**3.1.1 Antibacterial activity**

Lipopeptides have the most potent antimicrobial activity and have been a subject of several studies on the discovery of new antibiotics. The antibiotic activity is due to the ability of molecules of lipopeptide biosurfactants to self-associate and form a pore-bearing channel or micellular aggregate inside a lipid membrane (Carrillo et al., 2003; Deleu et al., 2008). Surfactin, in particular, has been associated with several physical and biological actions, such as antimicrobial, antiviral, anti-mycoplasma and haemolytic activities. It can penetrate into the membrane through hydrophobic interactions, thus influencing the ordering of the hydrocarbon chains and thus varying the membrane thickness (Bonmatin et al., 2003). Such membrane disruptions are a nonspecific mode of action and are advantageous for action on different cell membranes of both Gram-positive and Gram-negative bacteria (Lu et al., 2007). It has been suggested that such action by surfactin type peptides on membrane integrity rather than other vital cellular processes may perhaps constitute the next generation of antibiotics (Rodrigues & Teixeira, 2010). Similar bioactive fractions from the marine *Bacillus circulans* biosurfactant had antimicrobial action against various Gram-positive and Gramnegative pathogenic and semi-pathogenic bacteria including *Micrococcus flavus, Bacillus*  *pumilis, Mycobacterium smegmatis, Escherichia coli, Serratia marcescens, Proteus vulgaris, Citrobacter freundii, Proteus mirabilis, Alcaligenes faecalis, Acetobacter calcoaceticus, Bordetella bronchiseptica, Klebsiella aerogenes* and *Enterobacter cloacae* (Das et al., 2008). The chemical identity of this bioactive biosurfactant fraction showed overlapping patterns with that of surfactin lipopeptides and lichenysin. Mild antimicrobial action was also observed against methicillin-resistant *Staphylococcus aureus* (MRSA) and other MDR pathogenic strains. The biosurfactant was also found to be nonhaemolytic in nature thus indicating possible use as a drug in antimicrobial chemotherapy.

Very recently Huang et al., (2011) evaluated antimicrobial activity of surfactin and polylysine against *Salmonella enteritidis* in milk using a response surface methodology and showed *S. enteritidis* to be very sensitive to both molecules with minimum inhibitory concentrations of 6.25 and 31.25 μg/mL, respectively. The optimization of antimicrobial activity indicated that *S. enteritidis* could be reduced by 6 orders of magnitude at a temperature of 4.45°C, action time of 6.9 h, and concentration of 10.03 μg/mL (surfactin/polylysine weight ratio, 1:1).

In addition to surfactin, *Bacillus subtilis* strains produce a broad spectrum of bioactive peptides with great potential for biomedical applications, such as fengycin (Vanittanakom et al., 1986) and the iturin compounds: iturins (Besson et al., 1978; Peypoux et al., 1978), mycosubtilins (Peypoux et al., 1986), and bacillomycins (Peypoux et al., 1984), all of which are amphiphilic surface and membrane-active compounds with potent antimicrobial activities. Huang et al., (2007) reported that a lipopeptide antimicrobial substance produced by *B. subtilis* fmbj strain, which is mainly composed of surfactin and fengycin, was able to inactivate endospores of *B. cereus* through damaging the surface structure of the spores as seen by Transmission Electron Microscopy.

Lichenysin, pumilacidin and polymyxin B (Grangemard et al., 2001; Landman et al., 2008; Naruse et al., 1990; Yakimov et al., 1995) are other antimicrobial lipopeptides produced by *Bacillus licheniformis*, *Bacillus pumilus* and *Bacillus polymyxa*, respectively. Polymyxin B, in particular, due to its high affinity for the lipid moieties of lipopolysaccharide, has shown antibacterial activities against a wide variety of Gram-negative pathogens. Being a cationic agent, it binds to the anionic bacterial outer membrane, leading to a detergent effect that disrupts membrane integrity. Important nosocomial pathogens such as *Escherichia coli*, *Klebsiella* spp., *Enterobacter* spp., *Pseudomonas aeruginosa*, and *Acinetobacter* spp. are usually susceptible to polymyxins and considerable activity has been reported against *Salmonella*  spp., *Shigella* spp., *Pasteurella* spp., and *Haemophilus* spp. (Landman et al., 2008).

Another promising example of an antimicrobial lipopeptide that is under commercial development is daptomycin (Cubicin®). It has been approved for the treatment of skin infections by the FDA in 2003 (Giuliani et al., 2007, as cited in Seydlová & Svobodová, 2008). Daptomycin produced by *Streptomyces roseosporus* has been shown to be highly active against multiresistant bacteria such as MRSA (Tally & De Bruin, 2000, as cited in Seydlová & Svobodová, 2008). Another lipopeptide with antimicrobial activity and other interesting biological properties is viscosin, a cyclic lipopeptide from *Pseudomonas* (Saini et al., 2008).

Glycolipids, both rhamnolipids (Abalos et al., 2001; Benincasa et al., 2004) and sophorolipids (Kim et al., 2002; Van Bogaert et al., 2007) also have shown interesting antimicrobial activities (Fig. 5). Benincasa et al., (2004) reported that a mixture of six rhamnolipides homologues performed very well against *Bacillus subtilis* with a MIC of 8 µg/mL. Mannosylerythritol lipids (MEL-A and MEL-B) produced by *Candida antarctica* strains have also been reported to exhibit antimicrobial action against Gram-positive bacteria (Kitamoto et al., 1993).

*pumilis, Mycobacterium smegmatis, Escherichia coli, Serratia marcescens, Proteus vulgaris, Citrobacter freundii, Proteus mirabilis, Alcaligenes faecalis, Acetobacter calcoaceticus, Bordetella bronchiseptica, Klebsiella aerogenes* and *Enterobacter cloacae* (Das et al., 2008). The chemical identity of this bioactive biosurfactant fraction showed overlapping patterns with that of surfactin lipopeptides and lichenysin. Mild antimicrobial action was also observed against methicillin-resistant *Staphylococcus aureus* (MRSA) and other MDR pathogenic strains. The biosurfactant was also found to be nonhaemolytic in nature thus indicating possible use as a

Very recently Huang et al., (2011) evaluated antimicrobial activity of surfactin and polylysine against *Salmonella enteritidis* in milk using a response surface methodology and showed *S. enteritidis* to be very sensitive to both molecules with minimum inhibitory concentrations of 6.25 and 31.25 μg/mL, respectively. The optimization of antimicrobial activity indicated that *S. enteritidis* could be reduced by 6 orders of magnitude at a temperature of 4.45°C, action time of 6.9 h, and concentration of 10.03 μg/mL

In addition to surfactin, *Bacillus subtilis* strains produce a broad spectrum of bioactive peptides with great potential for biomedical applications, such as fengycin (Vanittanakom et al., 1986) and the iturin compounds: iturins (Besson et al., 1978; Peypoux et al., 1978), mycosubtilins (Peypoux et al., 1986), and bacillomycins (Peypoux et al., 1984), all of which are amphiphilic surface and membrane-active compounds with potent antimicrobial activities. Huang et al., (2007) reported that a lipopeptide antimicrobial substance produced by *B. subtilis* fmbj strain, which is mainly composed of surfactin and fengycin, was able to inactivate endospores of *B. cereus* through damaging the surface structure of the spores as

Lichenysin, pumilacidin and polymyxin B (Grangemard et al., 2001; Landman et al., 2008; Naruse et al., 1990; Yakimov et al., 1995) are other antimicrobial lipopeptides produced by *Bacillus licheniformis*, *Bacillus pumilus* and *Bacillus polymyxa*, respectively. Polymyxin B, in particular, due to its high affinity for the lipid moieties of lipopolysaccharide, has shown antibacterial activities against a wide variety of Gram-negative pathogens. Being a cationic agent, it binds to the anionic bacterial outer membrane, leading to a detergent effect that disrupts membrane integrity. Important nosocomial pathogens such as *Escherichia coli*, *Klebsiella* spp., *Enterobacter* spp., *Pseudomonas aeruginosa*, and *Acinetobacter* spp. are usually susceptible to polymyxins and considerable activity has been reported against *Salmonella* 

Another promising example of an antimicrobial lipopeptide that is under commercial development is daptomycin (Cubicin®). It has been approved for the treatment of skin infections by the FDA in 2003 (Giuliani et al., 2007, as cited in Seydlová & Svobodová, 2008). Daptomycin produced by *Streptomyces roseosporus* has been shown to be highly active against multiresistant bacteria such as MRSA (Tally & De Bruin, 2000, as cited in Seydlová & Svobodová, 2008). Another lipopeptide with antimicrobial activity and other interesting biological properties is viscosin, a cyclic lipopeptide from *Pseudomonas* (Saini et al., 2008). Glycolipids, both rhamnolipids (Abalos et al., 2001; Benincasa et al., 2004) and sophorolipids (Kim et al., 2002; Van Bogaert et al., 2007) also have shown interesting antimicrobial activities (Fig. 5). Benincasa et al., (2004) reported that a mixture of six rhamnolipides homologues performed very well against *Bacillus subtilis* with a MIC of 8 µg/mL. Mannosylerythritol lipids (MEL-A and MEL-B) produced by *Candida antarctica* strains have also been reported to exhibit

spp., *Shigella* spp., *Pasteurella* spp., and *Haemophilus* spp. (Landman et al., 2008).

antimicrobial action against Gram-positive bacteria (Kitamoto et al., 1993).

drug in antimicrobial chemotherapy.

(surfactin/polylysine weight ratio, 1:1).

seen by Transmission Electron Microscopy.

Fig. 5. Measuring antimicrobial activity for rhamnolipids, sophorolipids and SDS at various concentrations above and below the CMC for these surface active molecules against *Bacillus subtilis,* red circles showing clearing/inhibition zones.

Very recently, Nitschke et al., (2010) reported rhamnolipids produced by *P. aeruginosa* LBI with antimicrobial activity against several bacteria and fungi, including *Bacillus cereus*, *Staphylococcus aureus*, *Micrococcus luteus*, *Mucor miehei* and *Neurospora crassa*. Another glycolipid, flocculosin, a cellobiose lipid produced by the yeast-like fungus *Pseudozyma flocculosa*, was particularly effective against *Staphylococcus* species, including MRSA. Its antibacterial activity was not influenced by the presence of common resistance mechanisms against methicillin and vancomycin and it was able to eliminate *C. albicans* cells in a very short period of time (Mimee et al., 2009).

Trehalose lipids produced by *Tsukamurella* sp. strain DSM 44370 together with trisaccharide and tetrasaccharide lipids also showed some activity against Gram-positive bacteria, with the exception of the pathogenic strain *Staphylococcus aureus,* whereas Gram-negatives were either slightly or not inhibited at all (Vollbrecht et al., 1999, as cited in Franzetti et al., 2010b). Studies carried out to elucidate the molecular interactions between this biosurfactant and the lipidic component of the membrane showed that trehalose lipid increased the fluidity of phosphatidylethanolamine and phosphatidylserine membranes and formed domains in the fluid state and did not modify the macroscopic bilayer organization (Ortiz et al., 2008, 2009).
