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Soon after the introduction of methicillin in clinical settings, Methicillin-resistant Staphylococcus aureus (MRSA) were identified in the year 1961 (MRSA) and it remains till date one of the major multi-resistant bacterial pathogens causing serious healthcare-associated and community-onset infections (Barber, 1961; Gould et al., 2012). It is a superbug causing serious health and global issue which has shown increasing endemic and epidemic spread in the last four decades (Kuehnert and Kupronis, 2005; Frazee et al., 2005). Bioï¬lms are a population of cells growing on a surface, enclosed in an exopolysaccharide matrix that are notoriously difï¬cult to eradicate and are a source of many intractable infections (Lewis, 2001). Biofilms were first observed by Henrici in 1933 (Toole et al., 2000). It alters growth rate and transcribes genes that free floating organisms do not transcribe (Thomas and Day, 2007). By increasing the secretion of exopolysaccharide, these microorganisms adapt to the biofilm environment and thereby help the microorganisms to escape their killing by antibiotics (Toole et al., 2000).
This review will give an overview of the role of MRSA and microbial biofilms focussing on the mechanism of biofilm development, molecular/genetic basis of resistance in this organism and the challenges facing the control strategies worldwide. It also aims in suggesting ways of overcoming these challenges to limit the spread of MRSA and biofilm-associated health problems.
EPIDEMIOLOGY OF MRSA
The epidemiology of methicillin-resistantÂ StaphylococcusÂ aureus (MRSA) is continuously altering and their antibiotic resistance profiles diverge considerably throughout various regions and countries. The existence of MRSA strains was reported in the United States (US) with prevalence rate of less than 1%. (17) Since then, the endemic & epidemic outbreaks of the organism have been reported worldwide (18-20) but overwhelmingly from developed economies of the world. Presently, hospital of all sizes, other care centers and increasing number of different population groups at various communities globally are facing the problem of MRSA infections (1,7,21). Reports emanating from different parts of the world revealed increasing rates in the incidence of MRSA and population at risk. The epidemiological data from north America including Centre for Disease Control and Prevention (CDC) in the US showed that the prevalence of MRSA strains in both large and small hospitals located at different regions increased progressively over the years (21-25). Other studies conducted at various health care centers in the country revealed that out of all hospital bacterial isolates, the prevalence of MRSA, cumulatively increased from 6% in 1998 to 50% in 2002. (4) A similar study conducted earlier elsewhere in the country showed a 30% increase (from 20% to 50%) in MRSA prevalence within a two year period (i.e. from 1988 to 1990) (26). The published data from some countries of Europe and Asia presented identical scenario with significant increase in the outbreaks of MRSA infections. For instance, Mangeney and co-workers (27) documented MRSA prevalence of 33%-62% (in relation to S. aureus isolates) in their hospital wards in France. The report of De Sousa and colleagues (28) from Portugal though indicated downward trend in the outbreaks of MRSA infections but showed consistently high prevalence of MRSA during the last decade e.g. 65% in 1992; 49% and 47% in 1993 and 1994 respectively. In contrast, lower prevalence rates of MRSA have been reported in certain parts of Europe. For example, Harbath and co-researchers (29) reported 3% MRSA prevalence in their hospital in Geneva, Switzerland though further evidence showed that the MRSA were not nosocomial strains and could have originated from the community. In a similar study carried out in the UK, (30) the prevalence of MRSA was found to be comparatively low (<10%) but never the less significant in the population group studied. Identical scenario has been observed in Scandinavia and the Netherlands where incidence of MRSA appears relatively low compared to other European countries (31). In earlier studies carried out in Asia, a remarkable upsurge in MRSA prevalence has been documented in many areas, including Taiwan where an increase in MRSA prevalence from 4.3% in a period covering 1981-1986 was reported(32). In Japan(33) and Republic of Korea (34), MRSA prevalence of up to 54%
MECHANISM OF BIOFILM FORMATION
There are five steps of biofilm formation on medical devices.In steps 1 and 2, the identification and association with a surface is followed by strong adhesion. The time taken by this is 1 to 2 hours post - implantation. These reversible, non specific cellular associations occur through long and short range forces, e.g., van der Waal's forces, gravitational forces, hydrogen bonds, hydrophobic interactions, etc. In steps 3 and 4, microbial cells aggregate to form micro colonies. Thereafter, further growth and maturation of the biofilm takes place in the next 2 - 3 hours. Specific chemical reactions between the compound of the microbial cells and the substrate surfaces result in strong adhesion and irreversible molecular bridging. The biofilm which is formed can be of a flat or mushroom shape, which depends upon the nutrient source. Microbial polysaccharide and adhesin proteins promote the attachment of organisms to the substrate surfaces. In step 5, sloughing of the biofilm into small pieces occurs and these pieces move transiently to form daughter cells. The daughter cells which are thus formed travel down through the blood stream to various new attachment sites. Transitions to a sessile state of bacteria occur in response to the limitation of essential nutrients. Biofilm formation is commonly regulated by inter and intraspecies quorumsensing mechanisms. Availability of nutrients, chemotaxis towards the surface, the motility of bacteria, surface adhesion and the presence of surfactants, influence biofilm formation in microorganisms.
By using bacillus subtilis, Dr Stanley Wall has shown that a protein called Deg U regulates biofilm formation. Certain surface proteins, extracellular proteins, capsular polysaccharides and adhesins PS/A and autolysin (encoded by the atl E gene) regulate biofilm production. The ica genes also code for PS/A and intracellular adhesion. Biofilms may be formed by one or several types of microorganisms. Studies on polymicrobial biofilms which are formed by Candida albicans and Staphylococcus epidermidis indicate that biofilms which are produced, may protect the fungus from antifungal action. Many researchers have shown that the regulation of ica operon and the formation of biofilms depend upon various environmental factors like anaerobicity, carbon dioxide level and glucose and osmotic levels. Sodium chloride is a known activator of ica operon transcription. Some workers have found that sodium chloride mostly induced biofilm formation among methicillin sensitive staphylococcus aureus. They also found that biofilm production among methicillin resistant staphylococcus aureus was mainly glucose induced. There was a variable adherence of the microorganisms to the polystyrene surfaces in vitro. This may be due to the variation in different strains and due to the expression of the genes which are responsible for biofilm production. Another hypothesis is that, in the inserted catheter under in vivo conditions, several host proteins coat the catheter surface. The microorganisms lodge to the coat by using multiple receptors.,. The various factors such as surface area, the type of surface (rough/smooth), porosity, charge on the surface and surface hydrophobicity play a role in the formation of the biofilm. A rough surface is more favourable for the colonization of bacteria. The hydrophobicity in polymeric materials increases biofilm formation. Microorganisms get attached easily on porous surfaces. Electrostatic interactions cause biofilm cohesion. Cations contribute to the cross linking of the biofilm matrix.The extent of initial adhesion depends upon the adherence property of the receptacle, the duration and the number of bacteria coming in contact with the test surfaces and the fluid turbulence of the test media.
Exopolysaccharides are formed under selective pressure and are controlled by diffusible chemical signals (quorum sensing) of the cells within the biofilm. So, the biofilm is not homogeneous.,. A study showed that the addition of pheromones to biofilm forming Enterococcus faecalis yielded a high amount of biofilm formation. Another group of workers demonstrated a strong association between the biofilms which were produced by the clinical isolates of Acinetobacter baumanni and multiple drug resistance. The presence of extended spectrum beta lactamases (bla PER1) is likely to facilitate cell adherence.The prevalence and the expression of F-like conjugative pili, adherence fimbri and curly, which are known to promote biofilm formation in Escherichia coli K12, cannot totally account for the increased biofilm formation of non domesticated Escherichia coli in vitro.
BIOFILM ASSOCIATED INFECTIONS AND THEIR IMPLICATIONS IN HEALTH CARE
According to a recent public announcement from the National Institutes of Health, more than 60% of all the infections are caused by biofilms. As described by Prasanna et al, about 40-50% of adults had biofilm related gingival infections. Among 4000 infants with cerebrospinal- fluid shunts, 15-20% had biofilm related infections. 95% of the urinary tract infections were associated with urinary catheters. 86% pneumonias were associated with mechanical ventilation and 85% of the blood stream infections were closely related to intravascular devices.
THE SUGGESTED ROLES OF THE BIOFILMS IN PRODUCING INFECTIONS ARE -
a. Detachment of the cells - the cells may get detached from the biofilm. This may cause blood stream and urinary tract infections.
b. Resistance to the host immune system- Biofilm coated bacteria escape the damaging effect of the antibodies produced by the infected host cells.
c. Production of endotoxins - Gram negative bacteria which are encased in biofilms, produce endotoxin.
d. The generation of resistant organisms - Bacteria can transfer plasmids by conjugation within the biofilm. So, resistance factors may be exchanged through a plasmid.
Two types of biofilm associated infections can occur -
1. Foreign body infections
2. Native tissue infections
Foreign body infections - These are more commonly associated with the colonization of microbes on indwelling medical devices (IMD). IMDs may cause the haematogenous spread of infections throughout life if the devices are in place. For surgical IMDs, tissue damage and clot formation are associated with surgical implantation, thus causing increased microbial biofilm formation. For non surgical IMDs, e.g. Urinary catheters, colonization may occur from skin or through or around catheters, once they are implanted. Native tissue infections - Some biofilm related infections involve no foreign bodies eg. Urinary tract infections by uropathogenic Escherichia coli, cystic fibrosis by Pseudomonas aeruginosa, native valve endocarditis by streptococcus viridians, etc.,
MECHANISMS OF ANTIMICROBIAL RESISTANCE OF BIOFILMS
Microbial biofilms have been associated with a variety of persistent infections which respond poorly to conventional antibiotic therapy. This also helps in the spread of antibiotic resistant traits in nosocomial pathogens by increasing mutation rates and by the exchange of genes which are responsible for antibiotic resistance. Antibiotic therapy against device associated biofilm organisms often fails without the removal of the infected implant. An elevated expression of the efflux pump is another mechanism for the development of antibiotic resistance in biofilm bacteria. The specific up regulation of genes which encode antibiotic transporters, has been seen in biofilms which are formed by Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Physiological heterogeneity is another important characteristic which is observed in biofilm bacteria. This phenomenon affects the rate of growth and metabolism of the bacteria and is reflected by interbacterial quorum signals, the accumulation of toxic products and the change in the local micro environment. These so called persister cells are not resistant to antibiotics per se, but become resistant when associated with the biofilm. The overall healthcare mechanisms of the underlying antimicrobial resistance of biofilms are:
1. Trapping of antibiotics in the exopolysaccharide matrix - The exopolysaccharide slime causes a diffusion barrier by restricting the rate of molecule transport to the interior of the biofilm, or by chemically reacting with the molecules themselves. The exopolysaccharide which is negatively charged, restricts the penetration of the positively charged molecules of antibiotics by chemical interactions or by molecular binding. This also dilutes the concentration of the antibiotics before they reach to the individual bacterial cells in the biofilm, thus making the antibiotics less effective against microorganisms., 
2. Bacteria which are coated with biofilms escape the host immune system - Biofilm bacteria escape the damaging effect of the antibodies which are produced by the host immune system in response to infections.
3. Quorum sensing and genotyping adaptations alter the metabolism and decrease the growth rate of bacteria- A cell to cell communication in bacterial biofilms is established through chemical signaling. Small, diffusible molecules of class of N - acylated homoserine lactones (AHLs) are liberated by biofilm bacteria into their surrounding environment. These AHLs are associated with DNA binding proteins. As the amount of AHLsreaches a threshold level, it induces the transcription of specific genes throughout the population. The regulation of this type is known as quorum sensing (Requirement of a specific population of bacteria that is nessesary for the activation of the AHL - responsive genes). The cells lying deep within the biofilm have less metabolic activity and growth rates. This makes the biofilm organisms inherently less susceptible to antibiotics. Due to the consumption of oxygen and glucose, relative anaerobiasis is created at the deeper layers of the biofilm, where in order to survive, the microorganisms transform into slow growers and non growers. Older biofilms are relatively more resistant than newer biofilms.
After the attachment to a biotic or an abiotic surface, the bacteria undergo further adaptation, i.e, increased synthesis of exopolysaccharide and increased antibiotic resistance. They also develop an increased resistance to UV light, increased genetic exchange, altered metabolism and increased secondary metabolic production.,