Transcript LORUM IPSUM DOLORES 2008-2009 At vero eos et accumisto

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
60
70
80
70
60
50
Arenicin-3
Ciprofloxacin
Gentamicin
40
30
20
10
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
40
Arenicin-3 WT
SDS
PBS
30
2
4
0
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
HC50
10
Susceptibility data for Arenicin-3 - MIC susceptibility determination for a variety of Gram Negative (n:115)
No tested
60
20
10
0
1
0.
06
2
0. 5
12
5
0.
25
0.
5
50
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
64
12
8
25
6
51
10 2
24
Introduction
10 8
0
8
16
32
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