Um peptide concentration preventing regrowth of bacteria from the treated biofilm within 4 hours. In the second case, to determine viable cell counts of biofilms after peptide treatment, pegs from the challenge microtiter plate were removed and transferred to Eppendorf tubes containing 500 ml PBS. After sonication at room temperature for 15 min to break up the biofilm and remove bacterial cells from the peg, aliquots of bacterial suspension were plated on LB-agar plates for counting. Colony forming units (CFU) were expressed as percentage with respect to control (peptide-untreated biofilms). Minimum bactericidal concentration (MBCb) was defined as the lowest peptide concentration required to reduce the number of viable biofilm cells by 3 log10 (99.9 killing) [40].In vivo ExperimentsAnimal procedures were approved by the Ethical Committee of the Azienda Ospedaliera Universitaria Senese on November 18, 2010. Balb-c mice (20 g) were infected i.p. with lethal amounts of bacteria (see results) mixed in 500 ml PBS +7 mucin (mucin from porcine stomach, type II, Sigma-Aldrich). Bacteria were cultured overnight, centrifuged, mixed in sterile PBS, and measured by spectrophotometer. Possible further dilutions in PBS were sometimes necessary to obtain the right amount of bacteria. Groups consisted of 5 animals. Moribund animals were killed humanely to avoid unnecessary distress. Surviving mice were monitored for 7 days. Thirty INCB039110 site minutes after bacterial administration, peptides were inoculated i.p. with 0.5 ml PBS solution containing the indicated amount of peptide (see Results). Control animals received only PBS. P values were calculated using GraphPad Prism software.Anti-biofilm ActivityBiofilm formation was performed by adapting the procedure described in [39] using the Calgary Biofilm Device (Innovotech, Innovotech Inc. Edmonton, Canada). Briefly, 96-well plates containing the bacterial inoculum were sealed with lids 15755315 bearing 96 pegs on which the biofilm could build up. The plates were placed in an orbital incubator at 35uC (for P. aeruginosa and E. coli) or 37uC (for S. aureus) for 20 h under agitation at 125 rpm. Once biofilms formed, the lids were removed from the plates and the pegs were rinsed twice with phosphate buffered saline (PBS) toAuthor ContributionsConceived and designed the experiments: CF LL LB GMR AP. Performed the experiments: SP VL VC JB BL SB SS LL. Analyzed the data: AP GMR LB MLM ADG CF. Contributed reagents/materials/analysis tools: SP VL VC JB BL SB SS LL MLM. Wrote the paper: AP CF GMR LB.
Traditional and classical methods of genomics and microbiology allow researchers to study an 
individual microbial species obtained from the environment by isolating the organism into pure colonies using microbial culture techniques. However, this approach cannot capture the structure of the broader microbial community within the environmental sample, the relative representation of multiple genomes, and their interaction with each other and with the environment. Additionally, a large number of microbial species are very difficult, or impossible, to culture in vitro in the laboratory setting. The development of nextgeneration sequencing has advanced the field of metagenomics by enabling scientists to simultaneously study multiple genomes recovered directly from an environmental sample, thereby bypassing the need for microbial isolation through culturing (see [1] for a review). In a metagenomic experiment, a sample is purchase Indolactam V usually taken from a.Um peptide concentration preventing regrowth of bacteria from the treated biofilm within 4 hours. In the second case, to determine viable cell counts of biofilms after peptide treatment, pegs from the challenge microtiter plate were removed and transferred to Eppendorf tubes containing 500 ml PBS. After sonication at room temperature for 15 min to break up the biofilm and remove bacterial cells from the peg, aliquots of bacterial suspension were plated on LB-agar plates for counting. Colony forming units (CFU) were expressed as percentage with respect to control (peptide-untreated biofilms). Minimum bactericidal concentration (MBCb) was defined as the lowest peptide concentration required to reduce the number of viable biofilm cells by 3 log10 (99.9 killing) [40].In vivo ExperimentsAnimal procedures were approved by the Ethical Committee of the Azienda Ospedaliera Universitaria Senese on November 18, 2010. Balb-c mice (20 g) were infected i.p. with lethal amounts of bacteria (see results) mixed in 500 ml PBS +7 mucin (mucin from porcine stomach, type II, Sigma-Aldrich). Bacteria were cultured overnight, centrifuged, mixed in sterile PBS, and measured by spectrophotometer. Possible further dilutions in PBS were sometimes necessary to obtain the right amount of bacteria. Groups consisted of 5 animals. Moribund animals were killed humanely to avoid unnecessary distress. Surviving mice were monitored for 7 days. Thirty minutes after bacterial administration, peptides were inoculated i.p. with 0.5 ml PBS solution containing the indicated amount of peptide (see Results). Control animals received only PBS. P values were calculated using GraphPad Prism software.Anti-biofilm ActivityBiofilm formation was performed by adapting the procedure described in [39] using the Calgary Biofilm Device (Innovotech, Innovotech Inc. Edmonton, Canada). Briefly, 96-well plates containing the bacterial inoculum were sealed with lids 15755315 bearing 96 pegs on which the biofilm could build up. The plates were placed in an orbital incubator at 35uC (for P. aeruginosa and E. coli) or 37uC (for S. aureus) for 20 
h under agitation at 125 rpm. Once biofilms formed, the lids were removed from the plates and the pegs were rinsed twice with phosphate buffered saline (PBS) toAuthor ContributionsConceived and designed the experiments: CF LL LB GMR AP. Performed the experiments: SP VL VC JB BL SB SS LL. Analyzed the data: AP GMR LB MLM ADG CF. Contributed reagents/materials/analysis tools: SP VL VC JB BL SB SS LL MLM. Wrote the paper: AP CF GMR LB.
Traditional and classical methods of genomics and microbiology allow researchers to study an individual microbial species obtained from the environment by isolating the organism into pure colonies using microbial culture techniques. However, this approach cannot capture the structure of the broader microbial community within the environmental sample, the relative representation of multiple genomes, and their interaction with each other and with the environment. Additionally, a large number of microbial species are very difficult, or impossible, to culture in vitro in the laboratory setting. The development of nextgeneration sequencing has advanced the field of metagenomics by enabling scientists to simultaneously study multiple genomes recovered directly from an environmental sample, thereby bypassing the need for microbial isolation through culturing (see [1] for a review). In a metagenomic experiment, a sample is usually taken from a.