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Gram positive organisms are responsible for majority of bone infections. Treatment of osteomyelitis has been quite bit challengeable due to poor vascularity at the site of infection. Most of the aerobic micro organisms are catalase and superoxide dismutase positive. The main objective of this study is to create antibiotic loaded implants, which releases antibiotics during the presence of aerobic micro organisms, thereby preventing the formation of biofilms and improving the treatment efficacy.
Materials and Method:
Stainless steel plates were coated by dip coating method. The solution was prepared by using 15% of PHBV in Dichloromethane and to which the drug was added at a ratio of 5:1 (polymer: drug) respectively. Following which, spray coating of sodium formate was done over the polymer coating. Parameters such as invitro drug release, thickness of film, zone of inhibition were studied.
Coated plates with uniform thickness of 9µm were obtained. In all bacterial strains, S.aureus, C.sporogens, P aeruginosa, the zone of inhibition was noticed with a inhibited zone of 64mm for S.aureus and 19mm for C.sporogenes. The invitro drug release study showed that the total encapsulated drug was released at a constant rate, gradually over a period of 6 hours.
Antibiotic coated plate's offers a new perspective for treating implant related infections and also overcoming formation of biofilms. The study can also be applied for creating biosensors to detect micro organisms.
Key words: Infection, Dip coating, Ofloxacin, Biofilm.
Infection are defined as "invasion of the body by various agents -including bacteria, fungi, protozoans, viruses, and worms - and its reaction to our system can lead to tissue damage and disease" . They can be local, confined to one body system, or generalized as in the case of septicemia. Over the last few centuries, an infection tends to posses a great challenge in terms of preventing and treating it to our nature. For the past few decades, bacteria and, in particular those pathogenic for humans, have evolved towards developing antimicrobial resistance against various drugs.s Antimicrobial resistance is an issue great significance for public health at the global level [2,3]. Considered as sweet drugs, antibiotics are often prescribed inappropriately and inadequately and have thus become one of the highly abused agents . Bacterial pathogens causing acute infections are increasingly exhibiting resistance to the commonly used antibiotics and have great threat to public health .
For overcoming these challenges, the usage of antibiotic guidelines must be a step towards the up gradation among the antibiotic policies of many multispecialty and tertiary healthcare centres, thereby reducing the antibiotic resistance for next few decades. It is necessary to define an antimicrobial managing program as an ongoing effort by various healthcare institutions to optimize antimicrobial use among hospitalized patients to improve patient's outcomes, ensure cost effective therapy, and reduce adverse sequealae of antimicrobial use .
1.1 Mechanism of developing antibiotic resistance:
An antibiotic has to go through a number of steps in order to exert its antibacterial action. First of all it has to enter the cells (influx). Once inside, it has to remain stable and accumulate to inhibitory concentrations. In some cases it has to be activated to an active form. Finally it has to locate and interact with its target to exert its action. Alterations in any one or more of these processes can render the cells resistant to the antibiotic. All possible alterations have been realized in clinical as well as laboratory isolates of resistant bacteria.
Some of the known ways of developing resistance against antibiotics areas:
Antibiotic resistance by chemical alteration of antibiotics in vivo
Antibiotic resistance due to target alterations
Antibiotics and antibiotic resistance gene.
In Table 1 some of the common pathways of developing antibiotic resistance are mentioned [2,3].
In contrast to the traditional notion that bacteria are free living organisms, they exist as organized structures called biofilms, which consist of a self produced exopolysaccharide matrix in which bacteria cells are embedded. They are highly organized, surface adherent structures and permit the transport of nutrients and metabolic waste in and out. Several reviews on biofilms are available [6 - 8]. One of the characteristic properties of biofilms is their tolerance to a very high concentration of antibiotics . The apparent antibiotic resistance of biofilms associated cells is not due to mutation, since sensitivity reappears when the biofilms are disrupted and the cells are returned to the natural state. Moreover, it has been shown that biofilms associated cells are sensitive to several antibacterial agents [10, 11]. Based on various works, it was suggested that antibiotic tolerance of biofilms associated cells could be due to the presence of antibiotic insensitive persisters in the biofilms. According to model, antibiotic treatment of bio films eliminates most of the embedded cells, except the persisters which repopulate the matrix after the antibiotic is withdrawn, yielding a mixture of antibiotic sensitive cells (majority) and antibiotic tolerant persisters (minority) . This process repeats itself after successive antibiotic exposures, thereby the infection persist in spite of antibiotic therapy. Other factors such as reduced antibiotic influx due to matrix, and lower metabolic and growth rates of cells in the biofilms could also be involved [12, 13]. The other various models other than biofilms include persistence, swarming .
Overcoming the formation of biofilms has been a great challenge. Mechanism of biofilm-associated antimicrobial resistance varies between each organisms [15, 16]. A better understanding of the role of biofilms in infection and how in vivo biofilms respond to selected treatments requires more study .. Studies have been done to compare the effect of alcohols, povidone-iodine and hydrogen peroxide on biofilms of Staphylococcus epidermidis. They confirmed that hydrogen peroxide, at a concentration of 3% and 5%, and alcohols rapidly eradicate S. epidermidis biofilms, whereas povidone-iodine is less effective .
1.3 Infections and Drug Delivery Systems:
Infection are defined as a "homeostatic imbalance between the host and tissue and the presence of micro organisms at a concentration that exceeds 105 organisms per gram of tissue" . The emergence of infection is associated with a large variety of wound occurrences ranging from open injuries to chronic ulcers and complications following any surgery or implantation of any device .The main goal of treating the various types of wound infections should be to reduce the bacterial load in the wound to a level at which wound healing processes can take place.
Conventional systemic delivery of antibiotics for both prevention (prophylaxis) and curing suffers from the various drawbacks with various kinds of systemic toxicity s[22, 23]. Alternative by local delivery of antibiotics by topical administration, or even better by a local delivery device, address the major disadvantage of the systemic approach by maintaining control release of drug at the target site over a long period without much sideeffects . Antibiotics those already incorporated in controlled release device include vancomycin, tobramycin, amoxicillin,gentamicin, cephalotin, etc [22, 25]. The effectiveness of such devices is strongly dependent on the rate and manner in which the drug is released . Also it depends on other parameters such as nature of polymer, the type of degradation it undergoes, nature of drug that is loaded. The release of antibiotic at levels below the minimum inhibitory concentration (MIC) may evoke bacterial resistance at the release site and intensify complications [27, 28]. Furthermore, they are able to get attached to surface of implant leading to formation of an "protective bio- film layer- which is extremely resistant to both immune system and antibiotics" . These biofilms are considered the primary cause of implant associated infection. It has been found that killing bacteria in a bio film requires approximately 1000 times the antibiotic dose necessary to achieve the same results in a cell suspension .
Various biodegradable devices from both natural and synthetic polymers have been produced by different processes in recent years, for use as antibiotic carriers. Biodegradable polymers can release larger quantities of antibiotics and their degradation properties can be tailored for a specific application that will affect arrange of processes such as cell growth, tissue regeneration, drug release and host response .Synthetic bio-degradable polymers that have been reported for various antibiotic eluting devices include poly-(lactide-co-glycolide)copolymers [32-35], polycaprolactone [36,37], polyanhydrides [38-41], polyhydroxy-butyrate-co-hydroxyvalerate (PHBV) [42,43] and poly hydroxyalkanoates . Natural polymers such as collagen [45-49] and chitosan [50-52] are attractive, since they exhibit superior biocompatibility and facilitate cell growth. They are also inexpensive and readily available. Along with the nature of the drug, its molecular weight, water solubility and its solubility in organic solvent, melting temperature and its antibacterial spectrum must be known in order to design an antibiotic - eluting system . In general, these antibiotic eluting devices can be classified into musculoskeletal and orthopaedic related devices, wound dressings, periodontal devices, intravascular devices and vascular grafts.
1.4 Antibiotic loaded implant coatings:
Bacterial infections remains a major limitation of the utility of medical implants, despite sterilization and aseptic procedures, with reported infection rates in the range of 0.5 - 5% for total joint arthroplasties [54, 55]. Sources for infectious bacteria include the ambient atmosphere of the operating room, surgical equipment, and clothing worn by medical professionals, resident bacteria on the patient's skin and bacteria already in the body .
Antibiotic-loaded implant coatings present a straight forward approach for the prevention of implant-associated infections. They can provide an immediate response to the threat of implant contamination but do not necessitate use of an additional carrier for the antibacterial agent other than the orthopaedic implant itself. This is most relevant for 'cementless' implantation procedures that have gained popularity due to better early and intermediate-term results in young patients compared to cemented prostheses .
Unlike "passive" coating techniques that aims to reduce bacterial adhesion by altering the physiochemical properties of the substrate so that bacteria - substrate interactions are not favourable, "active" coatings are designed to temporarily release high fluxes of antibacterial agents immediately following the implantation. High local doses of antibiotics against specific pathogens associated with implant infections can thus be administered without reaching systemic toxicity levels with enhanced efficacy and less probability for bacterial resistance. Recent studies have raised the possibility of incorporating growth factors in order to promote tissue healing responses [58, 59].
The utilization of a bioactive ceramic coating containing hydroxyapatite (HA), calcium phosphate and other osteo conductive materials as antibiotic carriers offers the added value of providing the physiochemical environment and structural scaffold required for bone-implant integration. Invitro release of antibiotics from HA-coated implants has been reported for chlorhexidine, vancomycin, gentamicin, tobramycin and several other antibiotics [60-63], whose antibacterial efficacy was shown invitro by the formation of inhibition zones in agar plate testing. Calcium phosphate containing antibiotics were coated on titanium implants, where they showed that antibiotics containing carboxylic groups have a better interaction with calcium, resulting in improved binding and higher incorporation in to the calcium phosphate coating. Also, the longest antibacterial effect achieved still does not exceed three days .
The study of biodegradable polymeric coatings made from poly-lactic acid and its copolymers with glycolic acid is more established. Release profiles last from several hours to 12 days after exposure to an aqueous environment [65-67]. An additional advantage of such coatings is the relative ease with which the polymer can be applied to both alloys and plastics with polished, irregular or porous surfaces using a simple dip-coating technique . The implant can be dipped several times in a solution of polymer and antibiotics in an organic solvent to achieve a dense or thick polymer coating. The promising results displayed in an animal model for this type of coating and were first investigated in humans for internal fixation of open tibial fractures using gentamicin poly-( DL-lactic)-coated tibial nails. Gentamicin was not detected in the serum and no adverse events were observed during a one-year follow-up [58, 67].
The fluroquinolones are important antimicrobial agents that have demonstrated activity against a wide range of Gram positive and Gram negative organisms and have proved useful against micro organisms resistant to other antibacterial agents. Some examples include ofloxacin, cirpofloxacin, perfloxacin, levofloxacin. Ofloxacin - is a second generation of fluroquinolones with a 6-fluoro substituent and a 7- piperazinyl substituent on the quinolone ring structure.
They have excellent pharmacokinetic profile and attain appreciable concentrations well above their MICs in biological tissues [69-71].
Systemic chemical names:
Non proprietary names:
Empirical formula, molecular weight: C18H20FN3O4 , 361.388.
3. Elemental analysis
C,59.83%; H,5.5%; F,5.26%; N,11.63%; O,17.71%.
A pale yellow or bright yellow crystalline powder.
Mechanism of action:
Ofloxacin - a class of broad-spectrum antibiotic- that active against both Gram-positive and Gram-negative bacteria. It acts by inhibiting DNA gyrase, a type II topoisomerase, and topoisomerase IV, which is necessary to separate replicated DNA, thereby inhibiting cell division. As such some fluoroquinolones may cause injury to the chromosome of eukaryotic cells [72, 73].
According to various studies the bioavailability of ofloxacin in the tablet form is approximately 98% following oral administration. Most of the drug gets excreted via the kidney within duration of 48 hours of dosing. Fluroquinolones are one of the promising groups of antibiotics that are being used clinically for various pathological conditions. Ofloxacin has been proved to possess superior antibacterial activity both in vivo and has better pharmacokinetic properties as compared with ciprofloxacin and norfloxaicn.
The main objective of this study is to create antibiotic loaded implants, which releases antibiotics during the presence of aerobic organisms, by a simple degradation mechanism of the coated materials on implant, thereby preventing the formation of biofilms and thus increasing the treatment efficiency.