**3.2.1 Biofilms on medical devices**

Device-related infections are often identified as having a biofilm aetiology and biofilm formation can occasionally be facilitated by the host inflammatory response molecules which can make adhesion to the surface of the device easier (Hall-Stoodley et al., 2004). Almost all kind of surfaces are suitable to be colonized by biofilms (Donlan & Costerton, 2002). Biomedical devices are not the exception, biofilms are often found on the surface of urinary catheters (Stickler, 2008), central venous catheters (Petrelli et al., 2006), heart valves (Litzler et al., 2007), voice prostheses (Buijssen et al., 2007), contact lenses (Imamura et al., 2008), hip prostheses (Dempsey et al., 2007) and intrauterine devices (Chassot et al., 2008). Current biofilm preventive strategies are essentially aimed at coating medical surfaces with antimicrobial agents, a process not always successful (Basak et al., 2009; von Eiff et al., 2005). Bacteria in biofilms become highly resistant to antibiotics, and so they evade host defenses withstanding antimicrobial chemotherapy (Morikawa, 2006). Since nosocomial infections remain an important problem even for hospitals with strict infection control programmes, infection control measures remain highly sought after (Falagas & Makris, 2009). Development of successful technologies based on the biofilm formation and growth control is expected to be a major breakthrough in the clinical practice and preventive medicine.

To eliminate biofilm formation novel compounds capable of specifically targeting biofilm growth while causing no adverse toxicity to the environment of application are needed. Several reports have suggested that, in addition to their direct action against pathogens, biosurfactants are able to interfere with biofilm formation, modulating microbial interaction with interfaces (Federle & Bassler 2003; Merk et al., 2005; Neu, 1996; Rasmussen & Givskov, 2006; Rodrigues et al., 2006b, 2006c; Rodrigues et al., 2007).

Surfactin for example has shown to be an important part of a list of biofilm controlling agents. Surfactin is able to inhibit biofilm formation of *Salmonella typhimurium, S. enterica*, *E. coli* and *Proteus mirabilis* in polyvinyl chloride wells, as well as vinyl urethral catheters (Mireles et al., 2001). Many *Salmonella* species are important opportunistic pathogens of the urinary tract system. Recently, two lipopeptide biosurfactants, produced by *B. subtilis* V9T14 and *B. licheniformis* V19T21, showed the ability to selectively inhibit biofilm formation by pathogenic strains on polystyrene (Rivardo et al., 2009). In particular, *S. aureus* ATCC 29213 and *E. coli* CFT073 biofilm formation were decreased by 97% and 90%, respectively. V9T14 biosurfactant was active on the Gram-negative strain yet ineffective against the Grampositive while the opposite was observed for V19T21 biosurfactant. These effect were observed either by coating the polystyrene surface with these compounds or by adding the biosurfactant to the inoculum. The chemical characterization of V9T14 lipopeptide biosurfactant carried out by LC/ESI-MS/MS revealed that it was composed of 77% of surfactin and of 23% of fengycin (Pecci et al., 2010).

The activity of AgNO3 combined with the lipopeptide biosurfactant V9T14 has also been studied against a preformed *E. coli* biofilm on the Calgary Biofilm device (Rivardo et al., 2010). Results indicated that the activity of silver can be synergistically enhanced by the presence of V9T14, allowing a reduction in the quantity of silver used to achieve greater antimicrobial impact. The concentrations of silver in the silver–biosurfactant solutions were 129 to 258 fold less than the concentrations needed when silver was used alone. In another study, the V9T14 biosurfactant in association with antibiotics led to a synergistic increase in the efficacy of antibiotics in *E. coli* CFT073 biofilm innhibition and, in some combinations, to total eradication of the uropathogenic strain biofilm (Rivardo et al., 2011).

In another recent work, marine bacterial culture supernatants of *Bacillus pumilus* and *B. indicus* significantly inhibited the initial attachment process and biofilm formation and dispersal of mature biofilms of *Vibrio* spp. strains (Nithya & Pandian, 2010). The bacterial supernatants also reduced the surface hydrophobicity of *Vibrio* spp. which is one of the important requirements for biofilm development. Valle et al., (2006) observed that distinct serotypes of group II capsular polysaccharides, produced by the uropathogenic *E. coli* (UPEC strain CFT073) behaved like surface-active polymers that displayed anti-adhesion properties. The treatment of abiotic surfaces with group II capsular polysaccharides drastically reduced both initial adhesion and biofilm development of important nosocomial pathogens.

More recently, the effect of different temperatures on the anti-adhesive activity of surfactin and rhamnolipid biosurfactants was tested on polystyrene surfaces, regarding the attachment of *Staphylococcus aureus*, *Listeria monocytogenes*, and *Micrococcus luteus* (Zeraik & Nitschke, 2010). Surfactin inhibited bacterial adhesion at all tested conditions, and its activity increased with the decrease in temperature, giving a 63–66% adhesion reduction in the bacterial strains at 4°C. Rhamnolipids promoted a slight decrease in the attachment of *S. aureus* but were not as effective. The ability of rhamnolipid biosurfactant to inhibit adhesion of microorganisms to silicone rubber was also investigated in a parallel-plate flow chamber (Rodrigues et al., 2006c). The results showed an effective reduction in the initial deposition

To eliminate biofilm formation novel compounds capable of specifically targeting biofilm growth while causing no adverse toxicity to the environment of application are needed. Several reports have suggested that, in addition to their direct action against pathogens, biosurfactants are able to interfere with biofilm formation, modulating microbial interaction with interfaces (Federle & Bassler 2003; Merk et al., 2005; Neu, 1996; Rasmussen & Givskov,

Surfactin for example has shown to be an important part of a list of biofilm controlling agents. Surfactin is able to inhibit biofilm formation of *Salmonella typhimurium, S. enterica*, *E. coli* and *Proteus mirabilis* in polyvinyl chloride wells, as well as vinyl urethral catheters (Mireles et al., 2001). Many *Salmonella* species are important opportunistic pathogens of the urinary tract system. Recently, two lipopeptide biosurfactants, produced by *B. subtilis* V9T14 and *B. licheniformis* V19T21, showed the ability to selectively inhibit biofilm formation by pathogenic strains on polystyrene (Rivardo et al., 2009). In particular, *S. aureus* ATCC 29213 and *E. coli* CFT073 biofilm formation were decreased by 97% and 90%, respectively. V9T14 biosurfactant was active on the Gram-negative strain yet ineffective against the Grampositive while the opposite was observed for V19T21 biosurfactant. These effect were observed either by coating the polystyrene surface with these compounds or by adding the biosurfactant to the inoculum. The chemical characterization of V9T14 lipopeptide biosurfactant carried out by LC/ESI-MS/MS revealed that it was composed of 77% of

The activity of AgNO3 combined with the lipopeptide biosurfactant V9T14 has also been studied against a preformed *E. coli* biofilm on the Calgary Biofilm device (Rivardo et al., 2010). Results indicated that the activity of silver can be synergistically enhanced by the presence of V9T14, allowing a reduction in the quantity of silver used to achieve greater antimicrobial impact. The concentrations of silver in the silver–biosurfactant solutions were 129 to 258 fold less than the concentrations needed when silver was used alone. In another study, the V9T14 biosurfactant in association with antibiotics led to a synergistic increase in the efficacy of antibiotics in *E. coli* CFT073 biofilm innhibition and, in some combinations, to

In another recent work, marine bacterial culture supernatants of *Bacillus pumilus* and *B. indicus* significantly inhibited the initial attachment process and biofilm formation and dispersal of mature biofilms of *Vibrio* spp. strains (Nithya & Pandian, 2010). The bacterial supernatants also reduced the surface hydrophobicity of *Vibrio* spp. which is one of the important requirements for biofilm development. Valle et al., (2006) observed that distinct serotypes of group II capsular polysaccharides, produced by the uropathogenic *E. coli* (UPEC strain CFT073) behaved like surface-active polymers that displayed anti-adhesion properties. The treatment of abiotic surfaces with group II capsular polysaccharides drastically reduced both initial

More recently, the effect of different temperatures on the anti-adhesive activity of surfactin and rhamnolipid biosurfactants was tested on polystyrene surfaces, regarding the attachment of *Staphylococcus aureus*, *Listeria monocytogenes*, and *Micrococcus luteus* (Zeraik & Nitschke, 2010). Surfactin inhibited bacterial adhesion at all tested conditions, and its activity increased with the decrease in temperature, giving a 63–66% adhesion reduction in the bacterial strains at 4°C. Rhamnolipids promoted a slight decrease in the attachment of *S. aureus* but were not as effective. The ability of rhamnolipid biosurfactant to inhibit adhesion of microorganisms to silicone rubber was also investigated in a parallel-plate flow chamber (Rodrigues et al., 2006c). The results showed an effective reduction in the initial deposition

total eradication of the uropathogenic strain biofilm (Rivardo et al., 2011).

adhesion and biofilm development of important nosocomial pathogens.

2006; Rodrigues et al., 2006b, 2006c; Rodrigues et al., 2007).

surfactin and of 23% of fengycin (Pecci et al., 2010).

rates and in the number of bacterial cells adhering after 4h, for several microorganisms. Moreover, perfusing the flow chamber with biosurfactant containing solution followed by the passage of a liquid–air interface produced high detachment (96%) of adhered cells for several microorganisms. These capabilites have a lot of implications regarding biofilm formation and microbial contamination and establishments on such biomedical devices made of such compounds.

Antibiofilm activity was also reported for a glycolipid biosurfactant isolated from another marine bacterium *Brevibacterium casei* MSA19 against pathogenic biofilms *in vitro* (Kiran et al., 2010a). The purified glycolipid disrupted the biofilm formation under dynamic conditions and the biofilm-forming capacity of both mixed culture and individual human and fish pathogenic strains was significantly inhibited at 30 mg/mL glycolipid. Raya et al., (2010) analyzed the effects of rhamnolipids and shear on initial attachment of *Pseudomonas aeruginosa* PAO1 in glass flow chambers. The presence of rhamnolipids significantly reduced the initial attachment of PAO1, even at the low concentration of 13 mg/L. Prewashing the cells with a 100 mg/L rhamnolipid solution, however, did not affect the attachment significantly. The initial cell attachment increased with increasing shear at the very low shear range (up to 3.5–5.0 mN/m2), however the attachment could be minimized with further increase of the shear.

The biosurfactant Lunasan produced by the yeast *Candida sphaerica* UCP0995 completely inhibited the adhesion of *Streptococcus agalactiae*, *Streptococcus sanguis* 12, *Streptococcus mutans*, *Streptococcus mutans* NS, *Staphylococus epidermidis*, *Staphylococus aureus* and *Candida albicans* on plastic tissue culture plates at a concentration of 10 mg/ml and ≈92% inhibition of adhesion occurred for *Pseudomonas aeruginosa* (Luna et al., 2011). Lunasan, tested at the same concentration, also showed antimicrobial activity against the strains *Streptococcus oralis* (68%), *Candida albicans* (57%), and *Staphylococcus epidermidis* (57.6%). The same research group also described antiadhesive and antimicrobial activities of Rufisan, a biosurfactant produced by the yeast *Candida lipolytica* UCP 0988 (Rufino et al., 2011). Crude biosurfactant showed antiadhesive activity at ≥0.75 mg/L against most of the microorganisms tested (*Staphylococus aureus*, *Streptococcus agalactiae*, *Streptococcus mutans* NS) and the anti-adhesive property was proportional to the concentration of the biosurfactant while antimicrobial activities were also observed at higher biosurfactant concentrations.

In conclusion, the anti-adhesive activity of biosurfactants against several pathogens indicates their potential utility as coating agents for medical insertional materials that may lead to a reduction in a large number of hospital infections without the need for use of synthetic drugs and chemicals.
