text:
I was bored, as usual, and quite horny. I had been thinking about wearing sexy underwear. I have always worn boxers, but saw some photo of guys in thongs and bikini undies, and the idea intrigued me. I began to surf the web and saw through L.A. Frontiers, an ad broadcasting custom made thongs and briefs. A rush came over me and I decided to call. The man on the other end seemed friendly, yet not gabby. He told me he had about three dozen designs to look at, and he worked out of his home, so he said we would have privacy. I told him I would be over and got in my car, my heart racing.
He lived over in LaBrea, south of Wilshire Blvd. The apartments there were built in the 20s and 30s, mostly duplexes. He said he lived on the bottom floor. I arrived at his place, and waited a moment before I got out of the car. I had been through experiences like this before, but as always, wondered if I was doing the safe thing. But, as normal, I decided to be naughty and go for it. I walked up to the porch, trying not to breathe hard. I knocked on the door and heard him approach. He looked like he was in his 40s, middle eastern, face looking like he had had some work, no shirt, tight gym shorts and sandals. He reached out his to shake mine. It was very soft. I felt safe as I walked in.
He welcomed me and directed me to follow him. His apartment was big. We walked through the living room, down a long hallway, into a bedroom he had converted to a studio/sewing room. He had a bunch of thongs and panties laying out. He invited me to strip. As I undressed (and I loved how he told me to "strip"), he took each article of my clothes and hung them on a hanger, and placed them in his closet, even my shoes. Now, I was nude, a blank canvas for him to dress me.
He asked what I was looking for, and being bold, I told him, something feminine and sexy. He grinned at me and handed me a sheer panty, white, a panel in the front and a thong in the back. I put them on and pulled them up. They were tight, and see through. He then said something I was not prepared for: "These fit nice, especially because you are gifted with a tiny cock". It is rather small, but no one, man or woman had ever said that to me. I was so aroused that he would have the pluck to say that, and playfully pulled the panty down to expose myself to him. "Yes, I am tiny", I admitted. It amused me we now had the rules in place. Since he had seen mine, I decided it was only fair to see his. "You tiny too?", I teased. He peeled his shorts off completely. My eyes were saucers as I looked at his package. He must have been about eight inches, and six inches in circumference. It was the most magnificent penis I had ever seen in real life. It looked like it weighed ten pounds. I just stared at it. Then he said, "What do you think?" I just shook my head, letting him know it wasn't. By any means. He didn't put his shorts back on either. The other thing I noticed, was the fact he didn't have any hair on his body, armpits included, but he sported a rather thick bush. It did nothing to diminish his dick, but seemed odd. Still, it was hot to look at.
The next one he handed me was a series of strings, connected to a doily type front. It was more fem than the last, and made me feel even hornier. I told him I would take both and asked him what more he had. The next one was black lace, with a silk black panel. It was more conservative, but still racy. I wanted to play coy, and told him I didn't like it. He protested, saying the reason it didn't look good was because my cock was jabbing out. He reached into my underwear and pulled my cock so it was sticking up. I could not believe his chutzpah, or the fact I allowed it. But allow it, I did. It actually worked, and made the bikini more attractive. "All the men at the gym wear their speedos that way", he stated. I was thrilled to think I could hold my own, appearance wise, in the gay community.
I decided to take all three, and asked if I could try on the string and doily one again. He oblidged, and watched me look at myself. My tiny cock was not so tiny anymore, and neither was his. He was certainly not erect, but was more swollen, and about an inch thicker, and two inches longer. I was indeed impressed. While observing my body and this girly piece of clothing, I noticed how it didn't go with my hairy body. He sensed my negative feeling and asked what was wrong. I told him, jokingly, that my bikini line didn't accentuate his line of clothing. He replied that he thought that too. "Would you like a little manscaping, no extra charge?" It was though he read my mind. I didn't respond verbally, but only nodded my head. He smiled, and held out his hand. I reached for it, and he guided me down the hall, back into his living room. "Hang on, I'll be right back. Take off your undies". I obeyed and wondered what was next. Alan returned with a folded up massage table and set it up. "Hop on, and lie on your back." Again, I obeyed his command. The room was about eighty degrees and made me feel dreamy. He had left the room, and came back with a case, and two beers. He handed me one of them, and I drank it in one continuous gulp. He laughed, and handed me the second one. I don't know if it was his intention of getting me drunk, but I took the second, and took a long sip, handing it back to him. He drank from it as well, bonding us closer.
Before he started he told me his procedure. "I'm going to do a little trimming, and a little buffing. I'll be so glad to get rid of your horrible man look!" I wasn't sure how he meant that, but again, here I was with this small man with a penis like a banana. I was happy to be in his hands. He turned on the clippers and began to shear my legs. I was only thinking he was going to trim my outer pubic region, but the buzz on my legs felt good, and the beer was kicking in. I found out he was quite strong, being able to lift my limb to trim my calves and back thigh. I felt good letting him explore and groom me. I slipped into a dreamy trance while he did his work.
After a while, he stopped and said, "Now for a little buffing". I had no idea what that meant, but didn't care by this time. Alan had spent about an hour trimming, although it had just seemed like minutes. Before he started the buffing, he took the beer bottle and held it to my lips. I opened them and consented for him to feed me. I didn't know it, but I was falling under his control more and more. He then proceeded with the "buffing". It felt great. I constant vibration attacked me. I was afraid I was going to cum. I even laughed out loud and kidded that I might. He brushed my penis and replied, I'm not worried, there won't be much from that puny thing". I loved it.
When he turned off the equipment, I knew he had finished. He brushed off my body with his smooth hands. I was interested in seeing his work, and asked to see a mirror. He said he had an entire mirrored wall in his bedroom and helped me up. He continued to hold my hand again, as we walked down the hall. I noticed he had a really cute, tight butt with a dark crack caused by his olive skin. I found that very attractive.
When we came into the room and I saw myself for the first time, I thought I was going to faint. My knees actually buckled. I don't know how Alan did it, but he essentially shaved my entire body smooth. I had no hair anywhere except on my head. I couldn't believe what I was seeing. Alan had a smug look on his face. My jaw dropped. "You shaved off all my hair", was all I could sputter. "Why?, I pleaded. "Because your body hair and especially your pubes didn't jive with your dicklet. You look look much better now. Everyone at my gym would think you look normal in the showers". I reached down and felt my lower abdomen. It felt silky smooth. He commanded me to raise my arms over my head. My pits were hairless as well. Alan stood behind me and reached around to stroke my pits. "Doesn't that feel better? Don't you like the look? I think it really fits you!" With that, he continued to touch me, down my chest, playing with my nipples. "This is the new you", he announced. I was falling under his powerful spell. I hadn't counted on this to happen. "Yes", I replied, slightly moaning at the same time. His hands gently rubbed on my belly, and farther down, where my bush once was. His fingers then travelled back up, kneading my nipples. "Everything is more sensitive now. You feel things more". Then he turned me around and pressed his body against mine. He ground his cock into my crotch. "This is the best part. You feel the pubes of a real man. I nodded my head, unable to speak. "I knew you'd like that, from the moment I first met you". Then he turned me around, spread open my asscheeks, and put his cock in the crack like a hot dog. "But this is how you will feel my bush most of the time." I looked down, and saw my swollen cock leaking precum. I had never done that before.
Alan took his hands and placed them on my shoulders and began to press down hard. I succumbed and fell to my knees, powerless. I probably could have resisted, but definitely decided not to. He walked around and stood in front of me, motionless. I instinctively put my hands on his hips, opened my mouth and swallowed his entire cock. Its yeasty odor made me so hungry for it. My head bobbed back and forth. I suddenly could not get enough of his gland. He was now hard, so I knew I was doing the right thing. His bush ticked my nose, but that only made everything better. It made me think I was born only to service him.
Alan then pulled out, saying he didn't want to cum just yet, and gently rubbed my head. I held his hard member and kissed it. He smiled and said he wanted to finish grooming me, and to stand up. He put his gym shorts back on, which puzzled me. Again, he took my hand and led me out of the room. I love holding hands with him, making the bond even stronger. What I hadn't counted on was where he was taking me. We walked hand in hand down the hallway, back to the living, into his kitchen to the back door. His back door led to the backyard. I was naked. I stopped and looked at him with eyes of concern and anxiety. He shook his head and told me, "My neighbors are very tolerant". I wasn't sure what he meant, but the idea of publicly exposing myself made my hairless balls tingle.
We walked outside and he left me there while he went back in. He came back out with a pail, a couple of bottles, and a towel. We walked over to his hose, and he turned the faucet on and began to hose me off. Like bathing an animal. The water was cool, and I was hot, so I didn't mind one bit. What was really turning me on was that he was clothed and I was nude. Hairless as well, even more than nude. Alan got me all wet, then turned off the water and grabbed one of the bottles. He squeezed out a large dollop onto a body sponge and began to scrub me with it. The liquid had coarse materials in it. It was meant to remove the top layer of my skin. It didn't hurt, but made my skin tingle. He didn't miss a single section of my body. I loved how it felt, coupled by being nude in his backyard, potentially seeing a stranger. Speaking of which, an old man came out to empty his trash in the garage in back. Apparently he and Alan shared the same patio. He stopped and chatted while Alan scrubbed me. The old man smiled at me and said, "Client of Alan's?" I nodded my head. Even though Alan was bathing me within an inch of my life, I never felt dirtier. This old man looked at me like I was a large popsicle. I felt so sexy.
Alan knew it too, and valued his new prize. He spun me around slowly, to show me off. I was his trophy. I loved the feeling of wearing nothing, and being exposed. Nothing mattered at this point. My body tingled from the scrub, and felt cool. Alan opened the other bottle and squeezed a handful into his palm, then rubbed in all over me. It was the polish. It immediately soaked in and gave my skin glass like appearance. It' stung as it went on, then subsided. I couldn't believe how it felt. Alan approved.
He again took my hand and we walked back into his apartment. We went into his bedroom, and he gently led me to his bed, and laid me over it. My legs were still on the floor, but my chest and belly were on the bed. I had no idea what he was doing. Alan opened a draw, and took out a small tub, then told me to spread my legs. I obeyed, and felt him grab my cock and stretch it. He opened the tub and squeezed a small dollop onto the back of my penis head. Then he pressed the head to my perineum and held it there. Again, I had no idea what he was doing. After a minute, he let go, and told to remain in that position until he told me to get up. There I was, lying there, spread eagle. He could see everything. But I obeyed him.
After about five minutes, he told me to stand. I realized what he did. He superglued my cock to the back, so it looked like I no longer was male. My crotch was now a smooth panel. I didn't know what to say. Alan had a very smug look on his face. I have to admit, it turned me on. I had brought this on myself. I started all this. Alan was able to read me and finish the job. But he had stretched my cock so far, that I was no longer able to stand upright, but now my ass stuck out more, like I was wearing a humbler. I liked that too.
Alan gently pushed me back on the bed, and pulled my legs over my head. Before I knew what was happening, he began to lick my asshole. The feeling was unbelievable, and I knew it was his version of cunnilingus. The feeling was overwhelming and all I could mutter was the words, "Fucking yes". He was a master at it, and I could feel myself ready to cum. Before that happened, he stood up, peeled off his shorts and showed me his now rock hard cock. It looked like it was a foot long. He greased it up with Vaseline, then got on the bed and pressed the tip to my hole. I was terrified, hornier than I could ever remember. He pushed his cock into me, and began to fuck me slowly. I had been afraid it would be painful, but instead it felt great. I'm sorry I had waited this long. Alan stared at me while he fucked me, letting me know he was in charge. Then something happened that I had never anticipated. I had an orgasm. Different that before. Not as intense, but much longer duration. As I came, he thrust harder. I thought he was going to split me in two. Then, as I could feel him cum, he slowed down, but never pulled out. When he was done, he remained inside of me until he was soft. I realized he had cum inside me. Normally, that would been unsettling, but for some reason, I felt safe. I also felt really dirty that I not only allowed a man to fuck me, but I let him bareback me.
After we shoered together, we went back into the studio, and I put back on the doily string thong, my favorite. He then gave me some ultra tight gym shorts, and an orange spaghetti strap top. Essentially, women's clothes. He finished it off with a scarf over my scalp. I wore the same sandals I came in with. Those were the only male part of my attire. He called a friend and said he had a new toy he wanted to show off. He hung up and reached for my hand. I was off to start a new life.


text:
Miami International Art Fair (January 6-10) kicks off the 2010 international art fair season at the Miami Beach Convention Center (1700 Convention Dr., Miami Beach). Galleries from every continent will be presenting the best in contemporary art, photography, video, and installation art. The fair includes a lecture series featuring prominent experts in Latin American and International Contemporary Art.
From the MIA press release:
The Projects Gallery of Philadelphia will present work by the internationally acclaimed Cuban photographer, Marta Maria Perez Bravo, who represented Cuba in the III Havana Biennial and the XXI Sao Paulo Biennial. Projects will also be exhibiting artwork by: Florencio Lennox Campello, whose recent work has featured Cuba as the star of a series of mixed media paintings and etchings; Michael Janis, Co-Director of the Washington Glass School in Washington, D.C. and the Florida Glass Art Alliance's 'Outstanding Emerging Artist 2008-2009'; Cirenaica Moreira, who plays the lead role in her tableau-like photographs focusing on the performing arts; Sandra Ramos, one of today's most important and controversial contemporary Cuban artists; and Tim Tate, Co-Founder of the Washington Glass School, and 2003 Mayor's Art Award winner for 'Outstanding Emerging Artist for Washington, DC.'
Read the full MIA press release HERE
Washington Glass School artists Erwin Timmers and Anne Plant round out the show's featured artists.
Click HERE to jump to the MIA website
Rising Star: Anne Plant
Anne Plant "No Tillage"
Anne Plant's artwork has had a tremendous response and has garnered the attention of a number of galleries, curators and collectors. Anne is a scientist and is a resident artist at the Glass School.
Anne's evocative landscapes were first on exhibit at this year's Washington, DC "Artomatic"art fair and artDC Gallery's The Common Element show. She is now planning a show at the NIH (National Institutes of Health) Gallery, and the Miami International Art Fair, with Projects Gallery of Philadelphia.
Congratulations Anne!
2008 photo of Anne Plant working at Penland School of Craft
"As a professional scientist, I explore glass as a medium because of the myriad of forms and combinations that provide opportunities for experimentation. Combining the clarity and luminosity of the glass with imbedded imagery creates both beauty and story.
These works are created with ordinary window (float) glass. These images are constructed by painting parts of the scene onto different layers of glass, which are then fused together.
In this series, the physical depth of the glass layers is used to enhance the feeling of separation, distance and atmosphere. Anne Plant

subset: commoncrawl


text:
There are roughly three New Yorks... the New York of those who were born here... the New York of the commuter... and the New York of the person who was born somewhere else and came to New York in quest of something. Of these three trembling cities, the greatest is the last - the city of final destination... Commuters give the city its tidal restlessness; natives give it solidity and continuity; but the settlers give it passion.I saw the complete first season of this webseries at a local library with the director, one of the actors and one of the interviewees present. It is an interconnected story of various immigrants in NYC and was very good. I was worried that I might not like to watch the whole thing but it went by rather quickly, ending with me wanting more. Fitting viewing given recent controversies as alluded by an audience comment. Look forward to next chapter.
- E. B. White, "Here Is New York"
Various thoughts on current events with an emphasis on politics, legal issues, books, movies and whatever is on my mind. Emails can be sent to firstname.lastname@example.com; please put "blog comments" in the subject line.
About Me
- Joe
- This blog is the work of an educated civilian, not of an expert in the fields discussed.
Saturday, February 04, 2017
Three Trembling Cities
Labels: border issues, film, New York City


text:
Le Meridien ViennaStar Rating
State Opera
Star Rating Close
Our 4-Star classification designates those properties with well-appointed, deluxe accommodations, extensive amenities and comprehensive guest services. Expect attention to detail and a warm and hospitable staff ready to cater to your needs. These superb properties offer a truly refined getaway.
Le Meridien Vienna Hotel Package Deals
Le Meridien Vienna - 3 nights with flight$925+
State Opera
Classic Courtyard DoubleBook Now


text: On Sale Choose Options Compare Quick view 8" x 36" Aussie Exhaust Stack With 5" OD Bottom CHROME SA8-36SBC-5 Grand Rock MSRP: Now: $467.00 Was: $541.00 SA8-36SBC-5 8" x 36" Aussie Stack Reduced to 5" OD Chrome SA8-36SBC-5 What other exhaust parts will I need? Depending on your current exhaust system, you could need several truck pipes to complete the job. If you are needing to find out what... MSRP: Now: $467.00 Was: $541.00 Choose Options Compare Quick view


text:
- Home
- Student Services
- Alternative Learning
- Driver's Education
Driver's Education
Greenbush provides several levels of service for school districts to offer driver education locally. Coupled with state aid that is currently over $100 per student and the local decision to charge students fees, districts can offer these services in a budget neutral fashion. A Memorandum of Understanding (MOU) is made between the district and Greenbush on an annual basis to deliver the level of service that the district needs.
Option 1 - Online Classroom Course access ($70 per student)
With this option, Greenbush only provides access to the online software used for the classroom portion of the class. The district provides the teacher. Greenbush will provide training for the teacher to use the online software. The district will also provide the teacher for the in-car instruction.*
Option 2 - Online Classroom Course with Greenbush Instructor ($160 per student)
Greenbush provides both the online software as well as the teacher who will manage the software for the classroom portion of instruction. The district will provide the teacher for in-car instruction.*
Option 3 - Online Classroom Course with Greenbush Instructor and In Car Instruction ($280 per student)
Greenbush provides the online software, the classroom teacher and the in-car instruction teacher. The district will provide an inspected and insured car along with fuel.*
*The MOU provides greater details regarding the roles and responsibilities of both Greenbush and the school district.
Get started today!
Call or email Chris Kleidosty for further information on how we can develop a program that fits the needs of your students.


text:
Card Collection Vicktory Dogs
1 review
Inspiring, heartwarming and created with love - this card collection was made to celebrate the strength and resilience of the 22 dogs who were rescued from the property of NFL quarterback Michael Vick and brought to Best Friends Animal Society.
Featuring Victory dogs (as we affectionately call them) Curly, Cherry, Georgia, Handsome Dan, Lucas, Little Red, Mel and Mya. Each picture will remind you that all dogs are individuals worthy of a second chance, no matter what their breed or past. And that, together, we can end breed discrimination and Save Them All.
Artwork generously provided by Cyrus Mejia.


text:
In order for the Trinity University Police Department to maintain a safe and secure environment for the campus community, we need your help. See something. Say something →
In order for the Trinity University Police Department to maintain a safe and secure environment for the campus community, we need your help. See something. Say something →
An outdoor warning system was installed at Trinity University in July 2013. The system uses a siren followed with a message to alert the campus community to take shelter in the event of an emergency. The University plans to test the system once a month. The University is dedicated to ensuring everyone knows what to do when they hear the siren and message.
Click here to hear what the sirens sound like
The system test takes place at approximately 11:50 a.m. on the first Wednesday of every month. The tests last approximately one minute. You do not need to take any action during the test. If a severe weather warning is issued for the area on a scheduled test day, the test will be cancelled for that month.
You should continue your regular activities during a system test. You do not need to take any special actions during a test.
Monthly tests will begin with the announcement, "This is a test." If you hear the sirens, and do not hear, "This is a test," this means there is an emergency. A system test will only take place at approximately 11:50 a.m. on the first Wednesday of the month, and will never last longer than a minute. In the event of an emergency the siren and message will sound until the emergency is over, at which point you will hear an "all clear" announcement.
When you hear the siren and message you should follow the instructions given.
The sirens indicate emergencies that make it unsafe to be outdoors and require you to seek immediate shelter. The most likely cause of these unsafe outdoor conditions is severe weather, but other causes may include the unlikely occurrences of an environmental hazard or an armed individual on campus. The siren and message are only used for emergencies that impact or present a danger to our campus. For example, the siren and message will not be sounded for a tornado watch in the Bexar County area, but they will be sounded if there is a tornado on the ground that has the campus in its forecast path.
The Mass Notification speakers are located on top of two buildings on the Trinity campus. The speaker arrays are located on the roofs of Center for the Sciences and Innovation and the Thomas Residence Hall.
The sirens are loudest within the main campus. They are audible on both upper and lower campus, and are designed to be heard outdoors. You may not be able to hear the sirens if you are indoors or are listening to music using ear phones. In the diagram below, the green border represents the area in which the sirens are loudest.
Should you have any additional questions please contact the University Police Department at 210-999-7070.


text:
The gateway to the Historic Half! At the Healthy Lifestyle Expo presented by VA Runner, Historic Half Weekend participants pick up their packets including the official bib with timing device, competitor shirt and event program. Runners and supporters stop by selfie spots, meet celebrity guests, hear from experts in Speaker Square and visit over 60 vendor booths covering a range of fitness activities, wellness products and active interests.
When & Where
Free and open to the public, the Healthy Lifestyle Expo including Packet Pick-up will be held at the Fredericksburg Expo & Conference Center (2371 Carl D. Silver Parkway, Fredericksburg, VA 22401) on:
- Friday, May 17, 2019 from 4:00 p.m. to 8:00 p.m.
- Saturday, May 18, 2019 from 9:00 a.m. to 6:00 p.m.
Expo opening ceremonies commence at 3:30 p.m. on Friday. Located within Central Park shopping area and adjacent to Wegmans, the Historic Half Healthy Lifestyle Expo boasts plenty of free parking is available.
Featured Booths
- The interactive Mary Washington Healthcare booth with screenings, consultations, giveaways and a simulated treadmill climb of Hospital Hill
- Signup for a chance to be on Team GEICO Running at the GEICO booth.
- Pohanka Nissan sign-the-pace-car display
...and much more.
Packet Pick-up
All Historic Half Weekend participants are required to attend Packet Pick-up, located within the Healthy Lifestyle Expo to obtain the official bib with timing device and technical competitor shirt. Runners are required to bring the e-Card and a photo ID to Packet Pick-up. e-Cards are emailed to all participants in the weeks prior to the event.
Runners are highly encouraged to pick up their own materials and verify personal information.
For runners unable to attend Packet Pick-up, proxy pick-up is an available option. Runners may designate an individual to obtain their bib and event materials. The individual picking up the bib for a registered runner must present a hard copy of the completed e-Card with a copy of the runner's photo ID, as well as a copy of his/her ID. Please be sure to obtain the runner's preferred shirt size. e-Cards are emailed to the registered runner before the event. Parents may sign waiver and retrieve packets for their children under 18 years of age.
Packet Pick-up is not available on event morning.
Exhibitor Info
Connect with a motivated crowd at the Historic Half Healthy Lifestyle Expo! Sample, sell, promote, and demonstrate to more than 25,000 runners and guests from the national capital region, over 35 states and all branches of military. Held indoors, the Expo is free to the public, and conveniently located near restaurants, shops, and Historic Downtown Fredericksburg. Take action to participate in Fredericksburg's most impactful economic event and complete the Expression of Interest Form to begin the process of securing space at the Expo. A great spring weekend event with advantageously priced booths in a big time event setting. Expression of Interest Form will be available soon.
For more information, contact Kate Kovatch at firstname.lastname@example.com or 703.432.1894, or Marc Goldman at email@example.com or 703.784.2113
Explore and Discover Event Details
Fredericksburg VA
Located halfway between the capital of the US and the capital of the Commonwealth, Fredericksburg is a great spring weekend getaway. Come to #RunWithTheMarines, but stay for the shops, restaurants, historic sites and natural beauty.


text:
Machine Shop
We are proud to say we do our own in house machine work. this way we are able to control the cost, turnaround and quality to the best of our ability to serve our customers. With our Dynamics Research balance machine we are able to balance your rotating equipment to its proper "G" scale rating no matter the weight or length.
Waste Water Services
Over the last several years Armor Electric Repair has made itself know in the repair and service of municipal equipment. We service a wide variety of submersible pumps such as Hydromatic, Fairbanks, Flyght and Ebara.
531 Linkwood Road Rock Hill, SC 29730
Phone Number 803-366-6128
Fax Number 803-366-4740
Gear Reducer Repairs
Armor has been rebuilding gearboxes for many years and can handle your foreign and domestic needs. We fully disassemble, clean and evaluate your unit. All housings fits and shaft fits are inspected or looseness and wear. We do a dye test on your gears to look for cracks and abrasions.


text:
When it comes to Temporary and Direct Hire staffing Agencies in San Antonio there is just one recruiting employment staffing agency to turn to and that is Frontline Source Group. Our San Antonio team of professional recruiters offer staffing services for temporary, temp, temp to hire, contract to hire, project-based employment and direct hire opportunities to all areas surrounding San Antonio, Texas.
Staffing Areas San Antonio
|Frontline Source Group is one of the fastest growing San Antonio Temporary Agencies and Staffing Agencies for
5 YEAR Warranty on Direct Hire placements with our clients
We specialize in direct hire, contract, contract to hire, long-term projects, temp staffing, temporary and temp-to-hire placements. We are a staffing firm and offer staffing services to meet the needs of our clients. We work with companies throughout San Antonio, Texas and all of North America. We take the time and invest the manpower to visit with each candidate and express the process of screening for submittals to our clients on their open positions.
Our team of professional recruiters can solve your staffing shortage or provide new talent to help take your company to the next level. The time we take to understand your requirements is what makes us stand out above the rest. Not all Staffing Agencies in San Antonio are made the same and Frontline Source Group is not your average! When looking for the Best Temporary & Direct Hire Agencies in San Antonio Call 210-696-5627 for staffing results!
Why Choose Frontline?
We have a thorough screening process that delivers quality candidates perfectly suited to meet your needs. Click here to read more about this process.
Frontline is a member of the American Staffing Association. This membership demonstrates our commitment to operating within the highest ethical standards in the industry and to staying up-to-date on all the trends
We have been recognized as a leader in our industry, having received numerous awards for our exceptional service. Click here to view our accolades.
- Temporary Placements: If, within the first four hours of work, you are not satisfied with our temporary employee or their work, we will replace the employee and you will not be billed for those hours.*
- Direct Placements: We have a 5-year warranty. In case of concerns during the first 5-years of employment, we will credit a pro-rated amount of the fee based on the number of active service calendar days.*
*Certain restrictions apply
We'd love to speak with you about your staffing needs.
As one of the most rapidly growing San Antonio staffing agencies locally as well as across all of North America, we specialize in permanent placement direct hire staffing, contract, temporary, temp-to-hire as well as a variety of other placements. Our primary focus includes the following industries:
Accounting and Finance Bookkeeping Billing Account Clerk Staffing and Recruiting
Human Resources HR Payroll HR Assistant Staffing and Recruiting
Information Technology / IT Desktop Support Help Desk Staffing and Recruiting
Executive Assistant, Clerical & Administrative Assistant, Legal, Receptionist, CSR, File Clerk, Office Manager Staffing
Oil and Gas / Engineering, Systems Engineer Staffing and Recruiting
Please contact your San Antonio Temporary Agencies professional staffing branch near you now for local professional staffing services.
Frontline Source Group | San Antonio Staffing Agency
San Antonio Temporary Agency
I just called this morning very professional when i called. This is a place of business straight to point on how to a...
Written by: Brandon Morrison
Employment Agency
Date published: 4/23/2018
5 / 5 stars
4.1 based on 15 Google reviews
Staffing Awards
Useful info
It is difficult hiring in this tight job market so when you are looking for best individual to add to your team in San Antonio make Frontline Source Group your staffing agency today!
San Antonio Temporary Agency and San Antonio Staffing Agencies
Upload date: 2019-12-13
Interesting and Fun fact about San Antonio, Texas:


text:
Its has been know for quite some time now that Persona 5 is in development, Altus has been very secretive about the game. For instance, we still don't know if it will be on PS3 following the trend of having a main series game on all of the past Sony systems, on PS4 and Vita as a next generation title, or if it will be a multi-platform game like Persona 4 Arena. Producer, Katsura Hashino reminded fans of the game once again last weekend at a Dengeki games festival broadcasted exclusively on NicoNico.
The video bellow shows Hashino thanking the fans and says that the team at Atlus is working very diligently to bring the next Persona game a reality. He also talks a bit about Persona 4 Golden. The video isn't subtitled sadly.
We haven't received much information regarding Persona 5 but since the Persona series is rather lacking in new games this year outside of Persona 4 Arena finally coming to Europe, it seems that a promotional video showing the main cast is in order. Only time will tell when they will reveal anything, bringing the mysteries of the next installment of the Shin Megami Tensei Persona series to light.


text:
KOSHA swiss made - Halskette Drei Feder Anhänger B01N7T5DQT
MORE THAN JEWELRY, URBAN JUNGLE SHIELDS - According to Amerindians, bird feathers provide protection and attract beneficial energies. Each feather has very specific properties, the OIZO collection integrates these benefits in three combinations of words delicately engraved at the back of each pendent. "Vitality - strength - courage" "Love - joy - spontaneity" "Serenity - harmony - wisdom" COTTON CORD OR EARRINGS - The OIZO pendants can be worn alone, combined on a cotton cord or as earrings. SWISS MADE - The OIZO collection is made in Switzerland by craftsmen that work with a particular attention to details. INNOVATIVE SLIDING SYSTEM - The cotton cord is equipped with an innovative sliding system developed by KOSHA. It allows to set freely the length of the necklace and also to easily open the buckle to change or combine pendants. SIZES AND MATERIALS - The OIZO pendants are available in 3 sizes: 80mm, 65mm, 50mm and 5 finishes: STAINLESS STEEL High quality stainless steel combining resistance and flexibility. PEARLY WHITE High quality stainless steel with pearly white varnish combining resistance and flexibility. PURPLE High quality stainless steel with pURPLE varnish combining resistance and flexibility. BLACK GOLD Alloy of 9 carat gold and rhodium layered on a stainless steel base. RED GOLD Alloy of 16 carat gold and copper layered on a stainless steel base. Each piece is covered with a transparent protective lacquer. GIFT BOX - Each pendant of the OIZO collection is provided in a gift box manufactured using recycled cardboard and white silkscreen printing.
Sie können auch mögen
€331.64 €118.53
Sie sparen 64% !
€107.98 €51.75
Sie sparen 52% !
€129.07 €44.27
Sie sparen 66% !
Sterling Silber rh-plated Kinder 5–5,5 mm Süßwasser-Zuchtperle 3-station Armband – 10 centimete B07681F85B
€107.47 €49.08
Sie sparen 54% !


text:
Stead Lane is proud to sponsor the Pottsville Beach Football Club and help support the community. We gladly provide ongoing support to the Pottsville Beach Football Club with their website, which we assisted in the development of and set up the domain and hosting platforms for. Working to help the community is something we take pride in so being able to sponsor and support the Pottsville Beach Football Club is a position we are honoured to fulfil.
At Stead Lane, we specialise in web development, user experience, web design, digital campaigns and branding strategies. We are a results driven agency with a focus on exceeding expectations and delivering the best possible results. We can provide just about anything for your digital needs whether it be a beautiful Shopify website, a digital advertising campaign or SEO improvements. We have the capabilities for most projects no matter how complex or overwhelming they may seem.


text:
After some minor delays, Sony's upcoming adaptation of Valiant Comics' Bloodshot is scheduled to begin filming next month.
The production was initially expected to begin production this past May before being pushed back to July. Now with filming locations for the Vin Diesel vehicle all but officially secured, principal photography has received a reported start date for August 6.
RELATED: Vin Diesel's Bloodshot Film Enlists Fantastic Four Alum
Shooting is expected to take place at Cape Town Film Studios, the same South African sound stages where 2012's Dredd and 2015's Mad Max: Fury Road were filmed. Additional photography will take place in Eastern Europe with both Prague and Budapest scouted as possible filming locations.
First announced in 2015, Sony announced a partnership with Valiant Comics to create a cinematic universe based on their superhero titles. Leading the wave of planned Valiant films is an R-rated adaptation of Bloodshot, Valiant's lethal super-soldier with incredible healing abilities and the power to hack into tech using the nanites in his bloodstream.
RELATED: CBR TV: Dinesh Shamdasani on the Advantages of Valiant's Shared Universe
Originally planned for release in 2017, Bloodshot will tentatively be released next year. It is marks the feature directorial debut of Dave Wilson after a prolific animation career from a script by Eric Heisserer (Arrival). The Valiant Comics adaptation stars Vin Diesel, Toby Kebbell (Fantastic Four), Eiza Gonzalez (Baby Driver), and Sam Heughan (Outlander).
(via Omega Underground)


text:
VIDEO - Cadwell Park 2019: Josh Brookes wins Race Two
See moreSee less
British Superbikes
Races of the year: British Superbikes in 201994 views • 04/12/2019 at 11:07
Races of the year: British Supersport in 2019167 views • 04/12/2019 at 11:09
Redding roars to 2019 BSB title: Every finish of every race512 views • 24/10/2019 at 10:15
The moment Redding sealed BSB title despite Brookes' heroic efforts3,551 views • 20/10/2019 at 17:53
'Just what he needed!' Brookes sets up final-race showdown with Redding748 views • 20/10/2019 at 14:38
Emotional Scott Redding on his BSB title, promises a 'big celebration'2,068 views • 20/10/2019 at 18:24
Bike nearly hits Luke Mossey during scary crash at Brands Hatch5,346 views • 19/10/2019 at 23:33
Watch: Brookes takes BSB title to the wire with Race One win at Brands Hatch2,157 views • 19/10/2019 at 23:33
Rider loses bike as it flies out of the track and into trees11,830 views • 19/10/2019 at 23:33
0Read and react
0Read and react
More videos
'Foul!' - O'Sullivan plays shot with both feet off the ground to end break
'I'm a bit OCD with germs' - O'Sullivan on referee fist bump
'What a start' - O'Sullivan hits 111 in just five minutes
'What a pot!' - Trump wins match with outrageous pink
Watch Higgins equal Hendry record with century
The Big Take - 'Ronnie's blown out the cobwebs', says Neal Foulds


text:
Like collectible video game pieces? Then you will definitely want to bid on this hand crafted Samus Aran Action Figure. The Auction is for 1 custom 100% hand made, and hand painted Samus Aran action figure. More after the jump.
N4G is a community of gamers posting and discussing the latest game news. It's part of NewsBoiler, a network of social news sites covering today's pop culture.


text:
Don Quixote, much like Moby Dick or Gulliver's Travels, is such a familiar part of the canon, that it often seems unnecessary to actually read it. Most educated or culturally aware adults know about tilting at windmills, and the image of the ragged knight with his trusted Sancho Panza by his side, dreaming that impossible dream, so much so that it seems almost superfluous to tackle this large, old book. When they do, they might discover that this book is far stranger, more surprising and ultimately more transformative than all but a handful of works of art ever created.
Miguel de Cervantes wrote Don Quixote in two volumes. Most of the famous moments come in volume one, published in 1605, the story of an old man, intoxicated by the popular picaresque books about knights and chivalry, renames himself Don Quixote, and sets off on his mount, Rocinante and alongside his squire, Sancho Panza, to prove himself worthy of his love, Dulcinea (actually a neighboring peasant girl). The second volume came 10 years later, and was Cervantes' response to the popularity of an unauthorized sequel written by a different author. Where the first part is more light hearted and satirical, part two becomes more serious and philosophical, as a deceived Don Quixote grapples with his sanity and the nature of reality.
Don Quixote was among the first European novels, and it remains one of the most central works of the Western Canon. Shakespeare supposedly adapted a section of the novel into his lost play, Cardenio. Authors such as Kafka and Borges reimagined and reinterpreted the adventures of the Knight of the Sorrowful Countenance and his squire. Artists such as Gustave Dore and Pablo Picasso have created visual representations of the book, and both Orson Welles and Terry Gilliam famously struggled for years to create a filmed adaptation (appropriately enough, both attempts could be seen as the very definition of quixotic).
Find and reserve this book in our catalog.
Tags: Classics, Epic, Literary Fiction, Rob C.'s Picks


text:
One of the things that make traveling less pleasant is having to carry bulky luggage. The more items you carry, the more items you have to keep track and ensure they are not lost, stolen or left behind. Generic looking bags, for instance, can easily get picked up by other passengers in the baggage claim conveyor.
Of course, packing less means fewer or even no options, but if we pack fewer things for our journey, there are also benefits that come with it.
1. You avoid bag fees.
While most carriers, especially established ones that fly to dozens of destinations offer generous luggage allowance, some, like Frontier Airlines, charge fees even for carry-on luggage. It may sound ridiculous but without extra bag to bring along, you avoid paying such fees.
2. You have less waiting time.
With no check-in luggage to wait for at the arrival bag claim conveyor, you are among the first passengers to pass through the airport exits. This means you get to taxi queue first or assured you don't miss your onward flight.
3. You have more convenience.
Without extra luggage tucked along, you'll be more comfortable locating certain things such as your hotel or the correct train carriage while walking around. You'll also less likely inconvenience others in a crowded area as without the bulky luggage you occupy less space.
Traveling with less luggage can be done but certainly depends on how comfortable are we with less attachments. So the next time you travel, examine closely the things you usually bring if they were proven to be indispensable in your last trip. Otherwise, it's time to pack less.


text:
Your basket is currently empty.
Signup today to get regular news and offers delivered straight to your inbox.
Free delivery
Safe and secure checkout
Credit and debit card payments
Copyright © 2019 Info Stor Limited. All rights reserved. E & OE. Registered in England and Wales Number 06621886.
Registered Office: Devonshire House, 60 Goswell Road, London, EC1M 7AD, United Kingdom. VAT number: 934 4316 30.


text:
Balanced Scorecard West Africa is a management consulting firm incorporated in Nigeria and specialized in strategy and performance management training and consulting services. Our strategy and performance management services employ the award-winning Balanced Scorecard Nine Steps to success framework. The company is an Affiliate of the Balanced Scorecard Institute, (member of Strategy Management Group, USA) for West Africa and the leading provider of balanced scorecard training and consulting services in the region.
Why Choose Us?
- Stand up cross-functional BSC development teams.
- Launch BSC development organization & leadership readiness assessment.
- Change management & communication workshop.
- Scorecard development workshops.
- Scorecard roll out & implementation
Balanced Scorecard What It Is and Why So Many Organizations Have One
Why Strategy Fails
Why Strategy Fails is a clear in-depth explanation on why strategies fail. Watch the full length presentation or Download the pdf version for free below. Download Why Strategy Fails
Keeping our Focus Firmly on Result
Kayode Komolafe, in his article, "So, What is the Grand Vision?" (THISDAY of November 18, 2015), has again demonstrated the importance of starting with strategy in the management of the economy. By the way, strategy [...]
2019 Training Calendar
You can view the 2019 Training Course Calendar by clicking here
2019 BSP Brochure
Click here to download brochure
KPI Certification Program
Download the KPI Certification brochures by clicking here.
PuMP Brochure
Download the PuMP Performance Measurement brochures by clicking here.
The balanced scorecard training from BSWA remains the best training I have ever had.
The course is extremely useful for key personnel in any organisation implementing Balanced Scorecard
Balanced Scorecard, an amazing way to ensure that we are all aligned to achieve the same goal!
I feel like I have gone abroad to study. The course material and facilities are excellent
BSC is a must know for companies who want to take strategy to the next level
No better way to execute strategy than using the balanced scorecard!
Better informed, more understanding of FUG pensions vision and mission statement
However beautiful the strategy, you should occasionally look at the result
BSC a good performance measure tool when implemented properly
No training, no improvement


text:
Exhibits and playroom
Lutsu str 8, 51006 Tartu, ESTONIA
Ph. +372 736 1550
E-mail: email@example.com
Open Wed-Sun from 11:00 am to 6:00 pm. Play room open Wed-Sun from 11:00 am to 4:00 pm
Closed on Mondays, Tuesdays and National Holidays.
Barbie 60
fashionable, miraculous, fairy-like world will open for the Toy Museum visitors
as hundreds of Barbie dolls are displayed until the end of
the year.


text:
Football has a long and successful tradition at the College and in the Lent term we field five senior and six junior teams who play competitive matches every week. All coaches involved with our teams have Football Association qualifications and many have played at a high standard themselves. The 1st and 2nd Xls plus the U15A Xl play in the recently formed ISFA Mercian league, where they have performed well so far, finishing in the top half of the table.
In the Autumn term Sixth Formers can opt to play Football if they are not required to play Rugby and we regularly field two recreational XIs who play competitive games on Wednesday and Saturday afternoons. We also enter a team in the Independent Schools Football Association Cup.
Girls' Football is played in both the Autumn and Lent terms, with training on Tuesdays and Saturdays. There is a full programme of fixtures at U18 level and a smaller number of fixtures at U15 level. Matches are regularly played against independent and state schools as well as local clubs.
Among the highlights have been winning the ISFA (Independent Schools Football Association) South-West of England Schools Sixes at both U18 and U15 level and four overseas tours between 2012 and 2016. The 2014-15 season saw the U18 Girls win the prestigious Mediterranean Cup in Verona against teams from Italy, the Czech Republic and the Middle East, and reach the Semi-Finals of the ISFA National Cup. Most recently, the U18 Girls won the SSS Rome Cup in March 2016, competing against the best international schools in Italy. In the past three seasons, four of our girls have gained selection for the ISFA U18 National Development squad.
I started my Football career at Yeovil Town FC, before moving onto University where I played for both Cardiff UWIC and represented Wales at Student level. After leaving university continued playing in the Southern western Leagues before moving into teaching. I currently hold FA Level preliminary badge and have taught and coached football at various schools including Millfield, and Malvern College.
I have coached a variety of boys and girls' sports teams in my two and a half decades at Malvern College, having played Rugby and Football at Lincoln College, Oxford University, and taken Physical Education as my subsidiary subject during my teacher training at University College, Cardiff, gaining coaching qualifications in Football and Rugby. I have thoroughly enjoyed coaching Girls' Football at Malvern College since 2008, with particular highlights for me being the four highly successful overseas tours to Spain and Italy, participating in tournaments in San Sebastian, Verona and Rome.
Share this page:
Print This Page:
Malvern College, College Road, Malvern, Worcestershire, WR14 3DF, UK
t: 01684 581 500 | e: firstname.lastname@example.com
Click here for further details
High Visibility View
Printer view
Standard View
Cookie Policy
Privacy Notice


text:
Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (∼10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection.
Shiga toxin-producing Escherichia coli (STEC) is a foodborne pathogen that can cause severe illness such as hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in humans (1). Fresh leafy vegetables have been implicated in many foodborne disease outbreaks, including an STEC O157:H7 outbreak associated with consumption of fresh ready-to-eat bagged spinach in 2006 that caused 100 hospitalizations and five deaths (2, 3). In an unrelated outbreak, bagged baby spinach was recalled in 2010 due to STEC O157:H7 contamination (4). The Centers for Disease Control and Prevention reported that between 1973 and 2012, STEC was second only to norovirus in the number of outbreaks associated with leafy vegetables in the United States, of which 46 of the 49 STEC outbreaks were caused by a serogroup O157 strain (2). Although STEC did not cause the greatest number of outbreaks, it did lead to by far the greatest number of hospitalizations for a given microorganism, with 46% of the total hospitalizations due to all leafy-vegetable-associated outbreaks attributed to STEC. Illness due to ingestion of STEC-contaminated leafy greens led to nine deaths, including six from spinach (2). With a calculated infectious dose of only 10 to 100 CFU (5–7), the ability to detect very low levels of STEC contamination on fresh leafy vegetables is important.
Currently, the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA BAM) protocol is used for the detection of STEC on leafy vegetables (8). In this procedure, the produce is enriched in medium for approximately 24 h and then screened by multiplex real-time PCR for the stx1, stx2, and uidA (+93 single nucleotide polymorphism [SNP] mutation indicative of O157 strains) genes. Unlike other foodborne pathogens such as Salmonella and Listeria, it is not sufficient to detect merely the presence of the bacterial species when determining whether E. coli contamination presents a risk to public health. Many E. coli strains are not pathogenic to humans, thus necessitating at least partial characterization of an E. coli strain associated with fresh produce to determine pathogenicity. In fact, highly virulent hybrid pathotype strains have been reported, for example, the E. coli O104:H4 STEC/enteroaggregative hybrid associated with sprouts that caused an outbreak with a high percentage of HUS in Germany in 2011 (9). A detection method that incorporates the ability to simultaneously determine the presence of multiple virulence genes would be ideal. If virulence characterization of a contaminating STEC strain is desired beyond the presence of stx1 and/or stx2 in the genome, the strain must be isolated and used in conventional PCR assays (as practiced in the BAM protocol) or sequenced. These are multistep processes that trade time for strain-level information.
Pyrosequencing of 16S rRNA genes has been utilized for direct detection of microorganisms and their proportions in microbial communities without the time-consuming step of culturing individual bacterial species for identification. This method of determining the composition of bacterial communities has been applied to surfaces of ready-to-eat vegetables, including spinach (10–13). Using such an approach to determine the presence of foodborne pathogens in the bacterial community associated with fresh bagged leafy greens may provide the ability to detect pathogens more quickly than current methods permit. However, high-throughput metagenomic shotgun sequencing has been used to investigate the taxonomic composition of microbial communities and can provide higher-resolution taxonomic information than 16S rRNA sequencing, as it makes use of either clade-specific marker genes (14) or matches to k-mers unique to a database entry (15, 16). The discriminative k-mer methods of metagenomic sequence data classification have increased the speed at which data can be processed, which is an important aspect of a pathogen detection method. While 16S rRNA sequencing would allow detection of a possibly contaminating E. coli strain, metagenomic shotgun sequencing might allow classification at a higher resolution, e.g., E. coli phylogroup, and only shotgun metagenomic sequencing, provided adequate sequencing depth was achieved, would provide the data for virulence gene characterization needed to deduce the potential pathogenicity of the strain.
The objective of this study was to explore the sensitivity of a metagenomic shotgun sequencing method of detecting contaminating STEC on bagged spinach. Specifically, we addressed the questions of how much contamination is necessary for detection without enriching the spinach sample and how long an enrichment process is necessary for detection of a very low level STEC contamination with deference to the established FDA BAM culture protocol (8). In addition, because we were able to determine the bacterial community associated with bagged spinach using this method, we focused on bacterial community changes with regard to enrichment time and changes to the enrichment process.
Spiked E. coli strain.STEC strain Sakai (chromosome [BA000007.2], pOSAK1 [AB011548.2], pO157 [AB011549.2] [GenBank accession numbers shown in brackets]), used for spiking experiments, was grown overnight in modified buffered peptone water with pyruvate (mBPWp) medium. The overnight culture was diluted appropriately to add 274 μl containing the target number of spiked cells. Dilutions of the overnight culture were spread on Luria-Bertani plates to determine the level of artificial contamination of the spinach. STEC strain Sakai was chosen for spiking experiments, as a closed genome is available and this strain carries both stx1 and stx2 along with the other virulence factors utilized in this work. In addition, STEC Sakai is a serotype O157:H7 strain, the pathogenic STEC serotype causing the greatest number of outbreaks associated with fresh produce (2). The Sakai strain is associated with E. coli phylogroup E and is an O157:H7 clade 1 archetype (17).
Sample processing.Packages of ready-to-eat spinach (227 g) were purchased from a grocery store and stored at 4°C until use. Experiments were carried out using a slightly modified version of the BAM protocol (Fig. 1) (8) that included a greater volume of medium at the start of the experiment to allow for removal of aliquots during the course of the enrichment process. To 1,627-ml filter whirlpak bags containing 100 g spinach, 250 ml of mBPWp medium was added and then spiked with the appropriate CFU of STEC Sakai. The bags were manually massaged and then shaken at 37°C and 185 rpm for 30 min. For preenrichment samples, the bags were again manually massaged, and 20-ml aliquots were removed and pelleted. The bags were then incubated statically at 37°C for 5 h and manually massaged, and 5-ml aliquots were removed and pelleted. Three antimicrobial agents, acriflavin, cefsulodin, and vancomycin, were added to the remaining 225 ml medium in each bag to final concentrations of 10 mg/liter, 10 mg/liter, and 8 mg/liter, respectively. The bags were then incubated statically at 42°C for 18 h and manually massaged, and 1- or 2-ml aliquots were removed and pelleted. For samples in which aliquots were not taken at the preenrichment time point or the 5-h time point, the starting volume of the medium was adjusted appropriately. For samples taken between 5 and 23 h of enrichment, 2-ml aliquots were removed and pelleted. All pellets were stored at −20°C prior to genomic DNA (gDNA) extraction.
DNA isolation, sequencing, and mapping.Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Germantown, MD, USA) following the protocol for Gram-positive bacteria to ensure the inclusion of both Gram-positive and Gram-negative bacteria present on the spinach samples. Sequencing libraries were prepared with the Nextera DNA sample preparation kit (Illumina, San Diego, CA, USA) and sequenced on the Illumina MiSeq platform, generating paired-end 250-bp reads. The number of reads per sample averaged 11,596,284 ± 3,571,586. Raw reads were trimmed and mapped to the STEC Sakai genome using CLC Genomics Workbench v7.5.1, v8.0, or v8.0.1 (CLC bio, Boston, MA, USA). Mapping parameters used were 98% identity for 100% of the trimmed read length. The same mapping parameters were used for reference-based assembly. The coverages of the chromosome and two plasmids harbored by the STEC Sakai strain were determined from the mappings using CLC Genomics Workbench. For selected samples, reads were also mapped to the Spinacia oleracea cultivar SynViroflay genome (GenBank accession no. AYZV01). In this case, the mapping parameters used were 80% identity for 80% of the read length.
Determination of bacterial community.Metagenomic analysis for microbial composition was conducted using custom C++ programs developed to compile a k-mer signature database containing multiple unique 25-bp sequences per target entry (bacterial species, phylogroup, or serovar) and then identify bacterial species in a sample by the unique 25-mers using the trimmed sequencing reads as input. Determination of database entries was based on previous work (18), BLAST results using some sequence reads generated in this work as queries, and results from several of the samples utilizing MetaPhlAn version 2.0.0 (14). For each species of interest, each nonduplicated 25-mer from a reference whole-genome sequence was placed into a database. We removed any k-mers not found in at least two-thirds of a set of additional genome sequences of the same species, and we removed any k-mer found in genomes of other species. The resulting k-mer database used in this work contains 155 target entries, each consisting of approximately 40,000 (range, 967 to 155,819) unique k-mers. The coverage of each species' 25-mer set was determined by testing each possible 100-bp read in a reference genome against the 25-mer set and tallying the number of reads in the genome that are matched by a 25-mer. The database includes 52 different bacterial genera and can differentiate between E. coli phylogroups A, B1, B2, D, and E. An attempt to identify each read from the sequencing run was made by matches to the 25-mer database. Normalization is performed to correct for bias due to different numbers of k-mers used per database entry, and the results are tabulated as the percentage of identified reads (contribution to the microbial population of identified species) for each database entry. For those samples for which MetaPhlAn was utilized in determining additional bacterial genera/species to add to our database, the results from our custom programs for the percentages of bacterial genera/species in the microbial community were in agreement with those obtained from MetaPhlAn. However, we were able to perform classification at a higher level for some genera, and particularly important, within the E. coli species.
Molecular serotyping and virulence gene detection.The numbers of trimmed reads mapping to the stx1a, stx2a, γ-eae, and ehxA virulence genes, as well as the wzm, wzt, wzx, wzy, and fliC molecular serotyping loci were determined using SeqMan NGen v12.2.0 using the default reference-based assembly parameters for Illumina reads (DNASTAR Inc., Madison, WI, USA). The two-tailed t test was used to calculate P values.
Metagenomic data accession number.Metagenomic sequence data from this study were deposited in the NCBI Sequence Read Archive (SRA) under accession number SRP062607.
Bacterial community associated with bagged spinach.The metagenomic sequencing data were analyzed in three ways, namely, by mapping the reads to the Sakai genome, determining the ability to serotype and discover the virulence profile of the spiked strain from the data, and determining the microbial community using a k-mer signature database, including detection of the spiked STEC (as percent E. coli phylogroup E) within the community analysis. For unspiked preenriched spinach samples, the background values for percent reads mapping to the Sakai genome and percentage of bacterial community as determined from the k-mer analysis were 0.03% and 0.004%, respectively. Metagenomic data analysis was performed on unspiked spinach samples at preenrichment, 5-h, and 23-h time points to determine the native microbial community associated with bagged spinach and how the composition changes throughout the enrichment process. Spiked samples that were collected preenrichment also revealed information about the indigenous microbial community. Considering a contribution of greater than 0.1% to the identified bacterial population, analysis on preenrichment samples indicated 16 bacterial genera associated with the prepackaged spinach, including six different species in the genera Pseudomonas (P. aeruginosa, P. alcaligenes, P. fluorescens, P. mendocina, P. putida, and P. stutzeri) and three Exiguobacterium species (E. sibiricum, E. antarcticum, and E. undae). Most of the genera displayed low abundance, as only Pseudomonas spp., Pantoea agglomerans, Exiguobacterium spp., and Sutterella wadsworthensi were present at greater than 1% of the identified community in at least one sample. Preenrichment, the predominant bacterial species was P. fluorescens (more than 60% of bacterial population), with either P. agglomerans or E. sibiricum the second most abundant species. However, the microbial composition was observed to change with enrichment time so that after 5 h of enrichment, P. agglomerans became the predominant species in some samples, while Exiguobacterium spp. became the predominant species in others, Aeromonas spp. grew in abundance in some samples, and the percent P. fluorescens contributing to the bacterial community was greatly diminished. After 23 h of enrichment, more than 75% of the reads associated with identified species were attributed to Enterobacter cloacae, P. agglomerans had a lower percent abundance than at preenrichment, and the contribution of Pseudomonas spp. was less than 1%. E. coli phylogroup B2 was discovered in one bagged spinach sample at a low level of abundance, 0.109% of the population of identified species. Molecular serotyping identified E. coli O45 and O101 loci in this sample; therefore, there were two different strains in this sample. These strains did not possess the common STEC virulence factors stx, eae, and ehxA. In addition to the metagenomic community analysis, the reads from unspiked samples were mapped to the spinach genome. The results reveal that 41%, 6.3%, and 1.7% of the total reads could be attributed to spinach DNA for the samples at preenrichment, 5 h, and 23 h of enrichment, respectively.
Detection limit for preenriched samples.Spinach samples were spiked with STEC Sakai at levels ranging from 10 to 10,000,000 CFU/100 g spinach and processed preenrichment (Fig. 1). At the lower levels of contamination, 10 and 1,000 CFU, the spiked Sakai strain could not be detected (Tables 1 and 2). At the 10 and 1,000 CFU spike levels, the few reads mapping to the Sakai genome were at the rRNA-encoding loci, thus possibly due to very small amounts of an E. coli strain in the native bacterial community. At the 100,000 CFU spike level, the spiked strain was detected in the bacterial community analysis, and the reads mapping to the Sakai genome were more dispersed over the length of the genome, although the percent mapping was not above the background level (0.03%). The Sakai strain could be detected with greater confidence at the 1,000,000 CFU spike level; however, the virulence profile could not be reliably determined, as no reads mapped to the stx2a gene or one of the loci used for serotyping (Table 2). The Sakai strain was easily detected, and the serotype and virulence profile were determined at a spike level of 10,000,000 CFU. Some variation in detection sensitivity was observed between different bags of spinach. For example, the number of reads mapping to the stx2a gene varied between 6 and 26 reads among the three triplicate samples. Although the bacterial community found on the bagged spinach preenrichment varied in the percentages of minor species found, it was so dominated by P. fluorescens that biological replicates taken from different bags appeared quite similar (Fig. 2). Minor quantities, between 0.1 and 1.0%, of other species contributing to the overall community in at least one of the samples included Enterococcus faecalis, Lactobacillus sanfranciscensis, Listeria monocytogenes, Salinibacter ruber, Alistipes spp., Clostridium botulinum C, Lactococcus garvieae, Akkermansia muciniphila, Stenotrophomonas maltophilia, and Edwardsiella tarda (see Table S1 in the supplemental material).
Detection limit for samples enriched for 5 h.Spinach samples were spiked with STEC Sakai at levels ranging from 10 to 100,000 CFU/100 g spinach and processed at 5-h enrichment time (Fig. 1). Analysis of the metagenomic data revealed that the spiked strain could be detected in the community analysis at 1,000 CFU, and although many reads mapping to the Sakai genome were located at rRNA-encoding loci, there were also reads distributed over the length of the genome. However, information about serotype and full virulence profile could not be determined at this spike level (Table 2). At a spike level of 10,000 CFU, the metagenomic data contained sufficient reads associated with the Sakai strain to fully characterize the strain. In fact, an average chromosome coverage of 13× was obtained (Table 1). As was observed for the preenrichment samples, there was variable detection sensitivity between spinach bags. The number of reads mapping to the stx2a gene ranged from 22 to 128 for a spike level of 10,000 CFU. At a contamination level of 100,000 CFU/100 g spinach, significant chromosomal coverage was obtained from the metagenomic data, and the consensus sequence of the Sakai genome from a reference-based assembly contained only seven gaps totaling 265 bp. There was some variation between biological replicates in the composition of the bacterial community found in the samples after 5-h enrichment time and in the percent contribution from the spiked Sakai strain (Fig. 2). In addition, the metagenomic data revealed greater variation in bacterial composition in the samples enriched for 5 h in comparison to preenrichment. Along with the bacterial species included in Fig. 2, one of the samples (Fig. 2, column 2c) was found to contain 1.8% Yersinia enterocolitica contributing to the sample community. Although each contributed between only 0.1 and 1.0% to the overall population, other bacterial species, Bacillus spp. (other than B. cereus), Alistipes spp., L. garvieae, Leuconostoc lactis, and E. tarda were present in at least one sample (see Table S2 in the supplemental material).
Detection limit for samples enriched for 23 h.Metagenomic sequencing data were obtained from spiked spinach samples that were incubated for an additional 18 h at 42°C after the 5-h enrichment at 37°C for a total of 23 h (Fig. 1). Samples were prepared at STEC Sakai contamination levels of 10 and 1,000 CFU/100 g spinach. After 23-h enrichment, the spiked strain constitutes approximately half the bacterial community for a spike level of 10 CFU, resulting in a chromosomal coverage of 184× (Table 1). The metagenomic data contains a far greater number of reads than are necessary to determine the molecular serotype and virulence profile with confidence (Table 2). The consensus sequence of a reference-based assembly of the STEC Sakai genome was determined to contain only six gaps totaling 200 bp. A large shift in bacterial composition of the sample was observed, resulting in E. coli phylogroup E (spiked strain) and E. cloacae as the predominant species (Fig. 2). One of the samples (Fig. 2, column 3b) was found to contain 1.1% Proteus mirabilis contributing to the sample community along with the bacterial species included in Fig. 2. Other bacterial species, contributing between only 0.1 and 1.0% to the overall population, were present in at least one sample including Cronobacter spp., E. tarda, Klebsiella oxytoca, E. coli phylogroup B1 (serogroup determined to be O101), and Salmonella enterica serovar Heidelberg (see Table S3 in the supplemental material).
Determination of the shortest enrichment time to detect the lowest limit of contamination.Clearly, 5-h enrichment time is not long enough to detect the STEC Sakai strain at the low spike level of 10 CFU/100 g spinach, but the full subsequent 18-h incubation at 42°C is more than sufficient time. We sought to determine the minimum enrichment time necessary to detect 10 CFU. Sequencing samples were prepared at intermediate enrichment times, namely, 2, 3, 4, and 6 h of enrichment at 42°C after the 5-h incubation at 37°C. The spiked STEC Sakai strain appears to be transitioning between lag and exponential-phase growth at 7-h enrichment, at which point 0.22% of the reads map to the Sakai genome, and 0.322% of the bacterial community is attributed to E. coli phylogroup E (Fig. 3). The serotype and virulence profile cannot be reliably determined at 7-h enrichment, but by 9 h, there are hundreds of reads mapping to each virulence gene, and 22% of the metagenomic sample reads map to the Sakai genome. Intermediate to these time points, 8 h of enrichment yields an average of 2.85% reads mapping to the Sakai genome and average genome coverage of 10.5×. Furthermore, 5.647% of the bacterial community consists of E. coli phylogroup E, and the spiked strain is present in sufficient quantity to reliably determine the molecular serotype and virulence profile (Fig. 3 and 4). Enrichment beyond 11 h does not increase the ability to detect the spiked strain appreciably or to gain additional information about the strain, as it appears to enter stationary growth phase (Fig. 3). Although the quantitative results show disparity between the two methods of determining the contribution of STEC Sakai to the total sample, especially at 11 and 23 h, the time courses are consistent with each other. The bacterial population in the samples enriched for 8 h exhibited minimal variation and was dominated by P. agglomerans and Exiguobacterium spp. (Fig. 5). Although each contributed between only 0.1 and 1.0% to the overall population, other bacterial species were present in at least one sample; these species included Bacillus coagulans, L. garvieae, E. cloacae, E. tarda, S. wadsworthensi, and Citrobacter freundii (see Table S4 in the supplemental material).
Effects of changes in the enrichment process.After determining that 8 h is the shortest enrichment time for detection of STEC Sakai at a spike level of 10 CFU/100 g spinach, we sought to investigate how changes in the enrichment process would affect the detection sensitivity. To determine the effect of shaking, samples were shaken for 5 h at 37°C before increasing to 42°C for the remaining 3 h instead of incubating samples statically for 5 h at 37°C (Fig. 1). Shaking the sample had an effect on the proportional growth of the STEC Sakai strain compared to other bacterial species. While an average of 2.85% of the reads mapped to the Sakai genome from metagenomic samples that were incubated statically for 8 h, only 0.75%, significantly less (P = 0.042), mapped from samples that were shaken for the first 5 h of the 8-h enrichment process. Although the serotype and virulence profile could be determined, there were much fewer reads associated with the relevant genes (Fig. 4). For direct comparison, a pair of samples, one incubated statically and the other with shaking, were processed from the same bag of spinach (Fig. 5, bars 1c and 2a). The bacterial community analysis resulting from the metagenomic data suggests that Aeromonas spp. may grow preferentially at the expense of the spiked STEC strain when the sample is shaken. In addition, shaking appears to favor the growth of Paenibacillus. Other changes in population abundance were minor (see Table S4 in the supplemental material).
Another variation to the protocol investigated was the addition of antimicrobial agents after the first 5 h of incubation (Fig. 1). The bacterial community of a sample enriched for the entire 23 h, but without the addition of the antimicrobials, was determined to be comprised of 63% E. coli phylogroup E (see Table S3 in the supplemental material) compared to 67% processed with antimicrobials (Table 1); thus, the spiked STEC Sakai could be as easily detected and genomically characterized without the addition of antimicrobials as with them. In addition, this sample was determined to have 10% and 6.9% of the bacterial population comprised of E. coli phylogroup B1 (serogroup nontypeable) and Klebsiella pneumoniae, respectively (Table S3). Other E. coli phylogroups were detected in lower amounts, namely, E. coli phylogroups A (0.383%, serogroup O9) and D (0.102%). We sought to determine whether it was advantageous to add the antimicrobials if the enrichment time was only 8 h. The results indicate that the addition of the antimicrobials greatly affects the detection sensitivity at spike levels of 10 CFU and enrichment time of 8 h (Fig. 4 and 5). Only an average of 0.53% of the reads from the metagenomic samples prepared without addition of antimicrobials mapped to the Sakai genome, a statistically significant difference (P = 0.030) compared to 2.85% when antimicrobials were added. Relevant genes for serotyping and virulence profile determination had sufficient coverage; however, the coverage was significantly reduced (Fig. 4). A pair of samples was processed from the same bag of spinach for direct comparison of the associated bacterial community (Fig. 5, bars 1b and 3a). The results indicate a dramatic shift in bacterial population, with P. agglomerans, Exiguobacterium spp., and Pseudomonas spp. crowded out by B. cereus and Aeromonas spp. Another sample for which antimicrobials were not added is almost entirely comprised of Paenibacillus. K. pneumoniae or other E. coli phylogroups were not detected in the samples enriched for 8 h without antimicrobials. Other minor changes in population abundance were detected (see Table S4 in the supplemental material).
Variables associated with defining STEC detection limit.Although microbial community analysis of metagenomic samples using 16S rRNA sequencing has been applied to the study of bacterial communities associated with fresh vegetables, including spinach (10-13, 19), our study was aimed at expanding this concept to determining the sensitivity of metagenomic shotgun sequencing for detection of a pathogen contaminant associated with the spinach. We were able to define limits for detection of STEC on fresh bagged spinach at preenrichment and at 5-h and 23-h time points during the enrichment process following the BAM protocol (8) and a sequencing depth of 10,000,000 reads per sample (Tables 1 and 2). Our results demonstrate that the level of contaminating STEC necessary for detection preenrichment is 10,000,000 CFU/100 g spinach. This suggests that the overall microbial load associated with the ready-to-eat bagged spinach is too great for utilizing a metagenomic approach without some enrichment time to select for the contaminating STEC. In one study, plate counts from conventionally grown ready-to-eat packaged baby spinach determined the viable counts of culturable bacteria to be 108 to 109 CFU/g spinach (12), while in another study, total bacterial counts were found to vary widely from 103.9 to 108.2 CFU/g (20). In yet another study, Babic et al. found the initial populations of mesophilic and psychrotrophic aerobic microflora were each 106 to 107 CFU/g spinach but increased to 1010 CFU/g each during 8 days of storage at 10°C (21). Not only does this high indigenous bacterial load present difficulty in detecting very low levels of STEC contamination, but for preenriched samples, we determined that approximately 41% of the total metagenomic sample sequencing reads map to the spinach genome, thus necessitating even deeper sequencing to detect low levels of a contaminating pathogen. Similar to preenriched samples, our results indicate that the metagenomic method would be impractical at 5-h enrichment without deeper sequencing. However, following the complete 23-h enrichment process described in the BAM protocol (8), we were able to demonstrate that a low level of STEC contamination, 10 CFU/100 g spinach, could be detected. In fact, a reference-based assembly produced an almost complete genome, thus allowing full characterization of the contaminating STEC strain. Our results show that after 11-h enrichment, the STEC had completed the exponential growth phase, and therefore, further enrichment time is of no advantage (Fig. 3). By 8 h, the STEC strain could be detected, and the virulence profile could be determined (Fig. 4). In fact, so many reads mapped to the virulence genes at a sequencing depth of 10,000,000 total reads that it would be possible to detect and characterize the STEC without as great a sequencing depth, thus allowing for more samples to be processed in a single sequencing run. Alternatively, if time were not a factor, samples could be enriched longer than 8 h, and it would be possible to sequence an even greater number of samples contaminated with as little as 10 CFU/100 g spinach and still gain a significant amount of strain-level information. Our results illustrate the interplay between time, cost, and strain-level information gained when utilizing a metagenomic approach to pathogen detection. The limit of detection for a given enrichment time and number of total reads is dependent on the goal, i.e., whether mere knowledge of the presence of STEC is satisfactory or an assembly is required.
Assembly of the STEC genome from metagenomic sequencing data.The determination that a bag of spinach is contaminated with STEC does not require a draft whole-genome sequence of the contaminating strain; however, a genome sequence would provide essential information during an outbreak or traceback analysis. We performed reference-based assemblies using the metagenomic sequencing reads from several of our spiked samples. Our data suggest that the contamination level would need to be a very high value, 100,000,000 CFU/100 g spinach, to achieve ∼30× coverage of the spiked strain in preenriched samples. However, we were able to attain genome consensus sequences averaging only six gaps totaling 200 bp for samples spiked with 10 CFU/100 g spinach and enriched for 23 h. As with virulence gene detection discussed above, our results demonstrate that if samples spiked with 10 CFU/100 g spinach were enriched for 23 h, adequate genome coverage could be obtained for assembly with less than 10,000,000 reads per sample. Eliminating the need to first isolate a contaminating STEC from a sample enriched for 23 h by plating on selective medium and incubating overnight, followed by growth of a single colony isolate overnight for genome sequencing, can shorten the time necessary for obtaining substantial strain-level characterization by 2 days. At 8-h enrichment time, determined to be the shortest time to obtain the serotype and virulence profile of the spiked strain at an initial level of 10 CFU/100 g spinach, the average genome coverage was determined to be 10.5×. This result indicates that for 8-h enrichment, a contamination level of ∼30 CFU/100 g spinach would be sufficient to yield a genome sequence with ∼30× coverage. Because the spiked strain is just entering log phase growth at 8-h enrichment (Fig. 3), adding even 30 min to the enrichment time would make a significant difference as to whether a draft genome sequence of sufficient quality could be extracted from the metagenomic data for these very low contamination levels. Since the genome sequence of the contaminating STEC strain would be unknown in a produce screening or outbreak analysis situation, ultimately, it would be desirable to assemble the genome de novo, rather than as a reference-based assembly, as was possible in the present study. Extracting reads for the de novo assembly of a single genome from metagenomic sample reads is challenging, but it has been attempted with some success from a fecal sample containing STEC from an outbreak (22). These investigators used a method whereby they subtracted sequencing reads identified as either human or the microbial community found in healthy human fecal samples. Our results reveal that the percentage of total metagenomic sequencing reads matching the spinach genome declines quickly, from 41% to 6.3%, during the first 5 h of enrichment, thus for shorter enrichment times, subtracting the reads identified as matching the spinach genome could be useful. We have identified the bacterial species most often found associated with fresh bagged spinach at different time points throughout the enrichment process, and this information may prove valuable for future de novo assemblies if a subtractive approach is used to extract reads associated with the contaminating STEC strain.
E. coli virulence gene identification and typing.We detected native E. coli associated with the bagged spinach in three of the samples that had been enriched for 23 h. The fact that native E. coli was detected only after 23-h enrichment suggests that the initial number of E. coli bacterial cells on the bagged spinach was very low. The genomes of the E. coli O45 and O101 strains identified in an unspiked sample did not carry Shiga toxin genes or other common STEC virulence factors. The other indigenous E. coli strains identified in spiked samples belonged to E. coli phylogroups B1 (serogroups O101 and nontypeable), A (serogroup O9), and D (insufficient reads to determine the serogroup). Because these E. coli strains were found in spiked samples and the quantity was low, it cannot be definitively determined whether they carry the STEC virulence factors included in this study. The discovery of nonpathogenic E. coli associated with one or some of the spinach samples reinforces the need to identify virulence factors. This necessitates a greater number of reads mapping to the E. coli genome than would be necessary for detection relying solely on 16S rRNA sequencing of metagenomic samples. In the present investigation, only one STEC strain was used for spiking; however, we were able to determine the molecular serotype and identify the intimin gene allele type along with both Shiga toxin gene allele types carried by this strain. Determination of gene allele types and serotype is important, as they can be used to aid in determining the risk a particular contaminating strain poses to human health. Other virulence factors known to contribute to STEC pathogenicity could be included, and although we focused on STEC in this work, molecular markers for all of the E. coli pathotypes (1) could easily be incorporated into the analysis of the metagenomic data.
Indigenous microbial community associated with bagged spinach.Our study revealed that considering bacterial genera or species that contribute at least 1% to the population, the diversity of the bacterial community associated with unenriched fresh bagged spinach is low and that Pseudomonas spp., and in particular P. fluorescens, is the dominant bacterial species (Fig. 2). By pyrosequencing of the 16S rRNA gene, Jackson et al. also found that Pseudomonas spp. dominated (93.8%) the bacterial community associated with conventionally grown bagged baby spinach (12). Additionally, that study reported a lower operational taxonomic unit richness for baby spinach than for four types of leaf lettuce. Another study also reported that the most numerous 16S rRNA sequences associated with fresh washed spinach were identified as Pseudomonas spp. (9.2%); however, 13.7% of the sample was classified in the family Enterobacteriaceae (13). In contrast to our results, Leff and Fierer reported that although the microbial community on prepackaged spinach was dominated by Gammaproteobacteria, more than 50% of the sample consisted of bacteria belonging to the family Enterobacteriaceae rather than Pseudomonadaceae (4%) (10). In that study, Pantoea sp. was found with a relative abundance of 32.4%, and a low abundance of Paenibacillaceae and Exiguobacteraceae was discovered. Although the relative abundances are different, this is consistent with several of the bacterial genera we found in our work. Previous to the widespread use of molecular methods for determining bacterial species associated with fresh packaged spinach, Babic et al. used plating on media to differentiate and enumerate bacterial species (21). These investigators report that P. fluorescens was the most abundant species and that Pseudomonadaceae were found at numbers of approximately 2 log units greater than those of Enterobacteriaceae. In general, our community analysis results obtained from metagenomic shotgun sequencing analysis using discriminative k-mers are in agreement with microbiological and 16S rRNA sequencing methods as to the major genera associated with ready-to-eat spinach. However, we were able to obtain higher taxonomic resolution by differentiating presence and abundance contributions to the species level, and we were also able to identify low-abundance species. Lopez-Velasco et al. report changes in the spinach microbiome with storage, even at 4°C (13), Babic et al. report growth of both Pseudomonadaceae and Enterobacteriaceae with storage at 10°C (21), and others report changes in the microbial communities in ready-to-eat packaged vegetable salads with storage (19). Storage time and conditions may be factors that explain some relative abundance differences between studies, as well as season and type of irrigation as was reported for lettuce microbiomes (11).
Shifts in the microbial community affect STEC detection sensitivity.Enriching the spinach samples shifts the population in the microbial community so that initially low-abundance bacterial species may become more abundant and small differences in initial abundance may become magnified as exemplified by our results (Fig. 2). Along with some variation in abundance of species contributing at least 1% of the identified sample population between different bags of spinach, there was further minor variation revealed in the presence/absence of very low abundance bacterial species as noted by our results for bacterial species contributing between 0.1 and 1.0% to the total population. Clearly, 5-h enrichment at 37°C causes a dramatic shift in the relative abundances within the bacterial community (Fig. 2). The growth of some genera or species is favored over others, and there is greater variation in the abundances between biological replicate samples. It is expected that a complicated interplay between the presence and abundance of a particular member of the bacterial population in the sample and the other members will influence the resulting community at any given time during enrichment. The spiked STEC Sakai strain, as a member of the bacterial community, is also subject to the influence of the growth pressures from competing bacterial species. During the first 5 h, the spiked STEC is growing very slowly (Fig. 3), while it appears Pantoea agglomerans and Exiguobacterium spp. are multiplying at a higher rate and/or Pseudomonas spp. are unable to survive (Fig. 2). Despite the different bacterial population compositions of samples spiked with 10,000 CFU/100 g spinach and enriched for 5 h, the spiked STEC strain could be detected and characterized for serotype and virulence factors (Fig. 2 and Table 2). Not surprisingly, given the similar proportion of STEC Sakai to other bacteria in the samples and the fact that the STEC strain is just transitioning to exponential growth phase at 8 h, a comparison of the compositions of the bacterial communities in spinach samples spiked with 10,000 CFU and enriched for 5 h and that of samples spiked with 10 CFU and enriched for 8 h reveals considerable similarity (Fig. 2 and 5). However, by the end of 23-h enrichment, even at a low spike level of 10 CFU/100 g spinach, the STEC Sakai strain was able to outcompete other bacteria in the microbial community (Fig. 2). This may be partly due to the fact that the 18-h incubation is performed at 42°C, since some bacterial species, P. fluorescens included (23), do not grow as well as STEC does at this temperature.
Along with growth competition due to other bacterial species in the sample community, the growth of the STEC strain is affected intrinsically by the enrichment procedure. Baylis investigated the growth of 20 STEC strains under various conditions and reported that the growth of the STEC strains during enrichment was at least to some extent strain dependent and depends on a combination of factors, including temperature, media, and the concentration and combination of antibiotics (24). Ideally, these conditions would be optimized for the selective growth of STEC compared to the rest of the sample community. One change to the protocol we investigated in this work was the lack of addition of antimicrobials. Since a sample spiked with 10 CFU/100 g spinach and enriched for 23 h without the addition of antimicrobials was comprised of 63% E. coli phylogroup E, similar to the samples processed with antimicrobials (67%), the STEC Sakai strain appears to be able to outcompete other members of the community even without the aid of the antimicrobials if given enough time. Although the lack of addition of antimicrobials during sample processing did not appear to affect the ability to detect and characterize the STEC Sakai strain after 23-h enrichment, the lack of antimicrobials did greatly affect the ability to detect and characterize the spiked strain after only 8 h of enrichment (Fig. 4 and 5). Our results indicate that the antimicrobials inhibit the growth of the Gram-positive bacteria Bacillus cereus and Paenibacillus. Another change to the protocol that we tested, since E. coli grows well when shaken, was to shake the spiked samples rather than incubating statically for the first 5 h. Interestingly, this change led to a reduction in the percentage of the bacterial community identified as the spiked STEC strain when samples were taken at a total enrichment time of 8 h (Fig. 4 and 5). It appears that shaking favors the growth of Aeromonas spp. at the expense of the STEC strain. This exploratory work will be extended in future studies to investigate enrichment medium composition and incubation times and temperatures to ascertain the effect on the composition of the microbial community and the STEC detection level.
In conclusion, our work highlights the potential for utilizing a metagenomic approach to detect STEC on fresh produce, thereby eliminating the need to first isolate and culture a contaminating STEC strain in order to detect and characterize it. Assessment of the detection sensitivity using this approach has shown that strain-level characterization can be achieved even for very low levels (10 CFU/100 g spinach) of STEC contamination with less than the approximately 24-h enrichment currently used in the FDA BAM protocol (8). Along with determining STEC detection limits following the BAM protocol for enrichment, our metagenomic sequencing study was aimed at examining how the microbial community associated with spinach changes with modifications to the enrichment process, and we were able to determine this to the species level. We anticipate that this initial work will aid future work on optimizing the enrichment medium and/or protocol to enhance detection of STEC with a reduction in enrichment time. Knowledge gained in the present study about the bacterial community associated with spinach and how the population shifts during the BAM protocol will be a useful guide. In addition, this metagenomic shotgun sequencing approach can be applied to other foodborne pathogens.
This research was funded in part by the Oak Ridge Institute for Science Education.
- Received 10 August 2015.
- Accepted 15 September 2015.
- Accepted manuscript posted online 18 September 2015.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02601-15.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.


text:
Week ninety seven of Operation Graphite – theme: Atomic Age.
Atomic Age sci-fi might encompass a few sub genres, and many of the classic Atomic age stories and characters actually originated in the earlier, Golden Age of Sci-fi. While the genera is not as rigidly defined as Steampunk or Space Opera, there is something about the post war sci-fi that stands apart from other ages. Something about fear of the atom, paired with a new vigor for exploration and modernization make an interesting cocktail.
Contributing Atomic Punks:
Micah – Freak
Shock – Cosmic Rays
Thank you for pushing the limits of science.


text:
Iron-binding capacity
Iron-binding capacity is a topic covered in the Guide to Diagnostic Tests.
To view the entire topic, please sign in or purchase a subscription.
Medicine Central™ is a quick-consult mobile and web resource that includes diagnosis, treatment, medications, and follow-up information on over 700 diseases and disorders, providing fast answers—anytime, anywhere. Explore these free sample topics:
-- The first section of this topic is shown below --
Iron -binding capacity, total, serum (TIBC)
250–460 mcg/dL
[45–82 mcmol/L]
-- To view the remaining sections of this topic, please sign in or purchase a subscription --
McPhee, Stephen J., et al. "Iron-binding Capacity." Guide to Diagnostic Tests, 7th ed., McGraw-Hill Education, 2017. Medicine Central, im.unboundmedicine.com/medicine/view/GDT/619209/all/Iron_binding_capacity.
McPhee SJ, Lu CM, Nicoll D. Iron-binding capacity. Guide to Diagnostic Tests. 7th ed. McGraw-Hill Education; 2017. https://im.unboundmedicine.com/medicine/view/GDT/619209/all/Iron_binding_capacity. Accessed December 8, 2019.
McPhee, S. J., Lu, C. M., & Nicoll, D. (2017). Iron-binding capacity. In Guide to Diagnostic Tests. Available from https://im.unboundmedicine.com/medicine/view/GDT/619209/all/Iron_binding_capacity
McPhee SJ, Lu CM, Nicoll D. Iron-binding Capacity [Internet]. In: Guide to Diagnostic Tests. McGraw-Hill Education; 2017. [cited 2019 December 08]. Available from: https://im.unboundmedicine.com/medicine/view/GDT/619209/all/Iron_binding_capacity.
* Article titles in AMA citation format should be in sentence-case
TY - ELEC T1 - Iron-binding capacity ID - 619209 A1 - McPhee,Stephen J, AU - Lu,Chuanyi Mark, AU - Nicoll,Diana, BT - Guide to Diagnostic Tests UR - https://im.unboundmedicine.com/medicine/view/GDT/619209/all/Iron_binding_capacity PB - McGraw-Hill Education ET - 7 DB - Medicine Central DP - Unbound Medicine ER -