Tag Archives: biofilm

Biofilm on the septum/housing of a needleless connector. Image courtesy of Marcia Ryder, RN, MS, PhD

What are biofilms and how do they form?

Biofilm on the septum/housing of a needleless connector. Image courtesy of Marcia Ryder, RN, MS, PhD

Biofilm on the septum/housing of a needleless connector. Image courtesy of Marcia Ryder, RN, MS, PhD

Thousands of years ago, when no one was safe from famine, dangerous wild animals, or diseases, some of our early ancestors got a bright idea: let’s band together! By forming communities made of people with different talents and skills, the survival rate of individuals grew tremendously. There’s strength in numbers but let’s not pat ourselves on the back too hard — we’re not the only ones who got this idea. Ants and bees do it. Microbes too — they group together into communities called biofilms.

The microorganisms that form biofilms include bacteria, fungi, and protists. Perhaps the most common biofilm familiar to most is the dental plaque — that sticky, colorless film of bacteria and sugars that constantly forms on our teeth. That slime on the surface of water, particularly ponds, is also biofilm.

According to this paper, a bacterial biofilm is defined as “a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface.” In plain English, this means that bacteria sometimes join together, cling to essentially any surface, and form a protective matrix around the group. Indeed, we’ve found biofilms almost anywhere; on mineral, metals, inside our gut etc. In fact, biofilms have been around for at least 3.3 billion years. However, it’s in wet and moist environments that you’ll the most biofilms. They love wetness.

A vast number of pathogens are grouped as biofilms. Like humans, they’ve learned this configuration enhances their survival rate as they are better able to combat the cells of our immune system bent upon destroying them.

How biofilms form

The life cycle of a biofilm. Credit: Bay Area Lyme Foundation.

The life cycle of a biofilm. Credit: Bay Area Lyme Foundation.

The slimy films start forming when initially free-floating bacteria adhere to surfaces in aqueous environments and start ‘laying their roots’. To stay sticky, the bacteria excrete a glue-like substance that’s effective at anchoring them to all kinds of materials, from plastics to soil to medical implants such as pacemakers. This glue is known as an extracellular polymeric substance (EPS) and is comprised of sugars, proteins, and nuclei acids like DNA.

In time, layers upon layers of EPS are added. After a period of growth, a complex 3D structure emerges which is packed with water channels on the inside that facilitate the exchange of nutrients and waste products.

A fascinating thing about biofilm formation has to do with how the bacteria communicate. Pathogens can instruct each other where to position themselves through quorum sensing. Basically, this phenomenon allows a single-celled bacteria to sense how many other bacteria are there in its close proximity. If the bacteria senses there’s a dense population surrounding it, it will be inclined to join them. Remember, strength in numbers.

“Disease-causing bacteria talk to each other with a chemical vocabulary,” says Doug Hibbins of Princeton University.

“Forming a biofilm is one of the crucial steps in cholera’s progression,” said Dr. Bonnie Bassler, a microbiologist also at Princeton. “They [bacteria] cover themselves in a sort of goop that’s a shield against antibiotics, allowing them to grow rapidly. When they sense there are enough of them, they try to leave the body.”

Sometimes clumps of biofilm can break away from the main mass and establish themselves on a new surface. These new pioneers will continue to extend their slimy film until they form a new, bigger colony.

How big can a biofilm get

Most biofilms are very thin — just a few cell layers thick. That’s too thin to see with the naked eye. In fact, your kitchen counter almost certainly has a biofilm layer on it. You just can’t see it. Some biofilms, however, can grow many inches thick and are obviously noticeable. You’ll find these thick slime molds growing as algae on rocks in a streambed.

The thickness of biofilms depends on several environmental factors. Some organisms can produce large amounts of EPS and hence grow a thicker biofilm. Water flow is also an important factor or, to be more precise, shear stress is. If a biofilm forms in a creek where there’s a high flow of water, it ought to be fairly thin. Biofilms formed in slow flowing water, like a pond, can grow quite thick.

Why biofilms form

Biofilm on teeth, commonly known as dental plaque. Credit: Mead Family Dental.

Biofilm on teeth, commonly known as dental plaque. Credit: Mead Family Dental.

As mentioned, bacteria band together because as a community they enhance their chance of survival, but what threats do they face and how does living a slime mold protect them? Some of the stressors bacteria face are the lack of water, high or low pH, or the presence of ‘toxic’ substances, i.e. antibiotics or antimicrobials.

The EPS layers act as the first line of defense against these threats. It can prevent dehydration or shield the bacteria against UV light. When they come in contact with the EPS, antimicrobials, bleach or even metals become bounded and neutralized by the sticky EPS.

Antibiotics can certainly destroy biofilm but not always because biofilms employ another line of defense. For instance, despite antibiotic substances might penetrate the EPS layer, they can be met dormant bacteria. Because these bacteria lack cellular activity, the antibiotics don’t work their magic because there’s nothing to disrupt.

Another line of defense against antibiotics are the ‘persisters’ or special bacteria that do not divide. These bacteria produce substances that block the targets of many antibiotics, according to a 2010 paper. Compared to free-floating bacteria, those growing as a biofilm can be up to 1,500 times more resistant to antibiotics

Finally, living inside a community, often made of different bacterial species, means its members can reap the benefits that come with having a multi-skilled network. For instance, some biofilms are made of both autotrophic and heterotrophic microorganisms. The autotrophs produce their own food using photosynthesis and available organic material while heterotrophs don’t make their own food and require external sources of carbon. As such, in these biofilms, the microorganisms will often cross-feed. It’s a sort of division of labor.

Biofilms, humans, and disease

Scanning electron images of the surface of a mouse bladder infected with urinary tract infection show large intracellular communities of biofilm bacteria inside pods. Uninfected bladders appeared smooth, but infected bladders had bumps all over them. Credit: The Marshall Protocol Knowledge Base.

Scanning electron images of the surface of a mouse bladder infected with urinary tract infection show large intracellular communities of biofilm bacteria inside pods. Uninfected bladders appeared smooth, but infected bladders had bumps all over them. Credit: The Marshall Protocol Knowledge Base.

Biofilms seem to be able to form and cling to just about any external surface as long as it’s wet. This may naturally beg the question — does that mean they can form inside the human body as well? It certainly is wet enough and, indeed, we find that the answer is ‘yes’. According to the National Institutes of Health, more than 65% of all microbial infections are caused by biofilms. That might strike you as a lot but you need to keep in mind that the vast majority of infections are common like urinary tract infections, catheter infections, common dental plaque formation and so on.

However, biofilms can be involved in a range of diseases and medical problems. One example is kidney stones which are caused by biofilms. Some 15 to 20 percent of kidney stones form as a result of urinary tract infections, produced by the interplay between infecting bacteria and mineral substances from the urine.

Then there’s endocarditis, a disease that involves inflammation of the inner layers of the heart. Endocarditis seems to be triggered by a complex biofilm made from bacterial and host component located on a cardiac valve. This type of biofilm is known as a vegetation. The vegetation can disrupt valve function, produce a near-continuous infection of the bloodstream, and can block blood circulation through a process known as embolization.

Pathogenic biofilms also plague prostheses and various medical implants like artificial joints and heart valves or pacemakers. This first came to the medical community’s attention in the 1980s when bacterial biofilms were found on intravenous catheters and pacemakers.

“When people think of infection, they may think of fever or pus coming out of a wound,” explains Dr. Patel from the Mayo Clinic. “However, this is not the case with prosthetic joint infection. Patients will often experience pain, but not other symptoms usually associated with infection. Often what happens is that the bacteria that cause infection on prosthetic joints are the same as bacteria that live harmlessly on our skin. However, on a prosthetic joint they can stick, grow and cause problems over the long term. Many of these bacteria would not infect the joint were it not for the prosthesis.”

Biofilms have been poorly understudied until recently but evidence suggests they’re involved in many human diseases, including debilitating chronic infections. According to Dr. Trevor Marshall, a biomedical researcher at Murdoch University, Australia, some large microbiota of chronic biofilm like L-shaped bacteria can evade the immune system because, long ago, they evolved the ability to reside inside macrophages. Ironically, these are the very white blood cells of the immune system which are supposed to kill the invading pathogens. Marshall also says that biofilm infections occur with great ease in immunocompromised hosts.

Targeting biofilm infections

Research carried out over the past three decades suggests that biofilms are either extremely difficult or impossible to eradicate from the human body. What’s certain is that administering antibiotics in a standard manner (high dose, constant) does not work.

After high doses of antibiotics are administered, it may seem the biofilm infection has disappeared. However, it will reappear because the biofilm was not destroyed, only weakened. It seems that while antibiotics can penetrate the biofilm matrix and kill bacteria, a number of cells called ‘persisters’ are left behind. These are able to survive the onslaught of antibiotics and gradually allow the biofilm to form again.

Dr. Kim Lewis of Tulane University, however, says that it is possible to destroy some biofilms. His treatment involves using pulsed, low dose antibiotics to break up the biofilm. For instance, research suggests this technique is effective at destroying P. aeruginosa biofilm bacteria in a manner that is indistinguishable when the same antibiotic concentrations are administered to single planktonic cells.

When the low, pulsed dosing of antibiotics is applied, the first application eradicates the bulk of the biofilm cells, leaving the persisters behind. Because the antibiotics are stopped, the survival of the persisters is not enhanced. Lewis believes this causes the cells to lose their shape and biochemical properties, making them unable to restart the biofilm formation process. A second application of the antibiotic after a certain time should then completely eliminate the persister cells.

The efficacy of this method depends on the ability to manipulate the antibiotic concentration. Furthermore, not all biofilms can be broken down this way.

Useful biofilms

Biofilms can cause serious medical conditions and, as we’ve seen, they can be very difficult to get rid of. But there are instances when biofilms can be useful, for bioremediation purposes. Biofilms are used, for instance, in treating waste water or contamination with heavy metals or radioactive substances. Another practical use for biofilms is in microbial fuel cells. In such fuel cells, microbes that live on the surface of an electrode break down nutrients and transfer electrons through a circuit, providing electricity. Microbial fuel cells can be very useful if you need to remotely generate power for sensors in wastewaters or landfills.

Biofilms are right now the subject of intense research. Biofilms cause billions in damage every year due to disease, equipment damage, energy loss or contaminations, and as such finding ways to get rid of them is a priority. The resilience of biofilms is a big challenge and requires contributions from different sciences such as biochemistry, engineering, mathematics, and microbiology.

 

The Bacteria Files: Pseudomonas — What it is and why you should know about it

It may not be a household name on the level of E. coli or Salmonella, but this troublesome bacterium is very well known. In medicine and manufacturing, Pseudomonas is high on the list of things you don’t want to have nearby. It all comes down to one particular tendency it has: like a freeloader, a rash, or that one gray hair, once Pseudomonas shows up, it simply will not go away.

Pseudomonas is a genus of gram-negative, rod-shaped bacteria which use flagella for movement. They are typically found in soil and water as obligate aerobes (needing oxygen for survival), but also colonize plant and animal tissues. A colorful group, many of them produce blue-green pigments called pyocyanins. P. fluorescens, as the name suggests, produces a pigment that even glows in the dark. In a lab setting, seeing growth media turn green is a suspicious sign that the organism may be present.

Pseudomonas fluorescens under UV light. Image credits: Biotech Michael / Wikipedia.

A Hospital Hazard

When it comes to Pseudomonads as pathogens, P. aeruginosa is the star of the show. It likes to spend its time in hospitals, thriving on medical equipment, such as catheters and ventilators. It will settle on a surface, get comfortable, and form a large extended family — but it won’t be content to remain there. This microbial opportunist is simply waiting for the right moment to strike.

Supervillain that it is, it has quite a few accomplishments under its belt. Pseudomonas aeruginosa alone is associated with sepsis, pneumonia, dermatitis, urinary tract infections, and infections in cystic fibrosis patients and the otherwise immunocompromised. It’s also special within its genus because it is one of the only Pseudomonads that can maintain metabolism in the absence of oxygen. This is what enables it to stay functional in damaged lung tissue.

In cystic fibrosis, a genetic disorder causes cells to produce a much thicker, more viscous mucus than is typical. In the lungs, this blocks ducts and passageways, and generally makes it difficult for the natural defenses (such as cilia) to function. This is when your opportunist sees its chance. It enters with moist air from a contaminated ventilator, or simply from unwitting, unclean hands touching your face, and settles in the altered mucus of the lung. Now it can do the thing it’s most hated for: it forms a structure called a biofilm.

Scanning electron micrograph of P. aeruginosa. Image credits: Janice Haney Carr, USCDCP.

Once settled, the cells rapidly reproduce and then literally stick together, releasing cellular products to form a slimy matrix. In the end, you have layers and layers of bacteria forming on top of one another until all medication can truly do is cut away at the upper surface. And if the fact that it’s so anchored wasn’t enough, along with Staphylococcus aureus and Klebsiella species, P. aeruginosa is among the best known bacteria in the world for laughing in the face of antibiotics.

As it turns out, P. aeruginosa isn’t limited to only affecting humans from within. It can be the bane of any water bottling plant. Since they tend to live in springs, wells, and other natural sources, with one small error in the process, Pseudomonas could become attached to a factory line. And once the biofilm fully establishes on the equipment, no amount of scrubbing will make it go away. Heavy-duty chemical treatment is required. Their presence can be so pervasive, that manufacturers have been known to just toss out expensive equipment, rather than waste further resources trying to rid themselves of this plague.

Silver Linings

Still, it isn’t all bad news. Many Pseudomonads have made significant positive contributions to human activity as well — from antibiotics and research opportunities to even cleaning up our messes:

P. fluorescens, can be cultured to produce an antibiotic called Mupirocin, used in the treatment of highly resistant bacterial species. It is also found around plant roots and acts to protect them from fungal growths.

P. deceptionensis lives in the Antarctic and has provided multiple avenues of interest. Strain M1T has presented with a previously unknown internal structure called a stack, meanwhile strain DC5 produces silver particles during metabolic processes.

P. aeruginosa, P. putida and a few other species can decompose hydrocarbons and are used in bioremediation. In fact, a strain of P. putida is the first organism to be patented (with much difficulty) because of its potential for use in degrading toluene and naphthalene in soil, as well as converting styrene to a biodegradable plastic, without being a threat to human health.

Deception Island, Antarctica where P. deceptionensis was discovered. Image credits: W. Bulach / Wikipedia.

So, you see, even the worst of them can be used for positive things – it can break down hydrocarbons and is an excellent model for biofilm formation. As with most bacteria, the majority are harmless to humans. But it is always good to be informed and Pseudomonas is just one more thing you should know about.

New Silicone Technology Creates Super Slippery, Anti-Bacterial Surface

A new liquid-infused polymer can make sure that medical equipment is bacteria free by being extremely slippery. This technology, which involves silicone infused with a silicone oil also has a myriad of potential applications outside of medical equipment – in the oil industry, in air planes and cosmetics.

Harvard researchers have demonstrated a powerful, long-lasting repellent surface technology that can be used with medical materials to prevent infections caused by biofilms. (Image courtesy of Joanna Aizenberg, via Harvard University)

According to the National Institutes of Health, over 80 percent of all infections in the human body are caused by a build-up of bacteria. Bacteria accumulates into adhesive colonies called biofilms, which help them survive and protect them from outside threats. Common soaps don’t actually destroy the bacteria, but they make a slippery surface on your skin making it so that bacteria can’t attach themselves to you and fall off – this is the main idea here too.

Such bacterial biofilms tend to form on medical equipment, including surgery equipment heart valves, urinary catheters, intravenous catheters, and implants. Naturally, we don’t want that to happen – as it can be extremely dangerous. Now, a new study demonstrated a long-lasting repellent surface technology that can be used with medical materials to prevent infections caused by biofilms.

The new technology (liquid-infused polymers) can store considerable amounts of lubricant in their molecular structure, much like a sponge holds liquids. This lubricant can then travel to the surface, repelling the bacterian and blocking the environment in which it forms. The team led by Joanna Aizenberg from Harvard is now working on designing several such liquid-infused polymer systems which could be applied on various medical surfaces. However, super-slippery surfaces can have applications in more fields, including keeping glass clean, making better cosmetics and ensuring that ice doesn’t stick to airplane wings.

For this study, they used both a silicone material, and a silicone oil, which are non toxic and safe to use.

“The solid silicone tubing is saturated with silicone oil, soaking it up into all of the tiny spaces in its molecular structure so that the two materials really become completely integrated into one,” said Caitlin Howell, a Postdoctoral Researcher at the Wyss Institute and a co-author on the new findings.

To test the effectiveness of the super slippery surface, the study’s lead author Noah MacCallum, an exchange undergraduate student at SEAS, exposed treated and untreated medical tubing to Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus epidermidis, which are common pathogenic bacteria that form biofilms and are the most common culprits in blood and urinary infections. The experiment confirmed what scientists believed – that the surface greatly reduces biofilm adhesion and largely (though not totally) eliminated biofilm formation. The results give great hope for future applications and reducing infections, especially with drug-resistant bacteria.

“With widespread antibiotic resistance cropping up in many strains of infection-causing bacteria, developing out-of-the-box strategies to protect patients from bacterial biofilms has become a critical focus area for clinical researchers,” said Wyss Institute Founding Director Donald Ingber, who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital and Professor of Bioengineering at Harvard SEAS. “Liquid-infused polymers could be used to prevent biofilms from ever taking hold, potentially reducing rates of infection and therefore reducing dependence on antibiotic use.”

As for applying super-slippery surfaces to other fields, the authors have big plans.

. “We could apply liquid-infused polymers to other materials plagued with biofouling problems, such as waste-water management systems, maritime vessels or oil pipes,” said one of the study’s lead co-authors Philseok Kim, who was formerly a Senior Research Scientist at the Wyss Institute and is currently co-founder and Vice President of SLIPS Technologies, Inc.

However, before we can speak of actually implementing super slippery surfaces into waste water management or the oil industry, the technology has to prove its efficiency in experimental results. Still, the development shows great promise, and I’m certain we’ll be hearing more from it in the near future.

“Each technology in our portfolio has different properties and potential uses, but collectively this range of approaches to surface coatings can prevent a broad range of life-threatening problems, from ice accumulation on airplane wings to bacterial infections in the human body,” said Aizenberg.

 

Journal Reference: Noah MacCallum et al. Liquid-Infused Silicone As a Biofouling-Free Medical Material. DOI: 10.1021/ab5000578

biofilm

World’s water streams affected by pharmaceutical pollution

A new study stresses the overlooked hazards that dumped pharmaceuticals found in wastewater pose to the world’s freshwater streams. So far, the impacts and consequences on water quality and aquatic life are unknown or under researched, and the authors hope their findings might warrant more work in this direction.

Dr. Emma Rosi-Marshall, lead author of the study published in the journal Ecological Applications and a scientist at the Cary Institute of Ecosystem Studies, looked at how  six common pharmaceuticals influenced similar-sized streams in New York, Maryland, and Indiana. These were caffeine, ciprofloxacin, metformin, cimetidine, ranitidine and diphenhydramine. The synthetic compounds that end up in the world’s streams as a result of aging infrastructure, sewage overflows and agricultural runoffs are in much greater number, however, ranging from stimulants and antibiotics to analgesics and antihistamines.

The focus of the study was on biofilms or the slippery coating found on stream rocks, as they’re most easily recognized as. These coatings, made out of algae, fungi, and bacteria all living and working together, are center to supporting aquatic life and greatly influence water quality, as they recycle nutrients and organic materials, while also making up a fundamental food source for invertebrates, which at their own term form the basic food source for other animals, like fish.

biofilm

The authors’ findings suggest that the effects of waste pharmaceuticals are worrisome and need to be controlled. One of them, for instance, antihistamine has been found to dry out biofilms, while when exposed to diphenhydramine a 99 percent drop in biofilm photosynthesis was experienced. Diphenhydramine also caused a change in the bacterial species present in the biofilms, including an increase in a bacterial group known to degrade toxic compounds and a reduction in a group that digests compounds produced by plants and algae

“We know that diphenhydramine is commonly found in the environment. And its effect on biofilms could have repercussions for animals in stream food webs, like insects and fish. We need additional studies looking at the concentrations that cause ecosystem disruption, and how they react with other stressors, such as excess nutrients,” said  Rosi-Marshall.

Other substances’ influence on water biodiversity and quality were also found to have a measurable effects both alone and in combinations, using pharmaceutical-diffusing substrates. More work is required, however, for a broader picture of how various drugs, both alone and in mixtures, effect the freshwater stream environment. Results so far stress that a more thorough looks is required and considering most water treatment facilities in the world lack the necessary tools to filter out pharmaceuticals, the situation all of a sudden seems a lot more serious than at first glance.