[, Music, ], hello. Everyone welcome to the course on a medical biomaterials. Now we are going to talk about biofilm. Biofilm is a very important concept in biomaterial and it affects quite a lot of biomaterial design and life of a biomaterial. What is this biofilm? Well, the definition is it’s: a population of microorganisms concentrated at a solid-liquid interface, okay and surrounded by an extracellular polymeric substance. Okay, it’s called EPS matrix, so this biofilm is attachment on material surface. It could be a stainless steel or it could be a polymer or it could be a ceramic. It could be even the inside lining of vascular, graft. Okay, it may contain live cells, it may contain dead cells, it may can. It will contain proteins, sugars, polysaccharides metabolites that are metabolites that are produced by the bacteria secondary metabolized and also quorum. Sensing signaling molecules so biofilm might contain a lot of these okay and they are attached in at the early stages. The attachment is reversible. That means the material leaves, but as time progresses, the attachment becomes more and more difficult. Okay, both gram-negative and gram-positive bacteria forms biofilms on indwelling medical devices. That means like I said it could be a short duration device or it could be a long duration device. Biofilm can form even in the urinary catheters the first few hours bacteria can start to accumulate, which are reversible, but as time progresses, the attachment becomes very strong and the bacterial biofilm starts growing. So large number of bacteria can form this biofilm. These are the common bacteria found like to equalise Pseudomonas, Staphylococcus aureus, Staphylococcus epidermidis, Proteus, mirabilis, Klebsiella, pneumoniae, Enterococcus, streptococcus and so on. Actually so all these are bacteria found and biofilm whether it is a dental implant, whether it is urinary, catheters, urethral, catheters and so on. Actually, all these bacteria can form biofilms. I just want to show some pictures. You can see the bacterial attachment and growth in some of these dental area. Look at this fracture plate. Okay, there is an infection in that you can see these where the bacteria has settled down and formed biofilm and it’s more difficult to get rid of these type of bacteria and compared to bacteria, which is in the cell for more floating form. It has been found that almost you need ten times more concentrations of antibiotics to kill bacteria which are well established on surfaces like I said it could be a metal surface, it could be a polymer surface, it could be a ceramic and so on. Some more pictures to look at this. This is a prosthetic valve. You can see a lot of growth. Look at this is a neutral, stent. Okay, this is called a wjj pigtail return. Stand. You can see bacterial infection generally in the urinary region. You may have bacteria like e-coli or Proteus mirabilis there. So biofilm is very common in medical implants. It could be even within a few hours and it could last for years and years so addressing that. Biofilm is a very big issue in biomaterial research, because this biofilm can lead to infection infection can lead to immune compromising or failure of the biomaterial and so on. Actually, there are different strategies that are being practiced, then getting rid of biofilm. Some of them are successful. Some of them still needs a lot of research, and you cannot have one single strategy to get rid of all the biofilms, because, depending upon where the material is introduced, the type of the nature of the bacteria and the biofilm can vary quite a lot. These are some more biofilm pictures. These are scanning electron pictures of stuff, lock, focus, bio, Philemon, polyurethane surface. You can see this and this is separate. Pseudomonas aeruginosa biofilm one polyurethane surface, so you can see not only the bacteria or the EXO polysaccharide, so it forms a thick layer. Okay, so lot of materials, like I said in different environments, end up having biofilm. For example, if you look at catheters central venous, catheters made up of polyurethane environment this blood, but still you can have Staphylococcus epidermidis or Staphylococcus aureus or Pseudomonas capsular type of biofilm. Look at him. Oh, dialysis, catheters polytetrafluoroethylene is used here again. The environment is blood again, Staphylococcus aureus or gram-negative n erupts. Polyurethane is used in pulmonary artery, catheter x’. Again, the environment is blood. Sorry, you can have coagulase negative staph, lockup or Enterococcus Pseudomonas Actinobacteria netiquette Ito, like silicone or polyurethane or silicon. This is in the urine environment. You can have equal, me you can have internal pockets, you can have Proteus mirabilis, look at the peritoneal dialysis silicone. It will have an interaction with blood and fluids solids. You can have Staphylococcus epidermidis and Staphylococcus aureus. Internal feeding tubes like PVC or polyurethane generally fluids. Again, you can look at the type of bacteria here. Gastrostomy tubes made up of silicone-polyurethane intestinal fluids again into the caucus Staphylococcus e-coli again, endotracheal tubes, PVC, silicone stainless steel. Again, you can have a Staphylococcus, okay and track your tummy tubes. These are made up of PVC and silicone generally, its environment is air. You can have step, lock, occurs, epidermis and so on. So you can see the titanium stainless steel in spinal. It’S in contact with fluids. You can have a coagulase negative step, lockers or Mycobacterium tuberculosis penile implants, silicone it’s in touch with urine and it’s gram-positive rods. Cocci breast implants, silicone polyurethane. You can have again coagulants negative stuff, lacas orthopedic, whether it’s knee implant or hip, or dental defibrillators. Different types of materials we are talking about, mostly metal, is no cobalt chromium, aluminum, stainless steel gold is and then inorganic materials econia aluminum oxide, so they all have different types of bacterial contamination like Staphylococcus aureus, coagulase, negative staphylococci, gram-negative, anaerobes and so on. Actually, okay and finally devices different types of devices binary stand interacting device like copper, vascular, grafts, coronary stents, intraocular lenses; they all can end up having Staphylococcus aureus and epidermis and e-coli. So these are the common bacteria which goes and as I showed you in the past, two to three slides and the environment could be blood. They could be EURion, it could be fluids, different types of fluids where cranial fluid cerebral, spinal fluids and so on. So we have metals, non-metals, polymers, okay and ceramics. They all have this type of infection. Okay. So how? What are the possible entry points of infection? Periodontal disease, something related to the teeth? Catheters patients many times have urinary catheters inserted. So infection can happen. Implant surgery, a device is placed whether it’s an orthopedic, whether it’s a dental, whether it’s a knee where there’s a stent, ureteral, stent, Korea or cardiovascular trend. Okay, open wound: there is a wound that is open. A lot of bacteria Staphylococcus related bacteria can enter. So these are the point of entry and it’s been found that the most or the infection happens because of the implant surgery and almost sixty to seventy percent. Rejection in the early days of implant is because of infection. So if you can address the implant related infection in the first two to three weeks, then almost 70 % of the rejection of the implant could be overcome. Okay, so let’s look at this biofilm development and dynamics. So what happens is bacteria when it undergoes starvation and they can shrink and become suppose okay, so they become very, very small, ultra micro bacteria, so then they can attach to surface or they cannot be killed by antibiotics so attaching to the surface once they attach. There is a change in gene expression from sumo similar to biofilm for most okay. So there is a change in gene expression of those bacteria when it is in the same form as against amenities in the biofilm form. They can encase themselves really that’s like slimy matrix. We call it EXO polysaccharide lot of sugar, so there is a slimy matrix, that’s forming on top of it and they because of the reduced nutrient availability for bacteria, okay, which are at the lower level of the biofilm and nutrition diffusion, also slows down. So the black bacteria stop growing and they reach this type for starvation condition, and hence there could be change in the gene expression of that exchange molecular signal bacteria which are forming biofilms. They produce a chemical called quorum sensing and thereby they can change their gene expression and hence they can move from the society. The colonizer and the bacteria can also identify that they are a large number of population which are in the colonizing stage. Chemical gradients. There could be because of the biofilm, which is a matrix. There could be a gradient which can create microenvironments for different microbial species with different activities to be present. So there bacteria, which are on the top of the biofilm, which gets nutrients, which get oxygen that gets chemicals and you may have a different environment. So they will have a different activity and growth pattern when compared to bacteria, which are right at the bottom of the biofilm, which do not get Dean of nutrient, which star much longer which do not get enough oxygen. Okay, so antimicrobial drugs, antibiotics damage bacteria in the upper layer, but do not penetrate or present at low concentrations Li at the bottom, okay, okay, so they are below the misc’s when compared to the concentrations of antimicrobial available at the top of this yeah. So the bacteria which are at the bottom, okay, what happens is they become resistance to strains because they are getting a drug concentration much below they remain C, so shear forces can detach cells. So there are cells which are attached on the surface. Then there could be flow, shear forces, blood flow or urine flow, it can detach the cells and they can go out and again attach some aerials and again form a protective layer. So you can see there are so many things happening. It’S a very dynamic process. The biofilm growth maturation detachment is a very dynamic process and it is a very serious issue, especially in the area of bio-medical device. So there are two phases of pathogenesis of that: biomaterial Center infection, physico-chemical interaction between bacteria and surface molecular and cellular interaction. So first is the physical-chemical interaction between bacteria and surface modern is the molecular and cellular interactions. Lets look at it, so initially bacteria in the Brownian motion. They are moving around freely. They are in the society form, so they start to get attached because of non-bonded interactions. Like Vander Waal forces, gravitational forces, electrostatic forces, hydrophobic interactions, hydrogen bonds and so on. Okay, so they attach later on uncoated surface or there could be already a protein layer which is formed on which these bacteria can be attaching so either on uncoated or on protein correct. So the cells deposit, adsorbed and slowly they will get multiplied. Exo polysaccharides are formed, so they start forming big layer of EXO. Polysaccharide is called the matrix here. Okay, so they put the bacteria that are on the surface, get enough oxygen, and nutrient bacteria right at the bottom do not get as much because of the limitations of diffusion of both oxygen nutrients and other chemical chemicals. So they undergo gene genetic changes and compared to them and the antibiotic flow is also controlled because of diffusion, so the antibiotic may kill bacteria right at the top, whereas the concentration of antibiotic at the bottom may be so little. The bacteria here are not killed, so they get exposed to antibiotic concentration, much below the minimum inhibitory concentration, so they may start slowly developing resistance to antibiotics. Okay, so there could be antibiotic resistance bacteria at these layers, which have changed their gene pattern, which have started to grow slowly and so on. Okay, now because of shear forces because of the thickness of the surface of the biofilm, some bacteria may detach itself and start flowing downstream and again. This process of biofilm formation can keep happening. Okay, this is the process of biofilm. This can start in ours, and this can go right up to days. Okay, and some of these mature biofilm can be in weeks and go on forever and ever now, bacterial movement due to human attractants, the kima attractants could be amino acid. Sugar will ego peptides, okay, so this concentration gradient, then it could be cut, more taxes or her taxes, okay, chemotaxis, okay, which is diffusible, so the concentration gradient happens because of the diffusion of these chemoattractants, whereas there are some attractant which are bound to the surface. So the physical interactions are long-range interactions. They are not very specific. It could be greater than 50 nanometers between the cells and the surface, where the short-range interactions will be less than fine. Animators, hydrogen bonding ionic dipole interactions, hydrophobic interaction; they are all short-range, so these are all so we have two types of ingredients produced because of chemotaxis and have no taxes. Okay. That is why the bacteria starts moving towards surfaces, the molecular and cellular to the interaction between bacteria and surfaces. There are many proteins on the surface of the bacteria, okay, which may be doing this type of job, so the PS a this is a galactose-rich, capsular polysaccharide. It is in composed of beta 1 6 linked n as a tile like Osama amines residues with some oxygen-linked substance of succinate phosphate and acetate. Okay, then we have the SAA. This is lime associated antigen, composed of n-acetyl glucosamine. We have PIAA. This is a polysaccharide composed of beta one, six linked in acetyl glucosamine, with partly di seta lightly residues. Then we also have AP. This is an accumulation associated protein, so we see the proteins of the surface of the bacteria and the proteins on the surface of the material surface. There is an attraction, so the the bacteria may have a lot of nh-2 groups. The proteins and the surface proteins may have a lot of s or three groups, so there could be attraction. So this is the molecular and cellular interactions between bacteria and surface, whereas the physical chemical interactions could be because of presence of chemotaxis because of non-bonded interactions and so on. Okay, so P, si to take part in the bacterial material interactions. Okay, whereas a p IA and a gap in the cell-cell interaction, okay, soup, a is a polysaccharide consists of a composed of beta 1 6 link and acetyl glucosamine, whereas a is an accumulated accumulation associated protein, so that leaves into interactions between cell to cell. Whereas P Si and si a leads to interaction between these cells to surfaces, okay, so look at the biofilm mechanism. There could be environmental factors, there could be toxins, this could be the cell growth process and they with something related to the surface proteins. So environmental factors could be temperature, stress or starvation because, like I said, I’m bacteria at the bottom of the biofilm were almost under starvation condition. Okay, so bacteria is it’s a cell form it moves into biofilm, State, okay and there could be a reversible process. Also biofilm dispersion because of enzymes like proteases, which breaks the bond between the bacteria and the surface. We also have something called beta type of phenol soluble modeling’s. These are supposed to be very toxins produced during the biofilm formation, which can lead into a lot of infection. Now, phase of the cell growth, so the bacteria grows and some sometimes it reaches into stationary phase, okay and inside the biofilm they may be in the stationary phase. Then we also have the beta toxins so which are involved in the cell autolysis through a DNA which is also a reversibility process. Okay, so all these are involved in the mechanism of biofilm formation. So, what are the problems associated with bacterial biofilms? A lot of clinical challenges, chronic or recurrent infection, where the patient is a biomaterial, is placed inside the patient and the Maya material gets infected, so the patient gets into chronic infection or recurrent infection he’s given antibiotics and doesn’t get cured. They are given stronger antibiotics and so on. Actually, okay chronic inflammation infection later on there could be inflammation in the surrounding tissues, rapidly occurring antibiotic resistance because, as I said, the bacteria that are attached to the surface inside the biofilm slowly start acquiring antibiotic resistance. So they are not able to be killed, whereas bacteria, which are around the surface, get killed spread of infectious Mbali, so the infection starts spreading on remaining parts of the surfaces. Okay. So all these are problems associated with tiddle, my friend, so, for example, if you look at the frequency of occurrence of urinary tract infection, when you place urinary catheters like didn’t it turn, 20 percent of infection is because of this biomaterial-centered infection, percutaneous Korea, catheters short indwelling, Catheters temporary pacemakers – we are talking in terms of almost 10 percent max subcutaneous, like a peritoneal dialysis catheters, cardiac pacemakers around four percent soft tissues and memory tissues, intraocular lenses, circulatory system like poor, ecar valves, vascular, grafts, artificial heart, okay, bones total knee replacement around four percent Prosthetic hip due to the four percent. So, if you are thinking about having a long-term indwelling biomaterial like hip prosthesis or knee replacement or cardiovascular stings, or carry aware for them, diaphragm valves then long-term use of biomaterials MOCA medical devices. But you have to address this infection and it is the frequent cause of failure. Okay, these numbers were obtained from this particular reference. So, what are the factors influencing bacterial addition? Environment, if there is continuous flow or there is a stagnant flow? If there is a continuous flow, shear forces countryand biofilm formation, whereas if there is a stagnant, then this can lead into biofilm attachment material, surface characterization like surface chemical composition, surface roughness, hydrophilicity hydrophobicity, so on bacteria characteristic bacterial hydrophobicity, hydrophilicity bacterial surface charge. So all these 3 factors affect the biofilm and bacterial adhesion. So if you look at serum or tissue proteins – and there are many proteins like fibronectin, which promotes Staphylococcus addition to the substratum surface, we have albumin which inhibits bacterial addition to polymer-ceramic and metal surface. Okay, because blood will contain all these pull proteins by binding to the bacterial cells or changing the substrate surface to more hydrophilic. So if you have albumin, it will inhibit bacterial addition, whereas if you may have fibronectin, it promotes soft caucus or, as addition because albumin is very hydrophilic. So when the surface becomes hydrophilic, then bacterial addition is reduced. Fibrinogen, it promotes adherence of bacteria, especially staphylococci, to biomaterial. So fibrinogen also promotes thrombin. It also increases bacterial additions. It polymerizes fibrinogen in PPP and to Fabian, because fibrin strands surrounds and link the platelet aggregate to stabilize the thrombus, which also promotes vector addition. Then we have this PPP poor, platelet plasma, serum the addition of various coagulant negative stuff locos onto plasma current materials, much lower, then on to the untreated control surfaces. So generally many of these serum proteins accept albumin. Okay, increases practical addition like fibronectin fibrinogen, thrombin; okay. So, what is this so? This effect is due to albumin, well IgG and FN or less effective, and due to Roman effectiveness, fibrinogen can be displaced by other proteins present in plum plasma such as high molecular weight. However, PPP with thrombin increases bacterial addition, because thrombin enhances Bechtel addition, fibrinogen enhances back television frame, barrow nekton also enhances back to. In addition, no so platelets increases Staphylococcus addition in comparison to human serum albumin, especially in combination with PPP, thrombin. Okay, so platelets also increases the flow caucus so, as I said, generally PPP, serum and albumin inhibits, whereas all other proteins in the serum sort of enhances in different ways the bacterial addition. Now you may ask: what is this fibronectin? Okay? It’S a 450 440 kilodalton glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins okay. Similarly, if you look at what is albumin, the serum albumin is the main protein of human blood plasma and it’s water-soluble. What is this fibrinogen? It is also a protein produced by the liver and it helps in the blood clotting thrombin. It’S a serum protease that converts fibrinogen into fibrin in blood clot coagulation, so serum also plays a very important part in the addition of bacteria to material surfaces. Okay. So if you are having implants which are exposed to blood, then you have to consider these aspects also because the blood protein serum protein contains tissue proteins contain a lot of different types which may enhance or retard bacterial addition to various material surfaces. Okay, there are some bacteria that become persistence once they form biofilms; okay, so these persistence or persisters. Okay. These are a small subpopulation of bacterial cells that are dormant and extremely tolerant to antibiotics, because you have a thick extra polymeric, extracellular polymeric surface, that’s formed on the material with the bacteria live and dead cells. Antibiotics penetration becomes more problematic, so the antibiotics right at the bottom of the biofilm concentration is so low much below their high minimum inhibitory concentration, so they become those bacteria become tolerant to antibiotics. They also become persistent, they’re called persister cells. So these persistence, it’s a phenotype, expressed by almost all bacteria, including major pathogens. This can lead to chronic and lapsing infection. These persister cells can also tolerate high doses of bacteria seidel antibiotics, a small fraction of the bacterial population responds and others form resistance or tolerance towards antimicrobial agents. So that is one of the main, easy reasons in bacteria in biomaterial-related infection because of the formation of this persistent cells. Okay, we will continue about the persistence of bacteria in the next class in more detail. Thank you very much for your time. [, Music, ]