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F1-3986 Arenicin-3: A novel antimicrobial peptide showing potent in vitro activity against Gram-negative multi-resistant clinical isolates Contact information: [email protected] Phone: +45 44460663 D. SANDVANG, S. NEVE & H-H. KRISTENSEN Novozymes A/S, Bagsvaerd, Denmark Abstract Time kill for E. coli ATCC25922 Arenicin-3 in ca-Müller Hinton broth Results 10 10 MIC MIC testing of clinical isolates of multi resistant ESBL positive Escherichia coli (n:148) and Pseudomonas spp. (n:33) were performed by broth micro-dilution, according to CLSI-defined methodology, M7-A5 (2003), against Arenicin-3, ampicillin, gentamicin and ciprofloxacin. MBC testing was performed according to CLSI M26-A (1999), against Arenicin-3. Quality Control (QC): Results for all MIC testing were within the acceptable standards based on the CLSI recommended QC ranges for each comparator agent and the appropriate ATCC control strains. The second strain collection (Table) contained 115 different Gram negative bacteria to include a wide variety of species. These strains were mostly isolated from clinical material and the results for Arenicin-3 susceptibility were revealed in table. Haemolytic activity Freshly prepared human erythrocytes was used in the haemolytic activity test. The substance under investigation was added to the 8% erythrocytes to determine the influence of the substance on the cytoplasmic membrane of the red blood cells. The haemolytic activity was measured photometric by the absorbance of oxyhaemoglobin. CFU/ml Growth control 10 4 10 x MIC gentamicin 10 3 10 x MIC Arenicin-3 2 10 0 20 40 60 80 100 120 140 Time (minutes) The time kill curve shows that Arenicin-3 exhibits a very potent and fast bactericidal activity with a bactericidal action within 30 minutes compared with gentamicin which exhibited bactericidal action after 60 minutes. Pseudomonas (n=33) E. coli (n=148) Heamolysis of Arenicin-3 100 100 90 90 100 80 90 70 80 80 70 60 50 Arenicin-3 Ciprofloxacin Gentamicin 40 30 20 10 50 Arenicin-3 Ciprofloxacin Gentamicin 40 30 20 0. 06 2 0. 5 12 5 0. 25 0. 5 8 16 32 4 2 64 12 8 25 6 51 10 2 24 0.125 0.25 0.5 1 HC50 40 Arenicin-3 WT SDS PBS 30 0 2 4 2 4 8 16 32 64 128 256 512 1024 mg/L Arenicin-3 did not display any heamolytic activity on human reed blood cells. In fact, Arenicin-3 could not be distinguished from the negative control. Conclusion Minimal inhibitory concentration (µg/ml) for Arenicin-3 0.063 60 10 Susceptibility data for Arenicin-3 - MIC susceptibility determination for a variety of Gram Negative (n:115) No tested 70 20 10 0 1 0. 06 2 0. 5 12 5 0. 25 0. 5 60 MIC (g/ml) MIC (g/ml) The MIC testing of the E. coli and Pseudomonas strains clearly show the potency of Arenicin-3 with a MIC50 value below 0,5 μg/ml and a MIC90 value below 1 μg/ml for the E. coli strains. This should be compared to a MIC90 value for both comparators which is above 8 μg/ml. For the pseudomonas strains arenicin-3 showed a MIC 50 value below 0,125 μg/ml. The same trend were seen for other gram negative bacteria (table) Bacterial Strains Time Kill Time-kill assays were performed as suggested by CLSI guidelines (NCCLS, 1999) using a final concentration of antimicrobial agent at 10 times the MIC for the E. Coli reference strain. Bactericidal activity was defined as a reduction of 99.9% (≥3 log10) of the total count of CFU/ml (NCCLS, 1999). 10 5 10 1 The alignment shows the peptides related to Arenicin. Interestingly, the closest related peptide is Tachyplesin-1 identified in horseshoe crab with an identity of 57%. Tachyplesin-1 is also a beta-hairpin peptide stabilized with 2 cysteines bridges and is also highly charged with 6 positively charged residues. 0 Methods 10 6 10 % Heamolysis The Arenicin family consists of three members Arenicin-1 and -2 which were characterized by a Russian research group (Ovchinnikova et al., 2004) and Arenicin-3 which is a novel member of the family. Arenicin-3 was isolated from the marine lugworm Arenicola marina and shown to contain two disulfide bonds between Cys3, Cys20 and Cys7, Cys16. Together with 4 positively charged arginines, and 9 hydrophobic residues the peptide was shown to form a 21-residue amphipathic beta hairpin structure. Activity assays revealed that Arenicin-3 is highly active in vitro against a variety of Gram negative bacteria. MIC analysis showed that more than 90 % of the tested E. coli and Pseudomonas strains exhibited a MIC below 1ug/ml. 10 7 32 64 12 8 25 6 51 10 2 24 Introduction 10 8 0 8 16 Gram-negative bacteria including multi-resistant clinical relevant isolates of Escherichia coli, Klebsiella pneumoniae, Salmonella Typhimurium, Pseudomonas aeruginosa and Stenotrophomonas maltophilia were susceptibility tested to Arenicin-3. The results for isolates of both Enterobactericeae (n=148) and non fermentors (n=53) populations, MIC90 was < 1 mg/ml. The antimicrobial activity is markedly bactericidal (MBC~1-4xMIC), causing 3-log (99.9%) reduction in the viable bacteria population within 1-2 hours of Arenicin-3 exposure. In conclusion, Arenicin-3 has shown potential antimicrobial activity, even against multi resistant clinical isolates (ESBL positive, fluoroquinolone resistant, aminoglycoside resistant) GFCWYVCVYRNGVRVCYRRCN RWCVYAYVRVRGVLVRYRRCW RWCVYAYVRIRGVLVRYRRCW KWCFRVCYRG....ICYRRCR KWCFRVCYRG....ICYRKCR RRWCFRVCYRG....FCYRKCR RRWCFRVCYKG....FCYRKCR RSVCRQIKICRRR.GGCYYKCTNRPY XCRRLCYKQRC XCRRLCYKQRCVTYCRGR RGGGLCYCRRRFCVCVGR WC.RVCYR R....CYRRC 4 Results Sandworm, Arenicola marina) Sandworm, Arenicola marina) Sandworm, Arenicola marina) Horseshoe crab, Tachypleus tridentatus) Horseshoe crab, Tachypleus tridentatus) Horseshoe crab, Limulus polyphemus) Horseshoe crab, Limulus polyphemus) Scorpion, Androctonus australis) Spider, Acanthoscurria gomesiana) Pig, Sus scrofa) 50% consensus 10 9 2 Minimal inhibitory concentrations were performed according to the general guidelines for susceptibility measurements using micro-broth dilution provided by CLSI/ NCCLS (M7-A5) All isolates were tested by a standard time-kill methodology as described by CLSI document M26-A: Methods for Determining Bactericidal Activity of Antimicrobial Agents; approved guideline, with the exception of taking earlier time points than normal due to the rapid bactericidal nature of Arenicin-3. ARENICIN-3 ARENICIN-1 ARENICIN-2 TACHYPLESIN-1 TACHYPLECIN-3 POLYPHEMUSIN-1 POLYPHEMUSIN-2 ANDROCTONIN GOMESIN PROTEGRIN 1 Methods. aa-sequence Accumulated frequencies Arenicola marina living on sediments in the tidal water. Structural analysis showed that Arenicin-3 belonged to the beta-hairpin peptides. This class of AMPs are known to exhibit cidal activities towards a diverse number of microorganisms. Interestingly, susceptibility data on clinical isolates of Klebsiella pneumoniae, Salmonella enterica, Pseudomonas aeruginosa and Escherichia coli showed very potent activities of Arenicin. Origin Accumulated frequencies Background Arenicin-3 is an antimicrobial peptide isolated using Transposon-Assisted-Signal -Trapping from the lugworm Name 8 16 32 Enterobactericeae Citrobacter 7 2 5 Enterobacter 5 1 3 1 9 3 1 Escherichia coli 24 Hafnia alvei 1 Klebsiella . 9 Proteus mirabilis 3 Salmonella enterica 8 Serratia marcescens 2 Shigella . 2 Achromobacter . 3 Acinetobacter 4 Aeromonas 10 Alcaligenes 5 Pseudomonas Stenotrophomonas 24 8 1 11 1 4 4 1 1 2 1 1 6 2 2 2 1 2 6 • Arenicin-3 is very potent in vitro against multi-resistant Gram-negative bacteria. • Fast bactericidal action. • Low in vitro toxicity • Promising drug candidate 1 2 2 2 2 1 1 7 7 3 3 1 2 MIC determination were made in the range 0,06-128 (µg/ml) Arenicin 3 including gentamicin, ampicillin and ciprofloxacin as comparator antibiotics according to CLSI guidelines (M7-A5) and determined twice. MIC was performed on clinical isolates originating from Europe. Strain collection included sensitive and multi resistant isolates, most of which are know human pathogens 4 2 Reference 2 Ovchinnikova el al., FEBS Letters, Vol. 577 (1-2), 209-214, 2004