MIC changes in bacterial strains exposed to selection by the compounds containing <i>C</i>. <i>vicina</i> AMP complex or conventional antibiotics.

<p>(A) <i>E</i>. <i>coli</i> 774.1 (reference antibiotic cefotaxime). <i>E</i>. <i>coli</i> antibiotic sensitive strain 774.1 was exposed to selection by the AMP complex or cefotaxime in the course of 25 daily transfers as explained in Materials and Methods section. Resistance rate is expressed as fold change in MICs. 1 MIC unit is equal to the MIC value at transfer 1 (250 mg/L for the compound and 0.125 mg/L for cefotaxime, correspondingly). Selection by cefotaxime caused 16-fold increase of MIC while no signs of MIC change were found in the compound treated population. Difference in the compound versus cefotaxime effects on the resistance development was highly significant according to Wilcoxon test statistics (W = 276, n = 23, P<0.001). (B) <i>E</i>. <i>coli</i> 774.1 (reference antibiotic polymyxin B). The strain was exposed to selection by the compound or polymyxin B in the course of 15 daily transfers. 1 MIC unit is equal to the MIC value at transfer 1 (250 mg/L for the compound and 8.0 mg/L for polymyxin B, correspondingly). Difference in the compound versus polymyxin B effects on the resistance development was highly significant according to Wilcoxon test statistics (W = 91, n = 13, P<0.022). (C) <i>E</i>. <i>coli</i> 863.1 (reference antibiotic meropenem). <i>E</i>. <i>coli</i> antibiotic multiresistant meropenem sensitive strain 863.1 was exposed to selection by the compound or meropenem in the course of 15 daily transfers. 1 MIC unit is equal to the MIC value at transfer 1 (500 mg/L for the compound and 0.125 mg/L for meropenem, correspondingly). Difference in the compound versus meropenem effects on the resistance development was highly significant according to Wilcoxon test statistics (W = 78, n = 12, P<0.020). (D) A. baumannii 882.2 (reference antibiotic polymyxin B). <i>A</i>. <i>baumannii</i> antibiotic multiresistant strain 882.2 was exposed to selection by the AMP complex or polymyxin B in the course of 35 daily transfers. 1 MIC unit is equal to the MIC value at transfer 1 (500 mg/L for the compound and 2 mg/L for polymyxin B, correspondingly). Difference in the compound versus polymyxin B effects on the resistance development were highly significant according to Wilcoxon test statistics (W = 561, n = 33, P<0.001).</p

Resistance before and after selection by <i>Calliphora vomitoria</i>, <i>Lucilia sericata</i> and <i>Musca domestica</i> AMP complexes in <i>E. coli</i> 774.1 strain.

<p>*KR–ratio of MIC after selection to MIC before selection</p><p>Resistance before and after selection by <i>Calliphora vomitoria</i>, <i>Lucilia sericata</i> and <i>Musca domestica</i> AMP complexes in <i>E. coli</i> 774.1 strain.</p

Resistance development under selective pressure of cefotaxime, meropenem, polymyxin B and <i>C</i>. <i>vicina</i> AMP complex.

<p>*K_R−ratio of MIC after selection to MIC before selection.</p><p>Resistance development under selective pressure of cefotaxime, meropenem, polymyxin B and <i>C</i>. <i>vicina</i> AMP complex.</p

Antibiotic resistance spectra of bacterial strains used in selection experiments.

<p>Antibiotic abbreviations: Amc—amoxicillin/clavulanic acid, Ami–amikacin, Net–netilmicin, Gen–gentamicin, Ipm–imipenem, Mem–meropenem, Chl–chloramphenicol, Cip–ciprofloxacin, Cfp—cefoperazone, Cfp/sul–cefoperazone, Sul—sulbactam, Caz–ceftazidime, Ctx–cefotaxime, Cpe–cefepime.</p><p>*- no data.</p><p>Antibiotic resistance spectra of bacterial strains used in selection experiments.</p

Antibacterial activity, chromatographic, mass spectrometric and structural characteristics of active AMPs present in <i>C</i>. <i>vicina</i> AMP complex.

<p>Antibacterial activity, chromatographic, mass spectrometric and structural characteristics of active AMPs present in <i>C</i>. <i>vicina</i> AMP complex.</p

MIC changes in the course of selection by the combinations of antimicrobial agents.

<p>(A) Cefotaxime and polymyxin B combination. <i>E</i>. <i>coli</i> antibiotic sensitive strain 774.1 was exposed to selection by cefotaxime, polymyxin B or a mixture of polymyxin B and cefotaxime in the course of 15–20 daily transfers. Resistance level is expressed as fold change in MICs. 1 MIC unit is equal to 8 mg/L for polymyxin B, 0.125 mg/L for cefotaxime and 1.0 mg/L for a mixture containing cefotaxime and polymyxin B in ratio 1:32, correspondingly. Selection by cefotaxime, polymyxin B or a mixture of the antibiotics caused identical 8-fold increase of MIC. Differences in the mixture versus cefotaxime (W = 19, n = 6, P = 0.062) and polymyxin B (W = 19, n = 6, P = 0.062) effects on the rate of resistance development were statistically insignificant according to Wilcoxon test. (B) The compound containing <i>C</i>. <i>vicina</i> AMP complex and cefotaxime combination. <i>E</i>. <i>coli</i> strain 774.1 was exposed to selection by cefotaxime alone or cefotaxime in combination with the compound (50 mg/L) in the course of 15 daily transfers. Resistance level is expressed as cefotaxime fold change in MICs. 1 MIC unit corresponds to MIC value of cefotaxime at transfer 1 (0.125 mg/L). Delay of cefotaxime resistance development in presence of the compound sub-inhibitory concentration was statistically significant according to Wilcoxon test (W = 78, n = 12, P<0.02) and repeated measures ANOVA test (F = 16.465, η = 29, P = 0.001).</p

Chromatographic characteristics of naturally occurring compound containing <i>C</i>. <i>vicina</i> AMP complex.

<p>1 mg of purified complex isolated from bacteria challenged <i>C</i>. <i>vicina</i> larvae were subjected to reversed-phase HPLC fractionation with 1 min intervals as described in Materials and Methods section. Optical density of the fractions was measured in mAU units at 214 nm wave length. 53 fractions were individually collected, lyophilized and stored at -70°C until further antimicrobial activity and mass spectrometry analyses summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130788#pone.0130788.t002" target="_blank">Table 2</a>.</p

Numbers (mean ± standard error) of apoptotic (annexin V positive) and necrotic (propidium iodide positive, annexin V negative) human peripheral blood leukocytes per 100 gated cells after <i>in vitro</i> incubation with FLIP7.

<p>Numbers (mean ± standard error) of apoptotic (annexin V positive) and necrotic (propidium iodide positive, annexin V negative) human peripheral blood leukocytes per 100 gated cells after <i>in vitro</i> incubation with FLIP7.</p

FLIP7 and reference antibiotics effects on the <i>E</i>. <i>coli</i> ATCC 25922 and <i>S</i>. <i>aureus</i> 203 biofilms cell viability and thickness.

<p>FLIP7 and reference antibiotics effects on the <i>E</i>. <i>coli</i> ATCC 25922 and <i>S</i>. <i>aureus</i> 203 biofilms cell viability and thickness.</p

Planktonic and biofilm bacteria sensitivity to FLIP7 and reference antibiotics (TTC assay).

<p>Planktonic and biofilm bacteria sensitivity to FLIP7 and reference antibiotics (TTC assay).</p