*1.1.5. other conditions*

Similarly during acute phase of osteomyelitis, microscopical examination have shown biofilm formation on infected bone surfaces [33]. In chronic prostatitis, adherent bacterial colonies on the surface of prostatic duct have been observed on microscopical studies, even in culture negative cases [34].

### *1.1.6. Indwelling medical devices*

Biofilms can develop on indwelling medical devices like prosthetic heart valve, pacemakers, central venous catheter, urinary catheter, contact lenses, intrauterine devices etc. and can cause persistent infections which are usually lethal. Scanning electron microscopy clearly shows biofilm formation at the tip of urinary catheter kept for 7 days. On medical devices, biofilms are most commonly formed by coagulase negative Staphylococci (CoNS) especially *S. epider‐ midis* followed by *S. aureus, Enterococci, Pseudomonas aeruginosa* etc.

Biofilms can develop on both types of contact lenses i.e. soft and hard and also on contact lens storage cases. *Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, E.coli,* Candida species can adhere to contact lenses [35]. Evidence of biofilm on contact lenses and it's storage cases have been reported from patients with microbial kerattis [36]. The rate of prosthetic valve endocarditis (PVE) range from 0.5% to 4% [37]. Coagulase negative Staphy‐ lococci are the commonest early colonizers after surgical implantation of prosthetic valve whereas *Streptococcus viridans* most commonly colonize during late PVE (i.e. 12 months following valve replacement) [38]. Though *S. aureus*, Gram negative coccobacilli or fungi may also be responsible for PVE.

Infection with central venous catheter is a quite common device related infection. Biofilms have been shown by CLSM to be present outside the catheter or inner lumen [34].

*In S. epidermidis* biofilm initial adherence is by polysaccharide adhesin (PSA) and accumulation of cells is due to production of polysaccharide intercellular adhesin (PIA).) PIA is encoded by *ica* (intercellular adhesin) operon *ica* ADBC [39]. The *icaR* gene regulates *ica* operon. Production of PIA is also subject to ON - OFF switching (phase variation). Majority of clinical isolates of *S. aureus* also possess *ica* structural genes [40].

restrict diffusion of antimicrobial within the biofilm. Synergy between retarded diffusion and degradation by enzymes (e.g. β-lactamase) also provide effective resistance to antimicrobials. Fluoroquinolones are very effective in stopping the growth of a biofilm but restricted diffusion can protect the microbial cells within the biofilm [48]. All antimicrobials are more effective in killing rapidly growing cells. Penicillin & ampicillin do not kill non-growing cells as rate of killing is directly proportional to rate of growth for these two antibiotics. Even cephalosporins, aminoglycosides & fluroquinolones can kill rapidly dividing cells more effectively. Multiple drug resistance (MDR) pumps may play a role in biofilm resistance at low antibiotic concen‐ tration. Sometimes unknown MDR pumps might be over expressed in biofilm e.g. for chlor‐ amphenicol in *E. coli* biofilm. Moreover, the biofilms increase the opportunity of gene transfer beteen the microorganisms and can convert a previously avirulent commensal organism to a highly virulent pathogen. The enhanced efficiency of gene transfer in biofilms also fascilitates the spread of antibiotic resistance and virulence factors [49]. Though most of the research works deals with single species biofilms, multispecies biofilm amongst different bacteria and interkingdom biofilms between fungus Candida albicans and various bacterial species are also gaining importance in causing different diseases [50]. Biofilm formation is a major virulence factor for Candida albicans and Candida biofilms are difficult to eradicate due to their high resistance to antifungals. A recent study has reported that within the biofilm Staphylococcus aureus was attached uniquely with the pseudohyphae of Candida albicans. This synergistic interaction resulted in differential protein expressions which are actually virulence factors for Staphylcoccus aureus. This indicate C. albicans may enhance S.aureus pathogenesis [51]. Recently it has been reported that co-existence of S.aureus and C.albicans in a biofilm resulted in increased Vancomycin resistance in S.aureus [52] However antagonistic interaction has been

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reported between *Pseudomonas aeruginosa and Candida albicans* [53].

meant for planktonic cells.

*1.1.9. Biofilms and altruism*

of microorganisms.

cheap compared to genotypic method.

It is not possible to detect the antimicrobial resistance of biofilms by conventional methods of disc diffusion and broth microdilution as per CLSI guideline because these methods are only

Biofilms are like small cities and encourage altruism. Microbial cells within biofilm often sacrifice their maximum growth rate to use the available community resources more efficient‐ ly. In a biofilm atleast some of the microbial cells experience nutrient limitation and exist in a slow growing state [54]. In this process while individual cells are disadvantaged, the microbial community as a whole is benefited. Hence, it is said that biofilms are the colonial way of life

Detection of biofilms can be done by both phenotypic methods and genotypic methods. In phenotypic methods biofilms are detected by Congo red agar method(CRA),Plastic tube method(TM), Tissue culture plate method(TCP) and Confocal Laser Scanning Microscopy (CLSM). In genotypic method, usually Polymerase chain reaction (PCR) for amplification of microbial DNA, coding for biofilm formation is done. The phenotypic methods are easy and
