Immunological Features and Clinical Benefits of Conjugate ...

LONGEVITY
Complete a Life Complete a full life
All you have to do for this one is die. You probably have it by now, but if you're super attached to your first Bitizen, you can always save your Bitlife and play somebody else wastefully or hold out until they pass.
Octogenarian See your 80th birthday Nonagenarian 90th birthday Centenarian 100th birthday Super-centarian 110th birthday Mega-centarian 120th birthday
Get on a healthy diet and garden and meditate twice a year. I like Nutrisystem. It's expensive, but I have advice for managing that below...
Strong Genes Achieve a 500-year generation Long Lineage 1000-year generation Living Legacy 5000-year generation
Never don't have kids. I like leaving everything to the youngest child and playing as them, but that won't make your kids happy with you or your heir. Your call. Either way, it helps to have a couple Bitlifes going in case you get tired of living carefully. Sometimes you're gonna want to be more reckless, you know?
WEALTH
Millionaire Become a millionaire My Second Million Achieve a net worth of $2m
Now that we have Royalty and Sports, this is a lot easier. Traditionally, if you're hot (95%+), drop out of high school and get your GED ($1k, you can do that in a couple years of dog walking/freelance gigs) and wait for a singer or actor career. If not, work hard in school and go to the gym often. Check your parents' stats and if they're generous make sure you pass your drivers test (maybe even ask if you can get a nicer one! immediately sell your car, they lose value fast) and take a martial art. It's much cheaper if they pay for it ($1K per tier in some countries) and gets you in good shape. If you're athletic, grind at a sport from middle school onwards. If you're not, try some athletic-adjacent clubs and go to the gym and for walks often. Pets count as additional walks which you can take from age 8+. When you're in good shape you can get a soccer scholarship (which can become a/)or a professional sports contract. More sports tips below, same with other careers. Basically try to get famous, not through politics. Or be hot and marry rich/have rich parents who die/be royal.
Multimillionaire Achieve a net worth of $10m Rich Net worth of $20m Super Rich Net worth of $50m Stinking Rich Net worth of $100m
Get a couple million first, then invest it in real estate. Or do ads if you're famous and it won't ruin your career. Helps to be big on social media for influence on that stuff. Fix up 1M+ houses and flip them when they've hit a value of 2 or 3 million. If you've got great karma or you're a religious figure of some kind, exorcise some mansions. You can do it all that way, or keep grinding careers.
Bitionaire Achieve a net worth of $1b
It's hard to get here from 0. Helps to leave everything to your youngest kid before you die after living a long, fruitful life. You can let your kid "take over" your assets at any time without tax now, that's the best way to do it. Then as soon as they're 18 make them famous/invest in real estate and repeat.
CAREER
Actor Become an actor
Be hot. You can drop out at 16 and get your GED for $1k (ask your parents for money or do freelance gigs for a couple of years). If you're not, go to the gym and for walks often. Pets count as additional walks which you can take from age 8+. Grind at a sport in school if you can to keep your health well and get plastic surgery at 18. Generally if your appearance stats are low it's either a nose job or liposuction that will fix it. Always go to the best plastic surgeon. Marry rich if you have to or work for a couple years if you have to, but start the career as soon as you can. Always work 5 more hours a week than required and compliment your supervisor if their coolness is high. Sleep with people in Hollywood (bosses coworkers etc). Your spouse/parent will generally be mad if you're in rude magazines, so hold off on dating unless they're cool or make them deal with it.
Airplane Pilot Become an airline captain
Grind in school and keep your mental health well. No drugs but drink if you feel like it, just make sure you can go to AA or whatever. Always work 5 more hours a week than required and compliment your supervisor if their coolness is high. If you're rich and you've inherited an airplane or you can afford lessons, take them. Go to University for a science thing that isn't biology lol. Start your Pilot Apprentice job.
At Inner Peace Work 75 years as a monk
Follow my longevity tips above and don't party or drink or do drugs. Always be honest. Meditate. Don't date.
Candywriter Work for Bitlife
Be born in Tampa, United States. Go to university for Information Systems. You'll get the achievement right away when you're hired.
CEO Become a CEO
Go to school for Finance. Get a job. Work hard every year.
Dentist Become a dentist
Go to university for biology, then dental school. Work hard every year.
Doctor Become a doctor
Go to university for biology, then medical school. Work hard every year.
Fire Chief Become a fire chief
Stay in good shape. Work hard every year.
Jack Of All Trades Have 10 careers in one life
Work at retail and food service jobs for less than a year, then go to university to get even more opportunities. Keep going for different paths.
Judge Become a judge Lawyer Become a lawyer
Go to school for english. Go to law school. Work hard every year.
Last Resort Seduce your boss to save your job
Be hot. Work fewer hours than required at your job. Make sure your supervisor is attracted to your gender and low professionalism. When your boss tries to fire you, seduce them.
People Person
Start with your less popular coworkers and work your way up. Pay attention to their stats so you know what they want. Get hard-to-get people with Bitlife Bitizenships ($5).
Combat
Armed & Dangerous Kill someone with a learned martial art move
Get to the top level of a martial art, (especially in prison) pick someone old to attack. Start a fight with them.
Midieval Attack Get attacked with a midieval weapon
Kinda chance. Just keep picking fights. You can get into a lot of fights if you're rude at nightclubs or to people on the street. Sometimes if you attack your loved ones or enemies with a weapon they'll kill you with a sword or something.
No Grasshopper Earn the top belt in a martial art
Each martial art has 10 tiers. They can cost $1k+ if you're an adult so if you've got generous parents take advantage.
Sensei San Earn the top belt in every martial art
Have health above 50% when you take a martial arts lesson. Follow above tips. Parents will probably only pay for one set of lessons, so pay for the other arts yourself as an adult. At $10k per martial art, it will probably cost you $40k-$50k.
Disease
Addicted Sustain 3 addictions at once
Play Blackjack or go to the horse races often with mid-tier mental health. Get addicted to pills or some other hard non-psychedelic drugs. Start drinking last b/c it'll kill your health. Try not to let your Bitizen get depressed or you might die, lol. All addiction is dangerous so it may take a few tries.
Bubonic Plague Contract the bubonic plague
Have low health and luck out. I got it in the UK.
Foam at the Mouth Contract rabies
Try to take home every wild animal you see. One might bite you. If you succeed, take it to the vet. If it doesn't have rabies, release it. If it does, don't treat it! Take it home and bathe it until it bites you.
Sickly Contract 10 diseases in one life
Best if you're not vaccinated, but just have mid-tier health and be really social. All afflictions count.
Successful Rehab Have rehab cured at a rehab center
Go to fancy rehab if you can afford it. Do it from your military deployment to go AWOL.
Witchcraft Get cured of a disease by the witch doctor
Eye of newt and cow tongue are iffy. Always start with health at 100. They've fixed cancer and sickle cell for me.
Entertainment
BitBoi Watch Bijuu Mike on YouTube BTS ARMY Go to a BTS concert
Keep asking friends to watch YouTube/go to concerts every year until you get those options.
Movie Junkie Go to 5 movies in one life Moviegoer Go to a movie
Go to the movies every year. It's good for your relationship if you go with somebody.
Fame
Brightest Star Achieve maximum fame
Actor, model, writer, athlete career path. Keep doing every bonus thing (talk shows, books, pose nude, commercials) and verify on social media.
Centerfold Pose for Wank magazine
Agree to pose nude every time until you get it. I think this one has women mostly but I can't remember.
Endorser Get paid $2m for a commercial
Easy if you're a high paid actor or model doing an international commercial.
K-Pop Become a famous Korean singer
See my wealth advice. Follow it with the "background singer" career and start in Korea.
Fertility
DNA Donor Make 25 sperm donations in one life
This one is hard b/c you can only do it once a year and only until a certain age. So start at 18 and don't stop. I think you have to be American. Maybe UK and Canada too? Not legal everywhere. Try not to miss a year.
Fabulously Fertile Have 10 children in one life Fertile Myrtle Mother 25 children in one life
Meditate every year. Start at 18. You have to be cis. Eat healthy and exercise. Get boyfriends and have unprotected sex with them so you don't get STDs. You can be a mother up until like 51 if you're healthy and lucky. Keep having sex until you get pregnant.
Smart Seed Get artificially inseminated with lawyer sperm
Start at 18. You have to be cis. Be fertile (tips above). Keep pulling up the option to get artificially inseminated until a lawyer comes up. Don't listen to your partner if they don't want you to do it LOL.
Super Sperm Have 100 children in one life
Be a cis dude. Meditate. Be handsome. Have a million girlfriends. Use the dating app to keep dating young women. Don't abandon any kids but leave girlfriends as soon as they're pregnant. Hire every surrogate that will take you if it's legal. Sue them for the max ($200k) if they bail (not miscarry).
Three's Company Have triplets
Sometimes this happens if you're a dude with luck or while you're doing Super Sperm. Sometimes if you're a woman it's luck too or when you do IVF with your partner's sperm or other artificial insemination.
Military
Career Military Serve your full career in the military
Tips for staying alive below. Retire as soon as you can.
General Achieve the rank of general in the military
Be a good Army person. Grind at work like 5x a year. Keep in shape. Be nice to your seargeant.
Admiral Reach the rank of admiral in the military
Be a good Navy person. Grind at work like 5x a year. Keep in shape. Be nice to your seargeant.
Absent Without Leave Go AWOL in the military
Be deployed with an addiction and check into rehab. Whoops.
Excavator Clear 10 minefields
Be deployed, and use a minesweeper solver to not die if you suck at minesweeper.
Pet
Adopt Don't Shop Rescue every pet in the shelter
You gotta have a few houses. Then you're good. You gotta do it all in one year so have like a lot of houses. Like 5 at least. Tips for getting rich above.
Horsing Around Own 50 horses in one life
You gotta have a bunch of ranches. Buy a few horses a year. Tips for getting rich above.
Just Keep Swimming Buy a goldfish and release it.
You can do this one as a kid too if your parent gets you a goldfish.
Natural Selection Rescue every pet in the shelter
This one took forever. Just keep buying dangerous exotic pets and rescuing every dangerous animal you see. It's luck.
No Probllama Buy a Llama
Buy a ranch in Afghanistan. Go pet shopping.
Prison
Aftermath Escape prison in a riot Instigator Prison riot
Get good at Snake. Keep rioting. Works best in low security. Takes a couple tries, kind of luck.
Behind Bars Spend 50 years in prison True Lifer 75 years in prison
Do a murder in a country without the death penalty (Canada). Murder with full health at 18. Get a prison job. Meditate and work out every year. Keep your head down. Try half-heartedly to escape every once in a while so you don't accidentally get parole or something. But if you get out you can always go back. Rob a bank or something. But keep your health and behaviour up in case you get sick and need to go to the infirmary.
Gangsta Join a prison gang
Go to a medium or higher security prison.
Inmating Get a lover pregnant on a conjugal visit
Be a cis man with high fertility. Have a good relationship (80%+) with an 18 year old cis woman. Make sure she isn't on birth control. Do a small crime, get a prison job, and meditate. Request a conjugal visit.
Justice Get freed from prison by appeal
Be rich. Wait a couple years after you're sentenced for something non-violent.
Mercy Me Get granted clemency
Be a nun or a monk for 50+ years. Don't retire. Do a murder. Get a prison job. Meditate, work out, go to the library, and write letters to home. You won't know until the year you're scheduled to die, so hold on.
Midnight Express Get sentenced to Turkish prison
Be born in Turkey. Do a crime.
Theseus Escape a supermax prison
There are a ton of Bitlife prison guides. Do a murder and escape from death row.
Royalty
Executioner Execute 5 people
Be king. Or queen. Top dog, either way. It helps to have enemies or friends to make enemies.
Markle Marry into the royal family
Be a commoner in a country with royals. Be cute. Go on lots of dates. It'll pop up and be part of their name. They could be a viscount or whatever, no member of the royal family is too far removed.
Monarch Become a monarch
Start as prince or princess and inherit the throne.
Napoleon Get exiled to a distant land
Keep executing people. And do a bunch of disservice.
Reign Over Us Reign as monarch for 100 years
In a country where Prince/Princess is top monarch or where your king/queen parents are low health/dying, keep your health up until you're a super-centarian (see above).
Sports
Canton Get inducted into the football hall of fame
Be a great football player. Be famous. Play as long as you can. Keep being famous after football as long as you can. I stopped being famous at 40 and got inducted at 60.
Christiano Win the Ballon d'Or
Be a European soccer player. Keep winning championships (see below).
Full Ride Win an athletic scholarship
Start playing sports in middle school. Become captain of at least one team with a pro league.
Giggsy Win 13 career championships
You can train each stat up twice in a turn if you trade teams, but you'll lose respect, so pick your moments. Grind your whole life. Keep going to the gym. Trade teams when you guys start losing. Stay on top.
Hooker Yell at a leopard
Try out for professional rugby with high athletic stats. Choose Hooker as your position.
Lance Win a championship while doping
It's safest to dope the year after a drug test. Try it for your second or third championship.
Real Estate
House Hunter Make $2m from flipping a house
Buy a $2m house. Leave it to your kid. Sell it. See above.
Mansion Party Throw a party in a mansion Real Estate Mogul Purchase real estate worth $10m combined Trailer Party Party in a trailer
Pretty straight forward. If you're broke start with the trailer party. Then buy mansions. Advice for getting rich above.
School
Brothers Forever Get hired by a frat brother
Be a jock. See sports advice above. When you're in two sports at university, compliment the jocks' leader. Be good looking (plastic surgery if needed, see above) and google the answer to the question if you need it. Google high-level frats and pick one. Then when you get hired after school one of them might hire you!
Earning that A Seduce your teacher
Be really attractive and compliment your teachers who are attracted to people of your gender. Take the opportunity to sleep with them if it arises.
Naughty Child Get expelled from school
Be rude as hell to the principal/headmastedean
Swimming Star
Start swimming as young as you can and stay in shape. "Work harder" every year.
Social Media
Social Media Join social media Social Media Sharer Post Social Media Oversharer Post 5 times Social Media Star Get a million followers Check! Get verified
Join all social media platforms at 13. Be pretty and keep posting. Follow above advice to get famous in any public career to get more followers. Start with Instagram for verification around 100k. By the time you're a lead actosupermodel/etc you'll have 1m followers.
Vehicle
Antiqued Keep a car running for 200 years.
Buy a brand new car. Do maintenance twice a year. Pass it on to your kid (18+) and repeat.
Car collector Assemble a car collection worth $1m Lambo Buy a Lamborghini
Buy a lambo and a bunch of other fancy cars. Who cares. See advice above for money.
Not The Yellow One Buy a submarine
You need $5b for this to show up reliably.
Titanic Trouble Run into trouble on a yacht
Have a shitty yacht or shitty luck. Go for a bunch of rides.
Animal
Animal Rescue Rescue an animal
Helps to have 100% smarts. Read childrens books so you don't have to tap too many pages. It'll only take two or three.
Deaf Leapord Yell at a leopard
Buy a leopard from the exotic animals dealer and yell at it when it misbehaves.
Gorilla and the Fist Get decapitated by a gorilla
I had to buy so many gorillas from the exotic animals dealer to get one crazy enough to decapitate me. Just keep bathing it and letting it attack you every year until it kills you.
Unicorn Find a unicorn
Go for like 10 walks a year. Have good karma.
Hungry Hippo !!! NEEDED !!!
Apparently Egypt is good for this.
Lion Tamer !!! NEEDED !!!
Apparently Kenya is good for this.
Crime
Balcony Buccaneer Steal 100 packages in one life
It's a lot easier to avoid punishment by wielding your title if you're a monarch. This one took me ages as a civiliian.
Burglar Burgle 25 homes in one life
Play Snake well
Cold Killer Kill 10 people in one life Serial Killer Kill 25 people
Start with random homeless people. If you're a royal exert your title to avoid punishment. Keep buying your way out of prison as long as you can. Then start killing other prisoners, start with the oldest and work your way down to the strongest ones. Work out and meditate every year. Pay guards for protection if you can but you probably won't be fucked with if you keep strong and murderous.
Dillinger Rob 5 banks in one life
If you're royal you'll get away with it. Make sure you have a getaway car either way. Clown mask/closest equivalent and handgun/closest equivalent work best.
Scare to Death Scare someone to death
Do a murder but pick scare to death. Works best if they're old.
Bugatti Bandit !!! NEEDED !!!
Going Anywhere !!! NEEDED !!!
LOVE
Black Widow Widow 5 husbands in one life
Start using the dating app when you're 18 and go for old guys. Best if they don't have kids and if they're rich. Propose after you fuck when your relationship is at 100%. I like to be on birth control for this.
Golden Anniversary Be in a marriage for 50 years Diamond Anniversary Marriage for 75 years
Keep seeing movies together and fucking and complimenting each other. Cute as hell. Just marry young and try to both stay alive.
Fake It Propose successfully with a fake ring
Works best if you're rich and they love you and they're dumb.
Family Planner Convince a lover to go off birth control
Be a cis man. Be in a strong relationship with a cis woman. Ask her to go off birth control. Easiest if you're married to her.
Maiden Named Marry a man who takes your last name
Marry a man and don't change your last name. Kind of a luck thing. Make sure your relationship is strong.
Multigamist Get married 10 times in one life
Pre-nups and widowing make this easier but do you. Love them and leave them. If you're a young guy it's really easy to get older women to agree to marry you.
Stud Have 100 lovers in a single life
Hook up like crazy. Date all you can and fuck all of them. Use protection so you can stay alive.
Wedding Planner Agree to an arranged marriage
I did this in India as a woman with wealthy, religious parents.
Bejeweled !!! NEEDED !!!
General
All Along Have a parent who comes out of the closet
Could be luck. Or you can cheat it with a Bitizenship by making both parents gay and unreligious.
Begone Exorcise your own ghost
Be an exorcist. Buy a haunted house. Do what you do best.
Booty Call Have a successful Brazillian butt lift
Be healthy and have good karma. Use the best doctor. Cross your fingers. They still only work 1/3 of the time.
Cliff Diver Go cliff diving Hero Save someone's life Player Perks Accept a casino's hospitality offer Snake Snack Eat a snake ZAP! Get struck by lightning
Random event
Dignified Donor Donate a 1m+ heirloom to charity
Get your heirloom every day. Appraise it. Donate the first $1m+ one you get.
Flamin' Hot Survive 60 years on a Hot Cheetos diet
Get liposuction every couple of years and work out and walk a lot. Have no other conditions. Do your best. Get pets for more walks. Garden. Try to survive. Start at 18.
Flee the Country Emigrate to escape justice
Escape prison and emigrate
Frankenstein Survive 5 botched plastic surgeries
Keep going to the bad doctor. Go for risky procedures like butt lifts. Space them out to get your health back up.
Goat Grabber Join a goat grabbing team
Be athletic and join a goat grabbing team at school in Afghanistan
Human Dictionary Read the dictionary
So much tapping. But eventually it will show up in your books. Be strong.
Hyperthymesia Score 20 sequences on the memory test
The worst part of Bitlife. I did this one by writing 1,2,3 or 4 on a piece of paper according to which # square lit up with my right hand and doing the puzzle on my phone with my left hand. Still took like 5 tries and was really frustrating. Take breaks and come back with a clear head.
Jackpot Win the lottery jackpot
Keep your karma high and buy 10 tickets 5 times a year. You'll get it eventually.
Lowroller Get refused entry to a casino
Bet more money than you have on Blackjack. Once you're out of prison, try to come back. They'll turn you away.
Nightmare Wake up from a nightmare
As a pilot, buy a terrible plane. When it crashes, accept your doom. You might wake up.
Paranightmare Contract PTSD after a paranormal experience
Try to have bad mental and good physical health (a hard balance. Try gardening, dieting, and fighting with friends or loved ones) and then try to exorcise stubborn ghosts.
Perfection Achieve perfect stats
Pretty easy. Work out, get plastic surgery (lipo or nose job to start) and go for walks, read children's books (3 should get you to 100%) and go to the movies or on vacation.
Rich Justice Win a $1m+ lawsuit
Get fired from a really high paying job like CEO and win your lawsuit.
Run Bitizen! Win a bet on Bitizen There's Always Canada Emigrate to Canada Winnipeg, Eh? Visit Winnipeg
Wait until it pops up as an option
Say Goodbye To Hollywood Get deported from the United States
Move to the U.S. without permission. Get caught doing a minor crime.
Skeezy Get called "skeezy"
Be an asshole at nightclubs and in the streets. Fight with your friends and coworkers, insult them and start rumours.
Sweepstakes Win the sweepstakes
Set it up on a day where you'll be by your phone. Sign up every time you can.
Try & Stop Me Violate a restraining order
Stalk your ex. Do it again after they file a restraining order.
Ultimate Betrayal Your spouse leaves you following a gender reassignment
Have a terrible relationship with your heterosexual spouse. Get gender reassignment surgery.
Unethical Bribe a college official
Be rich and have dumb kids.
Roswell !!! NEEDED !!!
Sacrilege !!! NEEDED !!!
​

There is this statement in Uworld in the explanation of one of the immunology questions regarding the advantage polysaccharide vaccines have over conjugate vaccines. It states that with polysaccharide vaccines there is decreased incidence of replacement strains due to lack of mucosal immunity.
Can anyone please explain what it means?

Abstract

This is a preliminary characterisation of a bacteriocin, BacL49 produced by Lactococcus lactis ssp. lactis. This bacteriocin is significant due to its activity against Streptococcus iniae, a bacterial pathogen causing severe economic losses in the global aquaculture of various fish. Spot-on-lawn and microtitre plate assays were used to test antagonistic activity of the bacteriocin. BacL49 is heat and pH stable (100 °C for 60min, pH 2.5-9.5), and sensitive to proteinase K, a-chymotrypsin, trypsin and papain. BacL49 has a bactericidal mode of action and is produced during late log phase growth. BacL49 exhibits a broad activity spectrum against S. iniae, antagonising 93.75% (45/48) of S. iniae isolates collected from a variety of hosts and environments. The apparent molecular masses of the active protein components determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis are 5 and 54kDa. Molecular analyses were performed to locate the genetic determinants of BacL49. PCR of chromosomal DNA successfully amplified the structural gene encoding the precursor of nisin. Subsequent analysis of nucleotide sequences of the PCR products revealed it to be identical to the nis Z structural gene of nisin Z. There is a paucity of reports examining the inhibition of S. iniae by a lactococcal bacteriocin or even L. lactis as an aquacultural probiotic. This is one of the first studies to identify nisin genes in a strain of L. lactis exhibiting activity against S. iniae. BacL49 is a candidate biocontrol agent for mitigation of this important fish pathogen.
Keywords: Bacteriocin; Lactococcus lactis; Probiotic; Streptococcus iniae; Nisin Z
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Introduction

Streptococcus iniae is one of the most economically important bacterial pathogens causing streptococcosis in fish. Globally, economic damages have been in excess of US$100 million per year [1] with as much as 70% loss in annual production in certain fish cultures [2]. S. iniae causes disease and mortality in at least 30 species of fish in marine, brackish and freshwater environments [3,4]. Outbreaks of S. iniae typically occur in warm-water, cultured situations throughout the world including Australia, Bahrain, Israel, Japan, Korea, Spain, Italy, and the United States [4,5-10]. Epizootic incidents have also been reported in wild populations [11,12], the most notable being the 1999 and 2008 Caribbean fish kills [13,14]. Zoonotic infections of S. iniae, normally caused by percutaneous injuries sustained during raw seafood preparations, have been reported in Canada, China, Hong Kong, Singapore, Taiwan, and the United States [15]. Prevention and treatment of S. iniae in aquaculture remains difficult, particularly with the industry seeking safer alternatives to antibiotics and vaccines. Antibiotics, including erythromycin have previously been successful in treating streptococcus is in fish [16]. Nonetheless, the use of antibiotics in aquaculture is gradually being eschewed due to widespread development of antibiotic resistance in the environment and in cultured fish, which causes consumer concern [16-18]. In some cases, antibiotics are also believed to merely suppress clinical symptoms without eliminating the infection, thereby promoting the development of "carrier fish" [1].
Research devoted to creating effective vaccines for prevention of S. iniae suffered a major setback when a novel serotype of S. iniae caused severe outbreaks in Israel amongst vaccinated rainbow trout (Oncorhynchus mykiss) [19,20]. Since then, commercial vaccines against S. iniae have been marketed, but are very limited geographically [21,22]. Alternative vaccines have yielded successful results but are still undergoing testing [2325]. In spite of this progress, the task of effectively vaccinati Oceanogr Fish Open Access J 3(2): OFOAJ.MS.ID.555610 (2017) each individual can be tedious, expensive, and even stressful for the fish [26]. Vaccinations can be futile when applied to juvenile fish that may not be fully immunocompetent [27]. Additionally, S. iniae is capable of surviving in the aquatic environment without a host [11,28,29] and this was speculated to have been a factor in the evolution of the new serotype by allowing the bacterium to evade the immune response of the vaccinated fish [19]. Furthermore, the outbreak in Israel is evidence of serotype diversity that could enable S. iniae to eventually overcome yet another vaccine. Thus, the quest continues for a long-term solution that can be universally and easily applied throughout the aquaculture industry [25]. Chemical-free "green solutions" appear to be the next era of therapeutics for preventing bacterial epizootics in fish [17]. Probiotics, either in the form of whole cells or cell components such as bacteriocins, are anticipated as being effective replacements for antibiotics and chemotherapeutics to fight infectious diseases [30]. Bacteriocins are a group of proteinaceous molecules that are biologically active against bacteria that are closely related to the bacteriocin producer, while the producer is immune [31-33]. Ubiquitous across all bacterial genera, these ribosomally synthesized peptides confer a selective advantage to their producers [31,32,34]. Bacteriocins are recognized as potentially useful agents in the control of bacterialinfections due to their effectiveness, non-toxicity and relatively cheap production [32,35,36]. Only two published studies on bacteriocins have tested S. iniae as an indicator species [37,38].
An inhibitory substance, identified as bacteriocin BacL49, was found during experimentation with the library of S. iniae and other aquatic bacteria from James Cook University (JCU). The bacteriocin was produced by a strain of Lactococcus lactis ssp. lactis and was observed to inhibit a large spectrum of S. iniae isolates. L. lactis, along with the rest of the lactic acid bacteria (LAB) are considered the most prolific of all the Gram-positive bacteriocinogens [32,39]. A versatile species, variousstrains of L. lactis can produce an assortment of bacteriocins that are predominantly encoded on plasmids [40-43]. Some L. lactis strains produce the bacteriocin nisin, a small (<5kDa), membrane-active member of the Lantibiotic class of LAB bacteriocins capable of antagonistic activity towards a wide range of Gram-positive bacteria [44-46]. Nisin is heat-tolerant (115-121 °C) at low pH levels, making it an ideal preservative in pasteurised and acidic food products [45,46]. Being "generally regarded as safe," nisin has achieved worldwide recognition as a non-toxic food additive in over 50 countries [31,32,45]. As produced by various strains of L. lactis, nisin occurs as natural variants with designations A, Z, Q, and F [47-49]. 106 Unlike most plasmid-encoded bacteriocins produced by L. lactis, nisin is encoded on a conjugative transposon [50-52], a chromosomally- associated segment of DNA with the capacity to repeatedly insert into and mobilise plasmids and genomes [52,53]. Thus, one objective of this study was to identify the genetic determinants of the bacteriocin BacL49 through assessing the prevalence of plasmids in L. lactis subsp. lactis L49 and examining the bacterial chromosomal DNA for nisin genes. This study also provides a brief characterisation of bacteriocin BacL49 and highlights the potential of this substance as a green solution for S. iniae infections in fish.
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Materials And Methods

Source and identification of BacL49 producer. A lawn of S. iniae AS-04-1524#1 (JCU isolate S42; Table 1) was observed as a mixed culture, with one distinct colony type antagonizing the growth of the other. Both isolates were identified using a PCR assay for the lactate oxidase (lctO) gene of S. iniae using the primer combination LOX-1/LOX-2 [54], and a 16S rDNA PCR assay using universal primers 27F/1492R. PCR products were cleaned and sequenced by Macrogen Inc. (Korea). The 16S PCR assay returned a sequence 99% identical to Lactococcus lactis ssp. lactis in BLAST. According to sequencing, the antagonizing bacterium was identified as L. lactis L49, originally isolated from a moribund sleepy cod (Oxyeleotris lineolatus) at JCU. The inhibitory substance produced by L. lactis L49 was designated as BacL49.
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*Altered TAAHL accession number. Changes were made for unknown reasons by previous researchers; it is likely these isolates are from the correct animal accession but the final number has been altered (ie. -9 and -11). **Invalid TAAHL accession number.
Bacterial growth and isolation of BacL49. Unless otherwise stated, bacteria were propagated aerobically at 28 °C in heart infusion broth (HIB) or agar (HIA) produced by the addition of 1.5% technical agar to HIB. Bacterial lawns were made by seeding HIA plates with 1ml overnight growth of bacteria, removing excess culture and allowing lawns to dry at room temperature. To produce cell-free supernatant (cfs) containing BacL49, L. lactis L49 was grown aerobically at 28 °C for 10-12h, then centrifuged at 4300g for 5min and filtered through 0.45|im. Aliquots of cfs were stored at 4 °C.
135 Assays for antagonistic activity. The activity of L. lactis L49 against 48 isolates of S. iniae (Table 1) was determined by an altered deferred antagonism method [55]. Briefly, an overnight L. lactis L49 culture was streaked in a single line on an HIA plate with a sterile loop. Bacterial isolates and a control of uninoculated broth were then streaked with sterile loops in parallel at right angles to L49. Zones of inhibition were measured after incubation for 16h. For interest, streak plates were made to determine the activity of L. lactis L49 against B antibiotic resistant human pathogens including E. coli B597, E. coli 53e, community acquired methicillin-resistant Staphylococcus aureus and Streptococcus pneumoniae. For these human isolates, one plate was incubated at 37 °C with CO2 and the other was incubated aerobically at 37 °C to ensure sufficient growth of the pathogens. Zones of inhibition were measured after 24h.
146 Antagonistic activity of BacL49 was detected qualitatively on solid media using a spot-on-lawn assay [56]. Antagonistic activity was quantified in liquid media using a modified microtitre plate assay [57]. The indicator culture was grown to an optical density of approximately 0.2 measured at 540nm, and added to two-fold dilutions of the growth medium or cfs (treated or untreated) in duplicate 96-well round bottom plates. Plates were incubated aerobically for 3h at 28 °C and the optical density measured. Antagonistic activity was defined as the reciprocal of the dilution causing 50% growth inhibition (determined by optical density) relative to the control culture without cfs (AU = arbitrary units). S. iniae S23 was used as the indicator strain for all spot-on-lawn and microtitre plate assays due to its high level of sensitivity to BacL49.
Effect of heat, pH and enzymes on BacL49 activity. To determine the heat stability of BacL49 activity, cfs samples (pH 5) were heated at 100 °C for 10, 20, 30 and 60min. Samples were cooled to room temperature before spot-on-lawn and microtitre plate assays. To determine the effect of pH on BacL49 activity, cfs samples were adjusted to pH levels between 1.5 and 9.5 using 1N NaOH or HCl. Samples were incubated with agitation for 2h, then readjusted to pH 5 (the pH level of untreated cfs following incubation) before spot-on-lawn and microtitre plate assays. To determine the effect of different enzymes on BacL49 activity, cfs samples were adjusted to pH 7.0 and treated to a final concentration of 2mg ml-1 with the following enzymes: proteinase K (40 units mg-1), a-chymotrypsin (59.3 units mg- 1), trypsin (2.6 units mg-1), pepsin, papain (19 units mg-1), and catalase (1340 units mg-1). Samples were incubated at 37 °C with agitation for 2h, then at 100 °C for 5min to deactivate enzymes. Remaining antagonistic activity was measured using the spot- on-lawn assay. An untreated cfs sample was used as a control in all assays.
170 Kinetics of production and activity. The kinetics of BacL49 production by L. lactis L49 were investigated by measuring the growth of the bacterium and activity of BacL49 produced over the same period. Overnight growth of L. lactis L49 was added to HIB (3.75% volume) and the culture was incubated with agitation. The optical density of the culture was measured at 600nm every hour until 12h, then periodically to 28h. At each reading, 1ml culture was removed to produce cfs, which was stored at 4 °C. Antagonistic activity was measured using a spot- on-lawn assay following the collection of all cfs samples.
The activity of BacL49 was determined as either bacteriostatic or bactericidal by measuring the growth of indicator strain S. iniae S23 after the addition of BacL49. Early log-phase growth of the indicator strain was distributed into triplicate 10ml aliquots. One ml of cfs was added to two indicator cultures and HIB was added to the remaining control culture. Optical density was measured at 600nm over 24h. To measure cell viability in a treated culture, mid log-phase growth of the indicator strain was distributed into duplicate 10ml aliquots. One ml cfs was added to one aliquot and HIB was added to the control. Optical density measurements at 600nm were taken over time and 10-fold serial dilutions were grown on HIA to measure cell forming units (cfu) of the indicator strain in both cultures at 48h.
Protein purification. L. lactis L49 was grown overnight in HIB previously filtered through Millipore type HA filters (to remove unwanted proteins from the media). A portion of the cfs produced from this culture was treated with ammonium sulphate in two stages. For the primary precipitation, saturated ammonium sulphate solution was slowly added to 100ml cfs while stirring to 60% saturation at room temperature. The solution was agitated 12h at 4 °C, then centrifuged at 4500g for 30min at 4 °C. The precipitate was resuspended in 10ml sterile distilled water. A small portion was removed and stored at 4 °C for the activity assay. Saturated ammonium sulphate solution was added to the remaining primary solution while stirring to 80% saturation at RT. This solution was agitated 12h at 4 °C, then centrifuged at 4500g for 30min at 4 °C. The secondary precipitate was resuspended in 1ml (1% original volume) sterile distilled water and stored at 4 °C. The activity of the ammonium sulphate precipitated (primary and secondary) proteins was tested with a microtitre plate assay.
Approximate molecular size of BacL49 was determined by tris-tricine sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Aliquots of the partially purified protein samples and untreated cfs were prepared with TruSep tricine SDS sample buffer and run on 16% tris-tricine gels with a low MW prestained protein ladder (Fermentas, Australia) at a constant 150V. Gels were silver stained using a Silver Stain Plus staining kit (BioRad, Australia) according to the manufacturer's protocol 208.
Proteins separated by SDS PAGE were eluted from the gel using a modified protocol from Busarcevic et al. [58]. Following agitation at room temperature overnight, the eluted samples were centrifuged at 10,000g for 5min and the supernatants concentrated by vacuum at 33 °C for 20min. Two controls were used: one elution made with a slice of gel from an unused well lane (containing no protein) and one with elution buffer only. Eluted samples were assayed for antimicrobial activity with a spot-on-lawn assay.
DNA purification. Isolation of plasmid DNA was carried out using the Wizard® Plus SV Minipreps DNA Purification System (Promega, Australia). Low-copy number plasmids were assumed for all strains. Genomic DNA isolations were carried out using the High Pure PCR Prep Kit (Roche, Australia) for the detection of nisin structural genes. DNA isolation procedures were performed according manufacturer specifications with the addition of a lysozyme step for Gram-positive bacteria. Negative control preparations contained no bacterial culture.
DNA products were resolved by electrophoresis on agarose gels stained with GelRedTM and then visualised under UV light. Since L. lactis strains often harbour a wide size-range (2kb to about 100kb) of plasmids (Teuber and Geis, 2006), plasmid DNA products were visualised using two different DNA ladders. These included the 1kb GeneRulerTM (Fermentas, Australia) used with 1% (w/v) agarose gels at 90V and the Lambda Mix Marker 19 (Fermentas, Australia) used with 0.5% (w/v) agarose gels at 40V.
PCR analysis of the nisin gene. Oligonucleotide primers (Macrogen, Korea) were designed using the NCBI-ORF Finder and OLIGO 7 primer analysis software (Table 2) to target the nis A structural gene. Genomic DNA samples were prepared for PCR using GoTaq® Green master mix (Promega, Australia). As L. lactis L53 was found to contain a plasmid (results not shown), this isolate was included in the procedure. For negative controls, a tube of reaction mix containing indicator strain S. iniae S23 and one without sample DNA were included for the nisin structural gene protocol, and for the PCR protocol, respectively. DNA was amplified in a thermocycler (Eppendorf, Australia) set for denaturation at 94 °C, annealing at 55 °C and extension at 72 °C. PCR products were visualised on 2% (w/v) agarose gels at 120V, along with a 50bp GeneRulerTM (Fermentas, Australia). Sequence information (Macrogen, Korea) of the PCR products was analysed using Sequencher® 5.0 software. The resulting consensus sequences were compared to DNA and protein sequences contained in the National Center for Biotechnology Information (NCBI) database via BLAST search of "highly similar" sequences.
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Results

L. lactis L49 produced antagonistic activity against 93.75% (45/48) of S. iniae isolates in the JCU library (Figure 1). L. lactis L49 failed to inhibit the growth of isolates S32, S39, S47 and the human pathogens tested. Antagonistic activity of BacL49 remained following 60min at 100 °C and over a broad pH range (pH 2.5-9.5), though activity was weaker at pH levels higher than 5.5 (Table 3). The cfs also showed antagonistic activity against the indicator after exposure to pepsin and catalase, but lost activity when exposed to proteinase K, a-chymotrypsin, trypsin and papain. L. lactis L49 began producing BacL49 at the end of the log growth phase (Figure 2). BacL49 production reached a maximum at early stationary phase, but began to drop following600min growth and continued to decrease to the end of 24h.
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Activity measured as present (+) or absent (-) for enzyme tests. Absence antagonistic activity (-) correlates to senstivity (s) to the enzyme tested, and antagonism of the indicator (+) correlates to resistance (r) to the enzyme tested; Cfs: Cell Free Supernatant.
A Growth of the indicator strain was not only inhibited following the addition of BacL49, but the optical density of the culture continued to drop steadily over time without recovery, while the control culture grew normally to a high optical density. The cell viability of the treated indicator culture dropped 10fold in the first 30min following the addition of BacL49 and continued to drop over 70min while the control culture cfu increased 10-fold.
Ammonium sulphate precipitation successfully concentrated the bacteriocin (Table 3). The primary precipitate showed 8 times the activity (in AU) of the untreated cfs and the secondary precipitate showed at least 64 times the activity of the untreated cfs (1/512 was the highest dilution made of the substances tested in the microtitre assay).
Tris-tricine SDS PAGE allowed separation of the low molecular weight proteins, and multiple protein bands were clarified after silver staining of the gel (Figure 3). Following elution of proteins from the tris-tricine gel slices, only minor antagonistic activity was produced on the indicator strain by the small molecular weight band (5kDa) but strong antagonism was produced by the large molecular weight (54kDa) band.
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Following plasmid extraction, DNA samples of L49 showed faint bands that were too unremarkable to be denoted as plasmids (Figure 4). Based on this data, further plasmid experimentation was not pursued. PCR for the nisin structural gene resulted in products just over 100bp for isolates L49 and L53 (Figure 5). No bands were detected for S23 or the control. Sequence analysis of the L49 PCR product yielded a consensus sequence of the nisin structural gene (Figure 6) that was 99% homologous to the nis A structural gene and 100% homologous to the nisZ structural gene (Table 4). The protein BLAST search showed 100% homology with the Nisin Z precursor, followed by that of nisins F and A, and then nisin Q (Table 4). A deduced amino acid sequence of BacL49 was also obtained from the BLAST search, which showed that BacL49 was identical to nisin Z.
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Discussion

The inhibitory substance BacL49 produced by L. lactis L49 has been identified as a bacteriocin due to meeting the criteria of being a biologically active protein moiety with a bactericidal mode of action [33]. BacL49 can be produced in anaerobic conditions and is capable of diffusing through solid media (results not shown), which rules out hydrogen peroxide and phage activity, respectively, as the cause of antagonism. The protein nature of BacL49 was confirmed by its sensitivity to a number of proteases and the mode of action was confirmed as bactericidal by optical density and cell viability experiments.
BacL49 is heat and pH stable, though antagonistic activity was weaker when the bacteriocin was incubated in an alkaline environment. An acidic pH can be necessary for the retention of the cationic properties of bacteriocin peptides, which appear to be crucial for their antagonistic activity [59]. Most L. lactis bacteriocins, including nisin, are heat stable and tolerant to acidic conditions [21,60-62] but many show different enzyme sensitivities. A review of publications describing Nisin Z production by L. lactis strains isolated from various sources found that nisin Z is consistently sensitive to proteinase-K, but varies in sensitivity to trypsin, a-chymotrypsin, and papain. A few published descriptions of nisin exactly matched the protease sensitivity profile of BacL49 [63-65]. Interestingly, the only other described nisin isolated from fish differs from BacL49 in sensitivity to trypsin and a-chymotrypsin [37].
A bactericidal mode of action was confirmed by the reduction over time in optical density and Viable cell count of the BacL49 treated indicator culture. It is known that many antibiotics kill bacteria by targeting lipid II, thus blocking cell wall synthesis and leading to cell lysis by pore formation [44,66,67], and it is possible that BacL49 performs in a similar manner. The bactericidal mode of action of BacL49 not only confirms this substance is a bacteriocin, it indicates that BacL49 could reduce bacterial loads in fish or the environment by destroying S.iniae cells.
Production of antagonistic activity by the two differently sized peptide bands eluted from SDS PAGE gels (54kDa, and 5kDa) suggests that two different inhibitory substances are being concurrently produced by L. lactis L49. The two component lantibiotic lacticin 3147 consists of two 3-4kDa peptides, but both are required for antagonistic activity [68]. This required cooperation is apparently not the case with the two BacL49 peptides, as evidenced by their independent production of antagonism. It is also possible that cleaving of BacL49 is occurring; the large molecular weight peptide may be an unseparated quaternary structure of the small peptide.
The results of the plasmid extraction do not support the hypothesis that L49 contained plasmids. The faint bands observed after electrophoresis were not indicative of a typical plasmid profile. Chromosomal DNA extraction results suggest that L49 possesses the structural gene encoding the nisin precursor. Interestingly, equally strong bands for the structural gene were evident for isolate L53. Whilst this result was not expected due to isolate L53 only inhibiting 30% of S. iniae isolates in the JCU library (results not shown), it was also not surprising since various strains of L. lactis have been observed to produce nisin.
Based on the NCBI BLAST search results, BacL49 is likely nisin Z. The DNA sequence was shown to differ from that of nisin A exactly as stated in Mulders et al. [48], varying at position by a C to A transversion. Comparison of the deduced amino acid sequence with those of the other nisin variants, coupled with the protein BLAST results, substantiates the nucleotide results. Nisin Z is a natural nisin variant that has only been produced by strains of L. lactis, however these strains have been isolated from a variety of sources from different environments.
Producers of nisin Z have previously been derived from dairy [51,69] and vegetables products [70,71]. Recently, it has been shown that they are also associated with mangroves [72], marine fish [37], and now with freshwater fish. The source of the producing strain could have an impact on how and in what context it can be used most effectively.
Based on the results of this study, it is likely that BacL49 is encoded on a conjugative transposon. Like plasmids, some conjugative transposons can possess a very broad host range [73], allowing for the dissemination of various traits (e.g. antibiotic resistance) across different species [53,74,75]. Further research following this study should focus on isolating this mobile genetic element from L49, characterising it, and assessing its potential novelty and uses by genetic manipulation.
Other bacteriocin-producing L. lactis strains have been isolated from freshwater fish in the past [47,60] however these studies tested bacteriocin activity against S. aureus, L. monocytogenes, and other pathogens important in food spoilage and human infection. This study reports similar findings to that in Heo et al. [37], in which a strain of L. lactis subsp. lactis was isolated from the intestine of a marine olive flounder (Paralichthys olivaceus) and was found to inhibit S.iniae during in vitro experiments. Heo et al. [37] examined the in vitro effects of nisin Z against S. iniae by combining it with varying concentrations of NaCl. The authors concluded that the ability of nisin Z to inhibit the growth of S. iniae was synergistically improved when applied in conjunction with NaCl. This is significant for marine- based aquaculture. The producer of BacL49 was isolated from a freshwater fish species, thus it would be interesting to look for any significant differences in activity between BacL49 and nisin Z originating from a marine source BacL49 could have important applications in the aquaculture industry with regard to S. iniae.
The ability to retain its activity through heating processes would allow this bacteriocin to be readily incorporated into commercial fish food. The fact that BacL49 remains active over a broad pH range is also advantageous because S. iniae can establish in a variety of organs and tissues in infected fish. BacL49 could also be added directly into the culture water as a non-toxic means of biological control of S. iniae in the culture environment and be a supplement to vaccination procedures. This environmental treatment could address the issue of S. iniae cells surviving freely in the water and evading the immune responses of vaccinated fish. Differences in activity, such as increased inhibitory specificity, distinguish bacteriocins from classical antibiotics and are another advantage to their use in a cultured environment [76]. This specificity can reduce the risk of non-target bacteria (particularly beneficial ones) being antagonized and minimize the threat of resistance development. However, the broad activity spectrum of BacL49 on different isolates of S. iniae would prove to be an advantage due to the large variation that exists between strains of this pathogen [76,77].
The pathological effects of bacteriocins must be considered before they are used in an in vivo situation, and it would be beneficial to determine whether the bacteriocin is strongly antigenic [33]. However, most bacteriocins are not toxic to animals at effective antimicrobial concentration due to their specificity [78]. Despite the results of this study showing that high concentrations of BacL49 are easy to achieve, bacteriocin delivery or retention in fish tissues could prove difficult in vivo. If this were the case, L. lactis ssp. lactis L49 could be trialed as a probiotic. Great interest has been shown towards LAB as potential probiotics, as they are well-recognised for their bacteriocinogenic capabilities and presence within the normal microbiota of fish (typically the intestine) [30,79]. Not only can they withstand acidic stomach conditions, but they can grow and colonise the intestine of fish [80]. The piscine origins of L49 may contribute to its survivability and efficacy as a potential probiotic, though there are few studies examining L. lactis as a probiotic specifically against fish pathogens. Though L. lactis has not been documented as a fish pathogen and is regularly present in the aquatic environment and the intestinal tract of both freshwater and marine fish [71,81-84], it would be necessary to confirm isolate L49 as non-pathogenic to the species of fish undergoing treatment. The elucidation of the L. lactis genome and the fact that products from the bacteria are generally regarded as safe make the bacterium a unique candidate for genetically engineered live vaccines as well [85].
BacL49 is significant because it displays a broad activity spectrum for S. iniae isolates, implicating it as a new therapeutic or preventative agent for infections caused by this economically important fish pathogen. Nisin has had a long history as a safe food additive in the manufacturing of various foods for human consumption, thus BacL49 may also be integrated with fish food without serious concern over chemical residues. Purified BacL49 should be tested in vivo to determine antigenicity of the substance in fish, and the bactericidal action of the bacteriocin should be studied in depth to identify problems that may arise with bacterial resistance.
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The recommendation you will find here is yes, it is prudent to aspirate a needle when self-injecting IM. Even though the statistical odds are very low of a problem occurring if aspiration is not carried out, aspiration nevertheless potentially provides an extra layer of safety in return for just an extra 30 seconds of time.
What does aspirating an IM injection needle mean?
Practically speaking, aspirating the needle means that once an IM injection needle has been inserted and is in position - before any solution is injected - the syringe plunger is pulled back (creating negative pressure in the syringe body). A visual check is then made to see if any blood is drawn up through the needle into the syringe body. If any tiny specs of blood are noticed in the syringe body, it should be presumed that the needle tip is incorrectly positioned in a vein or artery - and the needle withdrawn without injecting any solution.
Most people think that air bubbles are the most significant hazard when injecting. However, even with IV injections, most likely, a significant quantity of air (3-5 ml/kg weight) would need to be introduced to cause a serious problem. Rather with intramuscular injections of oil based solutions there is a slight risk that oil might directly enter the blood supply and make its way to the lungs (or some other location). Note that the same risk does not apply to water based injection solutions such as for example, B12 injections.
Thus in the case of oil based estradiol injections of a typical dose a trans girl might typically inject (e.g. 10-20 mg), the risk doesn't particularly arise from the estradiol itself being injected into the circulatory system, but rather from the oil which is the carrier for the estradiol (entering directly into the circulatory system).
If oil does enter the circulatory system and make its way to the lungs, it can cause pulmonary embolism (PE), which is a serious condition resulting from a blockage of blood vessels in the lungs. If symptoms of PE are noticed after an IM injection (up to 36-48 hours after) then immediate medical assistance should be sought.
In past decades aspirating the needle when injecting intramuscularly was considered good medical practice (for doctors and nurses). However aspirating the needle is now considered less important in some countries, given the risk of pulmonary embolism arising from IM injection appears to be fairly low (see the paper cited below for more details about this).
However, it appears the few studies looking at the value of aspirating an IM needle are generally based on injections by doctors and nurses - not on those self-injecting. Therefore, it makes sense that those self-injecting IM do follow the procedure of aspirating the needle before injecting any oil based solution. As above, aspirating the needle only takes 30 seconds. At the minimum, it will mean 30 seconds of time has been used for no good reason - but maximally, it will mean that a significant extra layer of safety has been added to the complete injection procedure.
As noted in the paper citations below, it is important that aspiration is done with skill, so as not to cause additional issues.
Note that, as above, in some countries, aspirating a needle is still considered best practice for doctors and nurses.
Full paper: Aspiration in injections: should we continue or abandon the practice?
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333604/#__ffn_sectitle

From abstract: Aspiration during any kind of injection is meant to ensure that the needle tip is at the desired location during this blind procedure. While aspiration appears to be a simple procedure, it has generated a lot of controversy concerning the perceived benefits and indications. Advocates and opponents of aspiration both make logically sound claims. However, due to scarcity of available data, there is no evidence that this procedure is truly beneficial or unwarranted. Keeping in view the huge number of injections given worldwide, it is important that we draw attention to key questions regarding aspiration that, up till now, remain unanswered. In this review, we have attempted to gather and present literature on aspiration both from published and non-published sources in order to provide not only an exhaustive review of the subject, but also a starting point for further studies on more specific areas requiring clarification.

From Intro:

An injection is defined by the World Health Organization (WHO) as parenteral administration of medication through a skin puncture via a syringe, while aspiration is defined as the pulling back of the plunger of a syringe (for 5–10 seconds) prior to injecting medicine 1– 4. Aspiration is most commonly performed during an intramuscular (IM) or subcutaneous (SC) injection, and is meant to ensure that the needle tip is located at the desired site, and has not accidentally punctured a blood vessel.
Advocates of aspiration contend that it is a technically easy maneuver that is rapidly performed and well tolerated by patients with no increase in costs incurred. However, due to a paucity of available data, there is no evidence that this procedure is essential or truly beneficial.
A number of studies have concluded that a conventional syringe is a poorly controlled and non-ergonomic device during aspiration 21, 22. Possible lack of precision may result in local trauma and pain, prolonged procedure time, failed or incomplete procedures, accidental puncture of blood vessels or nerve bundles...

[The above point makes it clear that aspiration is a procedure which needs to be skilfully and carefully carried out, so as not to create other problems than that which it seeks to cover for.]
From methodology:

Published literature on injection technique advises aspiration before injecting a drug through different routes, i.e. IM 35, intravascular (IV) 36 or SC 37. However, it is important to note that emphasis has been placed on negative pressure being applied for 5–10 seconds for aspiration to be of benefit 1, 3, 4

From results:

IM injections: Aspiration prior to injection of medication through the IM route remains a part of most guidelines 4, 35, 38– 40. Nursing curricula and guidelines 4, 38, 39 clearly recommend aspiration as an essential step in IM injection technique. Guidelines originating in the UK recommend aspiration prior to IM injection of medications 35, as well as specifically as part of the Z-track technique of administering IM injections.
SC injections: It is apparent that there are opposing schools of thought when it comes to aspiration prior to SC injections. There are those that insist that aspiration should continue to be part of SC injection techniques for medication administration, and those who are convinced that aspiration is not necessary and has no real advantage; in fact, several disadvantages may be attributed to this step.
Injection of medication: The UK NHS 84 and Public Health Agency Canada 6 recommend aspiration before IM injection of medication.
Neither the ICN nor the Nursing and Midwifery Council 50, 51 (Europe and British Chapters) have made any kind of recommendation in their guidelines on administration of medication. The official website of the WHO’s Uppsala Monitoring Center (UMC) 85 does not list any warnings related to aspiration.
National nursing curricula in Nigeria 86 and Pakistan 87 do not mention aspiration before injection as a necessary step for IM and SC injections. Similarly, the syllabus for MSc Nursing in India did not elaborate on injection technique. Curricula for primary health workers in Nigeria 40 and nursing students in Pakistan’s foremost nursing school (Aga Khan University School of Nursing) do advocate aspiration 38 before injection. Similarly, the IndiaCLEN Model Injection Center Program advises aspiration prior to IM injection 88. None of the curricula mentioned above make any comment on the duration of aspiration.
The United States Pharmacopeia-National Formulary 89, British National Formulary 90 and Pakistan Pharma Guide 91 make no mention of aspiration before injection.

From discussion:

Aspiration prior to injection is just one part of the process of performing vaccinations, therapeutic injections and diagnostic/therapeutic procedures. The debate over its inclusion as an essential part of recommended techniques has driven this review, and is likely to continue in the absence of findings from randomized controlled trials. In most instances, general clinical or vaccination experiences guide global recommendations for aspiration. In others, anecdotal reports of adverse events form the basis for inclusion or exclusion of aspiration in standard injection techniques. The sheer number of injections given globally in the preventive and therapeutic sectors makes this omission even more surprising. This appraisal of current guidelines and literature has made it clear that the need for aspiration prior to administering an injection is dependent upon multiple factors, as elaborated below.
The drug that is being injected has a direct bearing on the decision to aspirate or not to aspirate. If the drugs to be given have potentially fatal consequences in the event of systemic administration (as in the case of immunotherapy), all possible precautions must be taken. This is even more important in cases where the drug is being administered electively by specialist staff. On the other hand, if there are no serious known sequelae to a drug being injected systemically – as in the case of vaccines – an argument can be made not to aspirate, especially since a huge number of immunizations are performed globally by vaccinators and health workers. Product inserts for 104 injectables on the WHO Essential Drug List were reviewed. Of these, only 3 inserts specified that aspiration should be performed prior to injection. Two of these inserts were for local anesthetic agents and the third was for Pneumococcal 7-valent conjugate vaccine. Other product inserts mentioned the importance of injecting into the desired site, but did not specify aspiration as a way of ensuring this. Clearer instructions must be stated if indeed potentially serious complications may occur if a drug or vaccine is inadvertently administered at a site other than that recommended.

From conclusion:

There is a shortage of consistent recommendations regarding aspiration before injection in published literature, regulatory guidelines, and medical and nursing school curricula. There is also no central and easily accessible place where one can access and review information and guidelines regarding the procedure. It is therefore important to bring all the evidence, published and otherwise, to the forefront for clinicians, researchers, regulatory bodies and device manufacturers so that they can make an informed decision. Based on our findings, the need for aspiration prior to administering an injection is dependent upon multiple factors. Systemic adverse effects profile and mode of delivery (IV vs IM and SC) of drugs plays a significant role in the decision to aspirate or not to aspirate. There is ample evidence that suggests that aspiration may not be required for IM and SC injections, while for IV injections the systemic side effects of the drug should be considered when aspirating before any injection.

Note that the last sentence in the conclusion suggests that aspiration may not be required. However, this is referring to injections in a medical/clinical setting with injections being carried out by trained nurses (i.e. it is not referring to trans girls injecting at home, on their own - away from medical assistance, if it might be required). Also, different injection locations for IM carry different risks. For instance IM into the upper, outer quarter of the glutes, can be considered higher risk than an IM injection into ventrogluteal muscle location (see below for a reference/discussion on this). Aspiration takes only 30 seconds, and given it provides a valuable additional layer of safety/security - it therefore just makes sense for a trans girl to aspirate the needle before injecting a medication.
Further comments and resources
Trans girl giving self IM injection with aspiration
[HOWEVER PLEASE NOTE: the trans girl in the video does the aspiration too quickly and doesn't sufficiently pull back the syringe plunger. When aspirating it is important that sufficient negative pressure is produced in the syringe body (so as to draw any blood present at the needle tip up into the syringe) - and secondly, that enough time is given for any (potential) blood droplets to flow up into the syringe body. As detailed in the study above: 'aspiration is defined as the pulling back of the plunger of a syringe (for 5–10 seconds)'.]

https://youtu.be/csSqhX3ErDw?t=7m33s

Website with images showing different injection site locations (see 'Glute' in left hand menu bar):

http://www.spotinjections.com/index3.htm

and a useful minute log vid, showing how to locate correct injection spot for glutes (i.e. buttocks):

https://www.youtube.com/watch?v=nRkuiszpsoQ

Re Needle sizing for IM injection
If the injection solution isn't too thick then a 25g needle can be used, which should be more comfortable than a 23g.
The important thing with IM, is that the solution is delivered deep into a muscle. At least for IM into the glutes, it's typically suggested to use a 25g x 1.5 inch needle. However this page https://www.chop.edu/news/make-sure-you-choose-proper-needle-length-when-vaccinating-your-patients, lists some various ways of determining what optimal needle length might be for a particular individual - and for different injection locations.
Wiki article on pulmonary embolism:
https://en.wikipedia.org/wiki/Pulmonary_embolism

Symptoms of pulmonary embolism are typically sudden in onset and may include one or many of the following: dyspnea (shortness of breath), tachypnea (rapid breathing), chest pain of a "pleuritic" nature (worsened by breathing), cough and hemoptysis (coughing up blood).[16] More severe cases can include signs such as cyanosis (blue discoloration, usually of the lips and fingers), collapse, and circulatory instability because of decreased blood flow through the lungs and into the left side of the heart. About 15% of all cases of sudden death are attributable to PE.[2]
On physical examination, the lungs are usually normal. Occasionally, a pleural friction rub may be audible over the affected area of the lung (mostly in PE with infarct). A pleural effusion is sometimes present that is exudative, detectable by decreased percussion note, audible breath sounds, and vocal resonance. Strain on the right ventricle may be detected as a left parasternal heave, a loud pulmonary component of the second heart sound, and/or raised jugular venous pressure.[2] A low-grade fever may be present, particularly if there is associated pulmonary hemorrhage or infarction.[17]
As smaller pulmonary emboli tend to lodge in more peripheral areas without collateral circulation they are more likely to cause lung infarction and small effusions (both of which are painful), but not hypoxia, dyspnea or hemodynamic instability such as tachycardia. Larger PEs, which tend to lodge centrally, typically cause dyspnea, hypoxia, low blood pressure, fast heart rate and fainting, but are often painless because there is no lung infarction due to collateral circulation. The classic presentation for PE with pleuritic pain, dyspnea and tachycardia is likely caused by a large fragmented embolism causing both large and small PEs. Thus, small PEs are often missed because they cause pleuritic pain alone without any other findings and large PEs often missed because they are painless and mimic other conditions often causing ECG changes and small rises in troponin and BNP levels.[18]

So I saw a figure in UWorld that said that an advantage of polysaccharide vaccines is that they have a decreased incidence of replacement strains due to a lack of mucosal immunity. I'm having trouble deciphering what this means. Can anyone shed some light on it? My understanding was that conjugate vaccines were generally considered better because they provoke a more robust immune response and involve T-cells as well, instead of just B-cells.

Authored by Hélio Ribeiro

Abstract

In the last years, inorganic nanostructures as carbon nanotubes, graphene, hexagonal boron nitride, and metallic nanoparticles have been applied in several biomedical and nanotechnology fields. These different types of nanomaterials also are awakened as new perspectives in prophylactic, diagnostic and therapeutic areas. However, the uses depends strongly on their chemical and physical surfaces that display an important role in their biocompatibility in different biological systems. A brief study of several types of nanomaterials, their modifications and biomedical application is the main contribution of this short communication.
Keywords: Nanotechnology; Nanomedicine; Nanomaterials; Carbon nanotubes; Graphene; Hexagonal boron nitride; Gold nanoparticles
Abbreviations: CNMs: Carbon Nanomaterials; h-BN: Hexagonal Boron Nitride; CNTs: Carbon Nanotubes; rGO: Reduced Graphene Oxide; Dox: Doxorubicin; PEG: Polyethylene Glycol; BNNTs: Boron Nitride Nanotubes; MDS: Molecular Dynamics Simulations

Introduction

Many studies about nanomaterials have been widely explored in recent decades in different scientific and technological areas. Several authors consider that the discovery of CNTs by Iijima [1] and graphene by Geim and Novoselov [2], stimulated the studies in nanotechnology. Concomitantly, the advances in probe, scanning, and transmission microscopies, it has also contributed to the discovery of new nanostructures and the understanding of others that were not yet well elucidated. It was expected exceptional physico-chemical properties and biocompatibility of nanostructures such as CNTs, graphene, h-BN, BNNTs and metallic nanoparticles, among others [3]. On the one hand, these materials have an enormous potential range of applications in nanotechnology, bioengineering and biomedicine [4,5], such as tumor markers[6], drugs delivers [7], bio-packaging [8], biosensing [9-11], adjuvant in vaccines [5-12,13], among others. However, the compatibility and dispersion of these nanoparticles in the medium of interest are fundamental to their potential applications [3]. The nanoengineering interfaces between host biological system and nanoparticles involves several challenges that need to be overcome. For instance, there-stacking or agglomeration processes of nanoparticles do not allow them to transfer their expected properties to the system, resulting in an inhomogeneous dispersion medium with minimum of biocompatibility. These undesirable processes can be overcome by physical or chemical modification methodologies of their surfaces, such as covalent or non-covalent functionalization. Thus, our choices will depend on the nanoparticles and the biological system in study. The covalent functionalization depends on bonding between the nanoparticles and the functional groups that were chosen, according to the selectivity [3]. Based on this approach, different organic or inorganic functional groups or nanoparticles can be anchored. For instance, it can be introduced on surfaces of oxidized CNTs or graphene oxide (GO), functional groups such as alkoxy (-OR), amino (-NH2), amine (-NHR), alkyl (-R) [14,15], heteroatom doping, metallic nanoparticles, biomolecules and biopolymers, among others. These modifications process alter significantly their interactions with the medium leading them to a large range of solubility in water, co-polymers or organic solvents [3]. On the other hand, non-covalent functionalization processes of nanoparticles are strongly dependent of their physical interaction with host system through intermolecular forces, such as van der Waals, hydrophilic, hydrophobic, hydrogen bonding and π-π interactions, among others [16]. Taking advantages of these physical interactions of molecules (conjugated, surfactants etc), they form homogenously dispersion into different medium with their controlled physicochemical and biological properties [17].
In this context, several studies have presented different types of covalent and non-covalent functionalization of these nanostructures with great technological demands, such as: in cellulose films or fibers [18,19], chitosan [20], polyethyleniminegrafted nanoribbon (for recognition of microRNA)[11], vinyl acetate co-polymer[17], octadecylamine [21], glucose oxidase biosensing [22], poly(ethylene glycol) [23], DNA [24], metallic nanoparticles, among others. Some examples of these modifications in CNMs can be seen in Figure 1a-1d.
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Large amount of nanostructures, zwitterions, supra molecular, clusters, including micelles, dendrimers, quantum dots, biopolymers CNTs, graphene, metallic nanoparticles, have been intensively investigated as biological agents in several biotechnological applications [25]. For instance, gold nano spheres and gold nanorods (Figure 2a-b), have represented the most attractive metallic nanostructures for biological application due to their biochemical features and low related toxicity. Gold nanorods biosensing is the modality widely used especially for the development of optical and electrochemical sensing platforms. The surface plasm resonance properties based on their sensitive spectral response in light absorption and scattering can change in the biological environment, allowing the monitoring of light signal, for instance in cancer cells[26]. One the most important gold nanoparticle application is in vaccine development [26]. There is a great evidence that these nanoparticles display adjuvant characteristics, promoting cell recruitment, antigen-presenting cell activation, cytokine production, and inducing a tumoral immune response [12]. Another relevant application based on physical properties of gold nanorods is their enhanced optical absorption in the visible and NIR region that has been proposed as an alternative for the localized ablation of target cancer cells without damaging other healthy cells, this technique is known as photothermal therapy [6]. Regarding the development of new strategies for cancer therapy, gold nanoparticles have shown promising perspectives to increase radiation effects mainly in tumor cells rather than in normal cells. This effect is due to the high Au atomic number that can lead to increased cross section probability under photons beams from ionizing radiation sources [27]. Nonetheless, gold nanoparticles present relative easy surface functionalization and several biological molecules currently used as immunotherapeutic, such as cetuximab and trastuzumab have demonstrated improved effects when associated with gold nanoparticles [28]. There is a plenty literature about biomedical applications of gold nanoparticles highlighting their importance and advantages to improve diagnostic and therapies for infectious and degenerative diseases as well as a `big deal` to the pharmaceutical industry in a near future [29].
Other important class of nanomaterial with great potential application in bionanotechnology are recognized as one or bi-dimensional, such as CNTs, BNNTs, graphene, h-BN,among others. These nanostructures present exceptional physical properties, besides good chemical stability, well-tailored biocompatibility and lower cytotoxicity [21,22,30]. For example, BNNTs and CNTs are tubular nanostructures with large aspect ratio, high mechanical strengths, and they have a potential application as nanocarriers for use as cancer drugs [25]. Weng et al., [21] demonstrated that the BNNTs functionalized with hydroxyl groups loaded ~300wt% of doxorubicin (Dox), a monoclonal antibody for targeting and a fluorescence marker for visualization, with application in cancer therapy [31], this nanocomplex exhibited higher efficiency to reducing LNCaP prostate cancer cellular viability than the free drug alone [21]. CNTs also can be used as carrier for drug delivery when they are tailored at entering the cells nuclei. Researches have showed that functionalized CNTs can cross the cell membrane, without cellular recognition as harmful intrudes [32]. In other studies, it was demonstrated that the Dox interacted with CNTs through π-π stacking following the functionalization with polyethylene glycol (PEG) to increase their blood circulation plasmatic halflife and to decrease their toxicity [33]. Likewise, CNTs samples, were modified with Dox, and the results showed that cancer cells efficiently took up this compound. While Dox was effectively released and accumulated in the nucleus, while CNTs remained in the cytoplasm.
This result indicates the high loading capacity of the CNTs due to its large aspect ratio and effective noncovalent interaction between them and drug molecules [25]. Gao et al. [19] showed by MDS studies that, DNA molecule in water environment can be inserted into CNTs (endohedral functionalization) through van der Waals and hydrophobic interactions. Based on their studies, they suggested that the encapsulated CNTs-DNA molecular complex can be used as DNA-modulated molecular electronics, biosensors, DNA sequencing, and gene delivery systems [32,34]. Zheng et al. [24] proposed an effective technique of dispersion of CNTs in water by their sonication process in presence of DNA. By MDS this research group suggested that DNA can binds to carbon nanotubes through π-stacking, resulting in helical wrapping in their surface[24] (Figure 1a).

Conclusion

In this short communication we highlight some experimental and theoretical works with different combinations of nanostructures and molecules, as well as metallic nanoparticles with potential bio and technological applications. However, the most important aspect for success and an optimal performance of these compounds is the choice of the best tailored functionalization process (bio-nano engineering) for each type of biological system. The physical-chemical modification is an essential step for relevant applications, leading to hybrid compounds chemically stable, tailored, well dispersed, and compatible with the biological environment of interest. Thus, it is possible to produce smart nano-systems with advanced applications in biotechnology and biomedical.
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