INTRODUCTION:

Persistant infections are due to the bacteria adhere to implanted medical devices or damaged tissue (1,2). A slimy layer, bio films is composed of bacteria hydrated matrix of polysaccharide and protein. Biofilm formation is important as it is associated with chronic nature of subsequent infection and with their inherent resistance to antibiotic chemotherapy because of the mode of growth. The diseases that are generally acknowledged to be associated with bio films are chronic lung infections in cystic fibrosis and peridontitis (3,4)various nosocomal infections such as those related to the use of central venous catheters (5), urinary catheters (6), prosthetic heart valves (7) and orthopaedic devices (8) are clearly associated with biofilms that adhere to biomaterial surface even though the microbial causes and host site varies these infections share common characteristics (11).

Bacteria in these biofilms evade host defences and withstand antimicrobial chemotherapy. Bio film based infections are rarely resolved even in individual with component innate and adaptive immune responses (11). Immune complexes and invading neutrophills causes collatrel damage to the tissues adjacent to the biofilm(9). Susceptibility tests with in-vitro biofilm models have shown the survival of bacterial biofilm after the treatment with antibiotics at concentrations hundreds or even a thousand time the minimum inhibitory concentration of the bacteria measured in a suspension culture (10). By killing free floating bacteria shed from attached population antibiotics might suppress some infections in vivo, those bacterial cell are still embedded in the film as they fail to eradicate. The biofilm can act as incidous of recurrent infection when antimicrobial chemotherapy stops. Until the colonized surface is surgically removed from the body, the biofilm infections usually persist.

ARCHITECTURE OF BACTERIAL BIOFILMS:

Biofilm growth of bacteria on the outer and inner surface of foreign body are the characteristic feature of foreign body infections. Biofilm growth also occurs on the natural surfaces as teeth (14), heart valves (15), in the lungs of cystic fibrosiss patients causing chronic bronchopneumonia (13), in the middle ear with the patients otitis media (16), in chronic osteomyelitis and prosthetic joint infections (17-19), in intravenous catheters and stents (20) and in chronic wounds (21,22). In a self produced biopolymer matrix, the microbes in biofilms ae kept together (27). or more species living in a socio microbiological way is present in A bacterial consortium (12,13,23,24). As it produces structural stability and protection to biofilm, the matrix is important. Planktonic bacteria that reversibly attach to a surface initiates a development of an invitro biofim, which may be covered by a layer of eg: proteins (14,25). Bacteria are still susceptible to antibiotics at this stage and this is in accordance with the success of perioperative antibiotics prophylaxis eg: alloplastic surgery (27). Multiplication of bacteria, irreversible binding to surface within next few hours, which forms micrcolinies on surface and begin to produce a polymer matrix around microcolonies (25). The biofilm grows in thickness and mushroom like or tower like structures are often observed in matrix biofilms under in vitr conditions. It shows maximum tolerance at this stage. It is followed by a stage where focal areas of biofilm dissolve and the liberated bacterial cells can then spread to another location where new biofilms can be formed. The liberation process may be caused by bacteriophage activity with the biofilms (26). Water filled channels are present in mature bifilms and thereby resemble multicellular organisms. Motile bacteria can use type IV pilli to mount or climb a biofilm formed by other bacteria and colonies, the top of biofilm resembling a hat (21). Important properties of biofilm growing bacteria are different from those of planktonic bacteria and this has a significant diagnostic and therapeutic consequences. Since they are located close to each other in aggregates surrounded by self produced matrix, the bacteria appears in biofilm infections. Biofilm can often be recognized by light microscopy in clinical specimens. DNA hybridisation technique can only be done for precise identification of all bacteria within biofilm and identification of all bacteria within biofilm and identification of components of biofilm matrix requires specialised staining technique(13). Tradiional sampling technique may not be sufficient to culture biofilm growing bacteria.

RESISTANCE MECHANISM:

The familiar mechanisms of antibiotic resistance, such as efflux pumps, modifying enzymes and target mutations (28) do not seem to be responsible for the protection of bacteria in biofilm. Even sensitive bacteria that do not have a known genetic basis for resistance can have profoundly reduced susceptibility when they form a biofilm. For example a beta-lactamase-negative strain of investigated the effect of genes encoding multidrug efflux pumps, such as the multiple antibiotic resistance (mar) locus (30). These studies did not show any important role for these genes in mediating biofilm resistance. The mar operon is not induced during biofilm growth of Escherichia coli, and mutants without mar have a resistance to ciprofloxacin similar to strains with mar when grown in biofilms. (31,32) In biofilms, strains of P aeruginosa that do not have the MexAB-OprM multidrug resistance pump also remained resistant to ciprofloxacin (33) Preliminary evidence indicates that conventional antibiotic resistance mechanisms are not sufficient to explain most cases of antibiotic-resistant biofilm infections. This evidence does not exclude the possibility that conventional resistance mechanisms, such as drug pumps, are expressed in biofilms and contribute to antibiotic resistance in the attached mode of growth. However, we should look beyond conventional mechanisms to understand biofilm resistance. Conventional antibiotic resistance can develop in biofilms treated repeatedly or for a long time—stable derepression of chromosomal _-lactamase contributes to the persistence of P aeruginosa biofilm infections(34) The mechanisms of resistance to antibiotics in bacterial biofilms are beginning to be elucidated(35) The first hypothesis is then possibility of slow or incomplete penetration of the antibiotic into the biofilm. Measurements of antibiotic penetration into biofilms in vitro have shown that some antibiotics readily permeate bacterial biofilms (36) There is no generic barrier to the diffusion of solutes the size of antibiotics through the bio film matrix, which is mostly water(37)However, if the antibiotic is deactivated in the biofilm, penetration can be profoundly retarded. For example, ampicillin can penetrate through a biofilm formed by a β-lactamase-negative strain of K pneumonia but not a biofilm formed by the _-lactamase-positive wild type strain of the same micro-organism (29) In the wild strain biofilm, the antibiotic is deactivated in the surface layers more rapidly than it diffuses. Antibiotics that adsorb into the biofilm matrix could also have a retarded penetration, which might account for the slow penetration of aminoglycoside antibiotics. (38,39) These positively charged agents bind to negatively charged polymers in the biofilm matrix. (40,41) The second hypothesis depends on an altered chemical microenvironment within the biofilm. Microscale gradients in nutrient concentrations are a well known feature of biofilms. Findings from studies with miniature electrodes have shown that oxygen can be completely consumed in the surface layers of a biofilm, leading to anaerobic niches in the deep layers of the biofilm.(42) Concentration gradients in metabolic products mirror those of the substrates. Local accumulation of acidic waste products might lead to pH differences greater than 1 between the bulk fluid and the biofilm interior, (43) which could directly antagonise the action of an antibiotic. Aminoglycoside antibiotics are clearly less effective against the same micro-organism in anaerobic than in aerobic conditions. (44) Alternatively, the depletion of a substrate or accumulation of an inhibitive waste product might cause some bacteria to enter a non-growing state, in which they are protected from killing. Penicillin antibiotics, which target cell-wall synthesis, kill only growing bacteria (45) This alternative possibility is strengthened by direct experimental visualisation of metabolically inactive zones within continuously fed biofilms. (46) Additionally, the osmotic environment within a biofilm might be altered, leading to induction of an osmotic stress response (47) Such a response could contribute to antibiotic resistance by changing the relative proportions of porins in a way that reduces cell envelope permeability to antibiotics. A third and still speculative mechanism of antibiotic resistance is that a subpopulation of micro-organisms in a biofilm forms a unique, and highly protected, phenotypic state—a cell differentiation similar to spore formation. This hypothesis is lent support by findings from studies that show resistance in newly formed biofilms, even though they are too thin to pose a barrier to the penetration of either an antimicrobial agent or metabolic substrates (48,49) Additionally, most bacteria in the biofilm, but not all, are rapidly killed by antibiotics (33,50) Survivors, which might consist of 1% or less of the original population, persist despite continued exposure to the antibiotic. The hypothesis of a spore-like state entered into by some of the bacteria in a biofilm provides a powerful, and generic, explanation for the reduced susceptibility of biofilms to antibiotics and disinfectants of widely different chemistries.

Bacterial Biofilm Infections:

Until the relatively recent development of vaccines and antibiotics, human societies have been beset by acute epidemic infectious diseases caused by the planktonic cells of such specialized pathogens as Vibrio cholerae and Yersinia pestis. Modern- day acute infections can often be treated effectively with antibiotics (except for cases of infection by a strain that is antibiotic resistant) and are not considered to involve biofilms. However, more than half of the infectious diseases that affect mildl compromised individuals involve bacterial species that are commensal with the human body or are common in our environments. For example, the skin bacterium Staphylococcus epidermidis and the aquatic bacterium Pseudomonas aeruginosa can cause devastating chronic infections in compromised hosts (51). Electron microscopy of the surfaces of medical devices that have been foci of device-related infections shows the presence of large numbers of slime-encased bacteria (52). Tissues taken from non–device-related chronic infections also show the presence of biofilm bacteria surrounded by an exopolysaccharide matrix. These biofilm infections may be caused by a single species or by a mixture of species of bacteria or fungi Biofilm infections share clinical characteristics. Biofilms develop preferentially on inert surfaces, or on dead tissue, and occur commonly on medical devices and fragments of dead tissue such as sequestra of dead bone (53); they can also form on living tissues, as in the case of endocarditis. Biofilms grow slowly, in one or more locations, and biofilm infections are often slow to produce overt symptoms (54). Sessile bacterial\ cells release antigens and stimulate the production of antibodies, but the antibodies are not effective in killing bacteria within biofilms and may cause immune complex damage to surrounding tissues (55). Even in individuals with excellent cellular and humoral immune reactions, biofilm infections are rarely resolved by the host defense mechanisms (52). Antibiotic therapy typically reverses the symptoms caused by planktonic cells released from the biofilm, but fails to kill the biofilm (56). For this reason biofilm infections typically show recurring symptoms, after cycles of antibiotic therapy, until the sessile population is surgically removed from the body (51). Planktonic bacterial cells are released from biofilms, and evidence supports the notion that there is a natural pattern of programmed detachment (51). Therefore, biofilms can act as “niduses” of acute infection if the mobilized host defences cannot eliminate the planktonic cell that are released at any one time during the infection (57).

THERAPY:

More work is needed to fully elucidate antibiotic resistance mechanisms in biofilms and develop new therapeutic strategies, but we have enough evidence to make some observations and suggestions. Clearly, there are multiple resistance mechanisms that can act together. Antibiofilm therapies might have to thwart more than one mechanism simultaneously to be clinically effective. Heterogeneity is a common theme of these resistance mechanisms; micro-organisms in a biofilm exist in a broad spectrum of states. First, cells might be exposed to different concentrations of antibiotic depending on their spatial location. Second, gradients in the concentration of microbial nutrients and waste products crisscross the biofilm and alter the local environment, which leads to a third, a small proportion of cells in a bacterial biofilm might differentiate into a highly protected phenotypic state and coexist with neighbours that are antibiotic sensitive. The proliferation of states that arises when these three types of heterogeneity are crossed means that any given antimicrobial agent might be able to kill some of the cells in a biofilm, but is unlikely to effectively target all of them. Most or all the antibiotics in current use were identified on the basis of their activity against growing cultures of individual cells. New screens of existing and potential antibiotics that select for activity against nongrowing or biofilm cells might yield antimicrobial agents with clinical efficacy against biofilm infections. As genes that mediate biofilm resistance to antibiotics are identified and their gene products characterized, these will become targets for chemotherapeutic adjuvants that could be used to enhance the effectiveness of existing antibiotics against biofilm infections. Because biofilm resistance depends on aggregation of bacteria in multicellular communities, one strategy might be to develop therapies that disrupt the multicellular structure of the biofilm. If multi cellularity of the biofilm is defeated, the host defences might be able to resolve the infection, and the efficacy of antibiotics might be restored. Potential therapies include enzymes that dissolve the matrix polymers of the biofilm (58) chemical reactions that block biofilm matrix synthesis, (59) and analogues of microbial signalling molecules that interfere with cell-to-cell communication, required for normal biofilm formation. (60) As the genetic basis for biofilm development emerges, the gene products identified as required for multicellular colony formation will become a potential target for chemotherapy. In other words, we believe that treatment strategies will target the formation of multicellular structures rather than essential functions of individual cells. We will learn to treat the persistent infections associated with biofilms when the multicellular nature of microbial life is understood

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Received on 15.02.2014 Modified on 03.01.2017

Research J. Pharm. and Tech 2017; 10(11): 4019-4023.

DOI: 10.5958/0974-360X.2017.00728.4