. parahaemolyticus.
Author Summary
Infections caused by Vibrio parahaemolyticus have increased significantly over the last two decades, with cases now regularly reported globally. The emergence of cholera at global scale has brought the attention toward other Vibrio diseases in developing countries. This was the situation in Peru, where the investigation of V. cholerae in hospitals and regional public health laboratories after the dramatic emergence of cholera epidemic in 1991 enabled the identification of other pathogenic Vibrio throughout the whole country. The submission of all these bacteria to the Instituto Nacional de Salud (INS, Lima, Peru) for characterization generated an extraordinary repository of records and isolates which have been decisive for sizing the impact of V. parahaemolyticus infections on the population. The present study addresses, for first time, the impact of the arrival of a non-endemic population of V. parahaemolyticus on the genetic structure and virulence attributes of local populations. The detection of the pandemic clone of V. parahaemolyticus to Peru in 1997 changed not only the epidemic dynamics of this pathogen, but also the population structure and genetic variation of native populations through population admixture, horizontal genetic transfer and homologous recombination between native and introduced populations of pathogenic V. parahaemolyticus.
Citation: Gavilan RG, Zamudio ML, Martinez-Urtaza J (2013) Molecular Epidemiology and Genetic Variation of Pathogenic Vibrio parahaemolyticus in Peru. PLoS Negl Trop Dis 7(5): e2210. doi:10.1371/journal.pntd.0002210
Editor: Jessica N. Ricaldi, Universidad Peruana Cayetano Heredia, Peru
Received: November 6, 2012; Accepted: March 29, 2013; Published: May 16, 2013
Copyright: © 2013 Gavilan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was partially supported by project 2007/CP381 from the Department for International Cooperation of the Regional Government of Galicia, Spain. This project finished in 2008. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that naturally inhabits marine and estuarine environments throughout the world. While many strains of V. parahaemolyticus are strictly environmental, some groups are pathogenic and may cause gastroenteritis in human [1]. V. parahaemolyticus is the leading human pathogen of bacterial food-borne diseases associated with the consumption of raw or undercooked seafood [2].
Recently, V. parahaemolyticus has gained notoriety due to global dissemination of infections [3]. The rise of infections was initially linked to the emergence of gastroenteritis throughout Asia associated with a single clone of the O3:K6 serovar in 1996 [4]. The subsequent detection of this clone causing infections in Peru [5], [6], and Chile [7] in 1997 indicated the pandemic expansion of O3:K6 clone infections. Afterwards, gastroenteritis cases associated with the O3:K6 clone were reported in many other countries and different areas of the world, such as the United States, Russia, Mozambique, Mexico, and Spain [3]. In addition to rapid dissemination of the pandemic clone, a parallel rise of human cases associated with other genetic groups has been reported in recent years. Specific genetic groups have been described as being associated with infections in particular geographic regions of the world. Consequently, the presence of local clones and serovars prevail among clinical cases, such as O4:K12 in the Pacific coast area of the United States [8], O4:K8 in the Peruvian coast [5], O4:K13 in Africa [9], and O4:K11 in the northeast of Spain [10].
The majority of clinical cases of V. parahaemolyticus have been associated with strains bearing the thermostable direct hemolysin (tdh) and/or TDH-related hemolysin (trh). Therefore, the presence of one or both hemolysins has been considered to be a conventional marker of V. parahaemolyticus virulence [11], [12]. However, it has been reported recently that not only the presence of hemolysins but also other virulence factors such as the type III secretion system (T3SS) have been involved in the cytotoxicity and enterotoxicity of V. parahaemolyticus [13], [14]. Whole genome sequencing of the clinical V. parahaemolyticus strain RIMD2210633 revealed the presence of two sets of genes encoding two different T3SSs, named T3SS1 and T3SS2, distributed in each chromosome [15]. A functional analysis of these two T3SSs revealed that T3SS1 is involved in cytotoxic activity, while T3SS2 has been related to enterotoxicity [13]. Consequently, the presence of T3SS2 has been consistently associated with pathogenicity of V. parahaemolyticus in humans. The T3SS2 found in the second chromosome of V. parahaemolyticus (T3SS2α) is part of a large pathogenicity island (PAI) of approximately 80 kb that also includes the tdh-genes. In the trh-positive strain TH3996, a novel PAI that is inserted in a distinctive pathogenic island containing a homologous T3SS2 (T3SS2β) has recently been described [16].
Comparative genome analysis of V. parahaemolyticus predicted that RIMD2210633 pathogenesis is associated with the presence of eight pathogenicity islands (VpaI) [17]. However, to date, VpaI that include T3SS2 have only been functionally characterized in infection models [13], [16]. Genomic analyses also revealed that different types of pathogenicity islands and mobile elements are the major structural differences between trh-positive and tdh-positive strains, including the pandemic clone [18].
Vibrio parahaemolyticus has not been routinely investigated in Peru and infections caused by this organism are rarely reported to the surveillance system. The mandatory investigation of V. cholerae in clinical laboratories after the dramatic emergence of cholera epidemic in 1991 in Peru contributed to the identification of other pathogenic Vibrio species. Vibrio strains isolated from hospitals and regional public health laboratories throughout the country were shipped to the Instituto Nacional de Salud (INS, Lima, Peru) for final identification and characterization. This extraordinary repository has been decisive for identification and evaluation of the impact of V. parahaemolyticus infections on the population, especially in remote regions and small villages along the Peruvian coast where seafood consumption constitutes the nutritional base of population.
The arrival of the pandemic clone to Peru in 1997 resulted in a major shift in the epidemic dynamics of V. parahaemolyticus in the region, replacing the seasonal and local self-limited infections attributed to native genetic groups by the generalization of infections exclusively caused by pandemic strains distributed across the country [5], [6]. Due to the environmental nature of V. parahaemolyticus, this overturn in population dominance and the subsequent population admixture may be expected to lead to an unpredictable impact on populations of pathogenic V. parahaemolyticus in Peru. To test this hypothesis, we investigated the population genetics, genomic variation and pathogenic islands distribution in a complete collection of non-pandemic strains recovered from clinical sources in Peru over the pre- and post-emergence of the pandemic clone. Pandemic and non-pandemic clinical strains representative of the entire period of study were subjected to serotyping and genotyping analysis by Multilocus Sequence Typing (MLST) and Variable Genomic Region (VGRs) analysis.
Materials and Methods
Epidemiological Data and Bacterial Strains
Peruvian V. parahaemolyticus strains were recovered from the collection of the National Reference Laboratory for Enteropathogens at the Instituto Nacional de Salud (INS) in Peru. This collection comprised 46 strains from the previously studied period of 1994 to 2005 [6] and 10 additional strains from the period of 2006 to 2007. To extend the comparison, a Chilean group, comprised of 13 strains from Antofagasta and Puerto Montt [19], [7], and 20 Asiatic strains, corresponding to 18 from Japan, one from Bangladesh and one from Thailand [20], [21], were included in the analysis (Table 1). The reference strain to V. alginolyticus ATCC17749 was also added to the panel of strains.
Detection of Genetic Markers and Serotyping
DNA extraction of V. parahaemolyticus strains was performed using an overnight culture in trypticase soy broth at 37°C using the Chelex-100 method [22]. Strains were confirmed by the presence of the V. parahaemolyticus species-specific genes toxR as described previously [23]. Additionally, the presence of the genes tdh and trh was determined according to procedures described by Tada et al. [24]. Group-specific PCR for the detection of the toxRS sequence of strains belonging to the pandemic clone of V. parahaemolyticus was performed as previously described [6], [25]. Serotyping Lipopolysaccharide (O) and capsular (K) serotypes were determined by a commercially available antisera scheme (Denka Seiken Corp., Tokyo, Japan).
Genetic Relatedness and Population Structure
MLST analysis was performed as previously described [21], based on internal fragments of seven housekeeping genes: recA, gyrB, dnaE, dtdS, pntA, pyrC, and tnaA. Sequences of both strands were determined by custom sequencing (Macrogen Inc., Seoul, South Korea). All chromatograms were assembled, manually edited and trimmed in Bionumerics 5.1 (Applied-Maths, Kortrijk, Belgium). Allele numbers were assigned to each strain by comparing the nucleotide sequence at each locus to all known corresponding alleles available at the V. parahaemolyticus MLST Database (http://pubmlst.org/vparahaemolyticus/). Novel sequence variants and sequence types (ST) were deposited in this database, and ST assignment was performed using the MLST website tools (http://pubmlst.org). Nucleotide sequences of MLST locus corresponding to STs generated from the study were also deposited in GenBank under accession numbers KC542949–KC543109.
Population genetic relationships among V. parahaemolyticus strains included in this study were performed based on the MLST allelic profiles by a minimum spanning tree analysis implemented in BioNumerics 5.1 software (Applied-Maths, Sint Maartens-Latem, Belgium). Strains were grouped according to priority rules adopted from the BURST algorithm [26], with the highest priority given to profiles with the largest numbers of single locus variants or double-locus variants in the case of a match. Clonal complexes were defined as groups with a maximum neighbor distance of one change and a minimum size of two strains.
Characterization of V. parahaemolyticus Variable Genomic Regions
Variable genomic regions (VGRs) were identified by comparative genome analysis based on a complete genome sequence (RIMD2210633) and two draft genomes of V. parahaemolyticus (AQ4037 and AQ3810) retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/). Genomic sequence data were analyzed by MUMmer 3.0 [27] to identify non-redundant genomic sequences. The identified sequences were listed as VGRs and used in further analyses. To determine the presence and distribution of VGRs among the strains included in this study, we carried out 31 different PCR assays using specific primers targeting the 23 regions identified by comparative analysis (Table 2). The PCR assays were performed in a 50-µl reaction volume containing 10 ng of genomic DNA, 1 U of Taq DNA polymerase (Roche, Mannheim, Germany), 0.2 µM of each primer (Sigma-Aldrich, Sigma-Aldrich, St. Louis, MO), and 200 µM of each deoxynucleoside triphosphate (Roche, Mannheim, Germany). PCR cycling conditions consisted of an initial denaturation step at 94°C for 3 min followed by 30 cycles of denaturation at 94°C for 50 s, 50 s at the annealing temperature, which was variable for each region (see Table 2), and extension step at 72°C for 1 min. A final extension step consisted of 10 min at 72°C.
Evolutionary Analysis
Phylogenetic relationships were inferred using ClonalFrame v1.1 software [28]. MLST loci sequences of unique STs were input into ClonalFrame using the default options. Two independent ClonalFrame runs were performed consisting of 500,000 iterations. The first 100,000 iterations in each run were discarded, and the phylogeny and additional model parameters were sampled every 100 generations in the last 400,000 iterations. The phylograms sampled from the two different runs were concatenated and summarized in a 50% majority rule consensus tree constructed by ClonalFrame GUI [28]. The convergence of the Markov Chain Monte Carlo (MCMC) in both runs was proven based on the Gelman-Rubin test as implemented in ClonalFrame [29].
To visualize potential associations between the phylogeny of housekeeping genes and the distribution of VGRs, we performed a transversal clustering analysis. For this purpose, the concatenated sequences of 67 strains were grouped by the mean of their phylogenetic relationship using ClonalFrame (50% majority consensus), while the VGR data were grouped using a UPGMA algorithm mean of their value (0 = absent and 1 = present), resulting in a data matrix ordered according to both phylogenetic relationship and VGR clustering.
Epidemiology Trends and Laboratory Investigation of V. parahaemolyticus
A total of 324 V. parahaemolyticus strain records were retrieved from the INS database from 1994 to 2007. These strains were obtained from gastroenteritis cases that occurred in different regions of Peru and were subsequently submitted to the INS in Lima for identification and storage. The overall distribution of V. parahaemolyticus cases over the 14 years of study (Figure 1) showed a characteristic seasonal pattern with annual peaks of incidence concurring with the warmest months. This epidemic pattern was uniquely disturbed in the course of the austral winter of 1997 when V. parahaemolyticus cases dramatically increased coincidently with an anomalous rise of temperature. The highest incidence of cases was observed from July 1997 to May 1998 with two peaks during September 1997 and February 1998. The epidemic dynamics were restored from 1999 onwards.
(A) Number of V. parahaemolyticus strains received at the National Institute of Health in Peru between 1994 and 2007 and environmental temperature over the same period. (B) Rate of pandemic versus non-pandemic strains over the study period; information on the dominant serotypes of each period is provided below the X-axis.
From the 324 cases recorded over the period 1994–2007, only 56 V. parahaemolyticus strains could be recovered from the INS collection. Distinct serovar dominances were detected over different periods of the study. From 1994 to 1996, infections were associated with serovars O4:K8 and O5:KUT. This serovar dominance abruptly changed during the winter of 1997, with the emergence of infections caused by strains belonging to the pandemic clone. Finally, an undefined pattern of serovar dominance was detected after the emergence of the pandemic clone in 1997–98. In this post-pandemic period, pandemic strains were identified together with O4:K8 strains as well as multiple serovars not previously detected (O1:K33, O1:KUT, O3:K30, O3:K58, O3:KUT, O5:KUT, O6:KUT and OUT:KUT). Pandemic strains represented the largest group of strains (n = 38, 67.9%) detected from 1997 onwards, and O3:K6 was the most frequent serovar among the pandemic strains (n = 31, 55.4%), although serovars O3:K58 and O3:KUT were also identified in the pandemic group. Serovar O4:K8 was the second most frequent group of strains over the whole period (n = 11, 19.6%), whereas the remaining serovars (n = 14) represented 25% of the total strains.
Genetic Structure of V. parahaemolyticus
The population genetic structure of V. parahaemolyticus strains representative of Peru, Chile, and Asian countries was analyzed by MLST (Fig. 2). MLST profiles of V. parahaemolyticus (n = 89) were categorized into 23 sequence types (STs). The recA gene of strains included within the ST265 (n = 8, O4:K8 serotype) showed an unexpected large PCR product of approximately 1500 bp (773 bp in the original) with a nucleotide identity diverging 18–19% from the characteristic sequence of V. parahaemolyticus.
A) MST of 89 MLST allelic profiles of the whole dataset included in this study. The colors are based on the origin of isolation. B) MST of the Peruvian subset that includes 56 strains. The colors are based on the serotyping group. Each circle represents an MLST genotype, and its size is proportional to the number of strains. The numbers along the branch links of MLST genotypes denote the amount of allelic differences.
The Peruvian group was split into 9 different STs (Fig. 2B). Minimum spanning tree analysis identified a single clonal complex consisting of ST88 (n = 3) and ST265 (n = 8) both of which included O4:K8 strains differing in a single locus. All of the strains belonging to the pandemic complex were grouped in ST3 (n = 24), whereas the remaining strains were included in 6 unrelated STs. Minimum spanning tree of the 23 STs resulting from the MLST analysis of the 89 strains showed a similar topography and group discrimination. Peruvian, Chilean and Asian pandemic strains shared the sequence of the 7 loci and were assigned to a single ST (ST3). Strains from Peru and Chile shared two additional STs: ST65 composed by O1:KUT strains and ST64 including strains belonging to O3:KUT and O1:KUT serovars. Three other clonal complexes (CC) were identified among trh+ Asian strains: one CC included STs 1, 83 and 82; a second group consisted of ST96 and ST91; and a third CC was shared by STs 87 and 14. The remaining strains were clustered in different and unrelated STs with differences in more than 5 loci.
Evolutionary History
The nucleotide sequences of 23 unique STs identified among the V. parahaemolyticus, as well as homologous sequences of V. alginolyticus (outgroup), were concatenated, and their phylogenetic relationships were inferred by ClonalFrame. The clonal genealogy inferred from the data revealed a star-like topology of V. parahaemolyticus delineated into two evident lineages, with the rest of the STs remaining unresolved (Figure 3). The pandemic lineage consisted of all of the pandemic strains from Asia, Chile and Peru (ST3), as well as other STs that included Asian pre-pandemic strains of diverse serotypes and the three variants of hemolysin-related genotypes. A second lineage consisted of trh+ strains of STs 1, 82 and 83, all of them from Asia.
A). The phylogenetic relationship of 22 V. parahaemolyticus STs was generated using ClonalFrame by 50% majority-rule consensus visualized using MEGA4. V. alginolyticus was used as an outgroup. Nodes A and B correspond to the recombination events presented in Figure 4B. Red, green and yellow circles denote the place of isolation from Peru, Chile and Asia, respectively. Coalescent units are indicated by the scale bar. B) ClonalFrame representation of the recombination event probability occurring in MLST concatenated is represented on the x-axis with the red line indicating the probability at each locus for an import event on a scale from 0 to 1 (y-axis). Each inferred substitution in the graph is represented by a cross, the intensity of which indicates the posterior probability for that substitution.
The genealogy showed the influence of recombination on the genetic diversification of V. parahaemolyticus with a central node (node A) from which all of the branches diverged. This specific topology indicated an unresolved phylogeny due to the non-identification of a common ancestor. ST88 and ST265, comprising all the O4:K8 strains were grouped in a single lineage. ST89 and ST93, which included Peruvian strains, also shared a common linage, as well as ST92 and ST63, sequence types composed of Asian and Chilean strains, respectively. For each branch of the reconstructed genealogy, ClonalFrame identified fragments that were likely imported. The influence of mutation and recombination events in the generation of polymorphisms was further investigated in the two cluster nodes (A and B) indicated in Figure 3A. Node A showed a high probability of importing events consistent with recombination substitutions, while divergence in node B, which included the Pandemic lineage, most likely originated as a result of point mutations (Fig. 3B). ClonalFrame analysis shows that the relative impact of recombination versus point mutation expressed as a ratio (r/m) was approximately 1.45, and that the relative frequency of recombination in comparison to point mutation (ρ/θ) was approximately 0.20.
VGR Distribution
To extend our analysis of the phylogenetic relationships and to understand the impact of genomic variation in the evolutionary history of V. parahaemolyticus, the distribution of VGRs was assessed by PCR assays in the 67 strains. A transversal clustering analysis was performed to visualize the evolutionary significance of the presence of genomic regions within the strain collection (Fig. 4).
Clustering was built using a ClonalFrame tree of MLST allele sequences of 67 V. parahaemolyticus strains, while the VGR data were grouped using a UPGMA algorithm, resulting in a data matrix ordered according to both phylogenetic relationship and the prevalence of VGRs. Solid boxes and open boxes in the appropriate column indicate VGR presence or absence, respectively. Peruvian subset names are shown in red.
ClonalFrame majority-rule consensus tree building with the whole set of strains revealed the same group structure as resolved by the clonal genealogy. However, analysis of VGR data showed a specific clustering of VGRs with three different groups (A, B, and C) (Fig. 4). Cluster A grouped five different genomic regions almost exclusive for trh+ strains with the exception of two tdh+ strains (U5474 and AQ3810). The presence of TTSS2β was only detected in strains bearing the trh gene. This cluster also included regions HGT20 and HGT22 encoding for the Type I restriction system, which was only identified in trh+ Asian strains. Cluster B included 6 genomic regions; two of them (HGT3 and HGT3A) correspond to ORFs coding the type 6 secretion system (T6SS1), which were present in all of the clinical strains with the exception of 4 tdh+/trh− non-pandemic strains isolated in Peru, Chile, and Asia. T3SS2α was singularly detected in those strains with genotype tdh+/trh− but was not present in those strains positive for the tdh and trh gene. Cluster C consisted of the majority of VGRs (20 genomic regions), which were uniformly distributed among almost all the pandemic strains. Some of these regions were partially present in all Asian strains phylogenetically related to pandemic strains, while those were less frequent in the remaining non-pandemic strains and distributed with an undefined pattern. Non-pandemic Peruvian strains showed a shift in the distribution of the VGRs included within this cluster from the pre-pandemic period to 1999 and afterwards. Genomic regions such as HGT6A (ribonuclease R), HGT12 (VPaI2), HGT9 (site-specific recombinase), HGT14 (hypothetical protein), HGT16B (phage genes), HGT13 (type IV pilin), HGT18 (hypothetical protein) and HGT19 (hypothetical protein), which are characteristic of pandemic strains, were exclusively identified in those non-pandemic strains from Peru isolated after the arrival of the pandemic clone in 1997. Finally, none of the VGRs investigated were found in V. alginolyticus ATCC17749.
Vibrio parahaemolyticus represents an intriguing food-borne pathogen and poses a significant threat to public health in Peru. However, despite this concern for public health, the knowledge of the molecular epidemiology and genetic structure of this pathogen remains incomplete.
Epidemiology of V. parahaemolyticus in Peru have been historically associated with sporadic outbreaks linked with seafood consumption. Since 1983, clinical strains were dominated by the presence of local serovars such as O4:K8 that was isolated from both clinical and environmental samples [30]. These infections were characteristically related to auto-limiting outbreaks detected along the coastal regions over summer months. This epidemic pattern shifted in 1997 with the unexpected arrival of pandemic V. parahaemolyticus to Peru [5]. The presence of the O4:K8 serovar in pre- and post-pandemic periods allowed for the identification of this group as the dominant population among clinical strains in Peru and the cause of recurrent outbreaks during the warmest months [31], [5]. The totality of strains recovered from infections in 1997 and 1998 belonged exclusively to the pandemic clone. After the period of the infections associated with the pandemic clone in 1997–98, the dominance of this clone in clinical infections began to decline and a mix of different serovars began to emerge. This specific epidemiological trend of arrival and rapid decline of pandemic V. parahaemolyticus infection in Peru clearly contrasts with the infection dynamics found in Chile, the neighboring country where infections associated with the pandemic clone started first in 1997 and subsequently in 2004, and where the pandemic clone has dominated the clinical isolations since the second epidemic radiation in 2004 [32]. The arrival of the pandemic clone to Peru provided a unique opportunity for testing the potential impact of an introduced genetic group on the structure and genetic variability of local pathogenic populations. One particular feature of the epidemiology of V. parahaemolyticus in Peru after the appearance of the pandemic clone was the sudden apparition of diverse serovars not detected previously. Serovars O1:K33, O1:KUT, O3:K30, O3:K58, O3:KUT, O5:KUT, O6:KUT and OUT:KUT had not been isolated prior to 1998. Similar results were found in a previous retrospective study carried out in Peru covering the 1993–2002 period [6], which reported the presence of serovars O3:K68, O3:K58, OUT:K6, O6:K18, O11:KUT, O11:K15 and OUT:KUT after 1998. The flourishing of serovars and genotypes in Peru may be related to the particular vehicle for the introduction and propagation of pandemic strains. The epidemic dissemination of this pandemic clone along the coast of Peru corresponded with the expansion and dynamics of the poleward propagation and the receding of tropical waters linked to the 1997 El Niño event [5], [33].
Another important aspect to be considered to understand the possible genetic impact of the pandemic clone on Peruvian local populations is the comparative analysis of phylogeny inferred from MLST sequences and the distribution of variable regions in the panel of strains. The pattern of VGR distribution forming cluster C showed a conserved presence for all regions among strains belonging to the pandemic clone, which provides an additional evidence for the highly clonal nature of this phylotype [34], [18]. On the contrary, a sparse presence of VGRs in cluster C was observed, showing an undefined pattern among non-pandemic strains. A detailed analysis of variations and phylotypes showed that VGRs in cluster C are only present in strains isolated after the arrival of the pandemic clone in 1997. This specific pattern of distribution and the connection between these strains and the arrival of the pandemic clone may suggest a common origin for all of these groups. However, the presence of a region coding hypothetical genes (VPA0434 and VPA0435) in one O4:K8 strain also raises the possibility of a local horizontal transfer from pandemic strains to local Vibrio communities in Peru.
Horizontal gene transfer appears to be the major force shaping both the genomic variation and virulence of V. parahaemolyticus, as evidenced in previous studies linking the acquisition of pathogenicity islands with the emergence of the pandemic clone [17], [18]. The results of transversal clustering analysis revealed that the presence of T3SS, T6SS and mannose-sensitive hemagglutinin (MSHA) pilus were clustered together in most clinical isolates showing a clear association with the pathogenicity of these strains, suggesting that these regions are conserved in pathogenic strains and could be a good marker of pathogenicity.
The evolutionary history of pathogenic lineages of V. parahaemolyticus has been analyzed previously by different approaches [21], [17], [35]. However, the previous analysis of population structure was not sufficiently integrated within the epidemic dynamics prevailing in a specific region so that there could be an adequate evaluation of the shift in genotype and population dominance over different periods. ClonalFrame genealogy inferred a star-shaped tree with long terminal branches showing a primary diversification affecting all of the STs, likely as a result of recombination events [36]. A defined lineage grouped all of the pandemic strains as well as most of the Asian genotypes showing a hierarchical pattern recently evolved from a common ancestor, likely due to the course of successive point mutations. The overall results evidenced the influence of recombination events in the diversification of most pathogenic V. parahaemolyticus genotypes.
Homologous recombination in housekeeping genes has been found naturally in Vibrio, and it is an important driver of diversification in this genus [37]. ClonalFrame analyses of the whole concatenated dataset showed rates of recombination of 1.45 and 0.20 for r/m and ρ/θ, respectively. These data suggest an intermediate rate of recombination among the strains characterized [38]. This estimate of recombination frequency suggests that recombination is relatively rare compared to other species, such as Streptococcus uberis (ρ/θ, 9.0) [39] and Clostridium perfringens (ρ/θ, 3.2) [40], but it is approximately similar to that observed for other groups, such as lineage I of Listeria monocytogenes (ρ/θ, 0.13) [41], This particular feature may be related to the exclusive use of pathogenic strains in the study, which are characterized by a clonal diversification [38].
To conclude, the results of this study describe the epidemiological impact caused by the introduction of the pandemic clone in Peru on the epidemiology and structure of the local population of V. parahaemolyticus. The presence of genomic regions characteristic of the pandemic clone in other non-pandemic strains provides early evidence of genetic transfer from the introduced population to the local communities. Additionally, the genetic relationships based on MLST and VGR analyses support the epidemiological connection between pandemic and non-pandemic strains isolated in both Peru and Chile. The phylogenetic and genomic analysis performed allowed us to determine the recent origin of the pandemic clone lineage, probably caused by successive acquisition of genomic regions, as well as the influence of recombination events in the diversification of non-pandemic pathogenic V. parahaemolyticus. Ultimately, these results provide a preliminary framework about evolutionary history of V. parahaemolyticus. Recent advances in high throughput sequencing are revolutionizing the field of population genetics of human pathogens. Application of fine-scale analysis based on whole genome sequences in future studies of pathogenic bacteria will contribute to improve our knowledge of the epidemic dynamics and routes of dispersion of Vibrio diseases [42], [43].
It is a pleasure to acknowledge the excellent technical assistance of Silvia Carles and laboratory professionals of Instituto Nacional de Salud, Peru. We also thank Mitsuaki Nishibuchi and Romilio Espejo for providing some of the strains included in this study.
Author Contributions
Conceived and designed the experiments: RGG JMU. Performed the experiments: RGG. Analyzed the data: RGG MLZ JMU. Contributed reagents/materials/analysis tools: MLZ JMU. Wrote the paper: RGG JMU.
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