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The activity of antimicrobial peptide S-thanatin is independent on multidrug-resistant spectrum of bacteria
Peptides 32 (2011) 1139–1145
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Peptides journal homepage: www.elsevier.com/locate/peptides
The activity of antimicrobial peptide S-thanatin is independent on multidrug-resistant spectrum of bacteria Guoqiu Wu a,∗,1 , Xiaofang Li b,1 , Xiaobo Fan b , Hongbin Wu b , Shenglan Wang b , Zilong Shen b , Tao Xi b,∗∗ a b
Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing 210009, China Biotechnology Center, Department of Life Science and Biotechnology, China Pharmaceutical University, Tongjiaxian 24#, Nanjing 210009, China
a r t i c l e
i n f o
Article history: Received 3 March 2011 Received in revised form 17 March 2011 Accepted 17 March 2011 Available online 29 March 2011 Keywords: Antimicrobial peptide Thanatin Antimicrobial activity Clinical isolate
a b s t r a c t In this study, the activity of S-thanatin (an analog of antimicrobial peptide derived from thanatin) against different bacterial pathogens frequently which can cause therapeutic problems was tested. The result showed minimal inhibitory concentrations (MICs) of S-thanatin against all isolates of the Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella ornithinolytica and Klebsiella oxytoca were in the range of 4–16 g/ml, no matter which antibiotic the bacterial was resistant or susceptible, while almost all MICs to Gram-positive bacterial were >128 g/ml except Enterococcus faecium. S-thanatin was more effective toward Gram-negative strains, especially for Enterobacter and Klebsiella. The MICs of S-thanatin were no significantly different in the same species regardless of antibiotic sensitive or -resistant isolates to single or multiple antibiotic (P > 0.05). Likewise, no notable difference could be observed between E. coli, K. pneumoniae, E. cloacae, E. aerogenes, K. ornithinolytica which were sensitive to S-thanatin (P > 0.05). It was implied that the antimicrobial activity of S-thanatin was independent on multi-drug resistance spectrum of bacteria. © 2011 Elsevier Inc. All rights reserved.
1. Introduction One of the most remarkable features of new antimicrobial drugs is their ability to kill microbes efficiently which can cause serious therapeutic problems in clinical therapy, in particular multidrug-resistant (MDR) bacteria has become a severe global health problem for the constant increase of the pathogens resistant to conventional antibiotics [17,19]. In spite of that the whole situation presence fomented severe therapeutic problems, very few antibacterial therapeutic compounds of novel mechanism have been approved over the last 40 years [14]. Thus, there is still an urgent need to develop new antibacterial drugs with potential to kill those resistant pathogens. Fortunately, the naturally occurring peptides termed antimicrobial peptides seem to solve this problem. Antimicrobial peptides (AMPs) have attracted a great deal of attention, which seem to be promising drug candidates for drug-resistant bacteria. Some AMPs are isolated from a wide variety of organisms including plants, prokaryotes, invertebrates
∗ Corresponding author. Tel.: +86 02583272355x808. ∗∗ Corresponding author. Tel.: +86 02583271389. E-mail addresses: guoqiu [email protected] (G. Wu), xitao [email protected] (T. Xi). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.03.019
and mammals [3], and they are important components of natural defenses for most living organisms to resist invading pathogens. They are relatively small, cationic and amphipathic with variable length, sequence and structure [5,7,23,24]. Some of them have already been evaluated in clinical trials [7]. Many AMPs likely contribute to format pores in the plasma membrane then lead to extensive membrane rupture eventually result in energy depletion and microbial lysis [1]. Osmotin, a tobacco pathogenesis-related protein of family 5 (PR-5), has antifungal activity in vitro and in vivo [33], and the action mechanism of this osmotin is heightened by its structural and functional similarities with AMPs. They are considered to be components of the related innate immune response of plants and animals [15,25]. Although many AMPs have the ability of damaging the bacterial membrane, some other bacteriostatic and bactericidal modes of action have been described, which AMPs can affect bacterial growth by binding DNA, inhibiting DNA replication, blocking gene expression or protein synthesis, as well as interfering with other enzymatic activity [1]. Thanatin (GSKKPVPIIYCNRRTGKCQRM), a cationic AMP with antiparallel -sheet structure from 8th amino acid to the Cterminus constrained by disulphide bonds, was isolated from the hemipteran inset Podisus maculiventris, it shows broad antimicrobial activity against bacteria and fungi [8,11]. In this study, S-thanatin, an analog of thanatin, showed higher activity against the Gram-negative bacteria, lower toxicity and better tolerance with cations and PH conditions [27]. The study was designed to
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G. Wu et al. / Peptides 32 (2011) 1139–1145
investigate the activity of S-thanatin on clinical isolates resistant to conventional antibiotics with different structures. 2. Materials and methods 2.1. Bacterial strains and growth conditions A total of 97 clinical isolated bacterial strains were used in this study. The multi-drug resistant strains were isolated from human clinical specimens: (1) Gram-negative bacteria: Escherichia coli (7 strains), Klebsiella pneumoniae (7 strains), Acinetobacter baumannii (8 strains), Stenotrophomonas maltophilia (5 strains), Pseudomonas aeruginosa (7 strains), Enterobacter cloacae (6 strains), Serratia marcescens (5 strains), Enterobacter aerogenes (7 strains), Proteus mirabilis (6 strains), Klebsiella ornithinolytica (4 strains), Klebsiella oxytoca (4 strains); (2) Gram-positive bacteria: Staphylococcus aureus (7 strains), Staphylococcus epidermidis (7 strains), Staphylococcus haemolyticus (9 strains), Enterococcus faecium (5 strain), Streptococcus agalactiae (3 strains). These isolates were obtained from the Center of Medical Laboratory of Zhongda Hospital, Southeast University, China. 15 reference strains from American Type Culture Collection, respectively: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC 19606, Stenotrophomonas maltophilia ATCC 13637, Pseudomonas aeruginosa ATCC 15442, Enterobacter cloacae ATCC 13047, Serratia marcescens ATCC 14041, Enterobacter aerogenes ATCC 49701, Proteus mirabilis ATCC 12453, Klebsiella ornithinolytica ATCC 31898, Klebsiella oxytoca ATCC 43086, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Staphylococcus hominis ATCC 29970, Enterococcus faecium ATCC 29212. All strains were stored frozen in 10% glycerol at −80 ◦ C. 2.2. Preparation of S-thanatin S-thanatin was synthesized by the solid-phase methodology with 9-fluorenyl-methoxy-carbonyl as protecting group, and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using an appropriate 0–60% acetonitrile gradient in 0.05% trifluoroacetic acid. Molecular mass was determined by electrospray mass spectrometry using an API instrument (Perkin Elmer SCIEX) as a quality control of synthesis [27]. The peptide in reduced form was taken up in oxidation buffer (1 mg/ml) [100 mM ammonium acetate (pH 8.5)], allowed to refold for 3 days at room temperature under stirring and purified by RP-HPLC [27]. After freeze-drying, S-thanatin was dissolved in phosphatebuffered saline (PBS, PH 7.4) to give a stock solution of 1280 g/ml in −20 ◦ C. 2.3. Other materials MH broth culture was from Oxoid Crop England, polypropylene 96-wells microtiter plates were from CORNIN Crop. All other chemicals used were of reagent grade. 2.4. Drug susceptibility about bacterial strains AST-GN13 cards for Gram-negative bacteria, AST-GP67 cards for Gram-positive bacteria and GN/CE strips with the VITEK2 system (bioMérieux, Marcy l’Etoile, France) were used to confirm isolated bacteria identities and susceptibilities to antimicrobial agents. 2.5. Antimicrobial assays of S-thanatin To test antimicrobial activity of S-thanatin, a microdilution assay was performed in polypropylene 96-wells microtiter plates (PSMicroplates were sterile) [2,4,21]. A 10-l aliquot of the purified
peptide were added to 100 l of a logarithmicphase culture of the tested bacteria which was diluted to 105 –106 colony forming units (cfu) per ml. Control wells were inoculated with 10 l of phosphate-buffered saline. Final concentration of S-thanatin in the test well was 0.5 g/ml, 1 g/ml, 2 g/ml, 4 g/ml, 8 g/ml, 16 g/ml, 32 g/ml, 64 g/ml, and 128 g/ml, respectively. Microbial growth was measured as an increase of optical density at 630 nm (OD630) by a microplate reader (MRX, Dynex) after incubation at 35 ◦ C for 16–20 h. MICs are expressed as where S-thanatin being the lowest concentration causing 100% of growth inhibition [4]. Each concentration was tested in triplicates. 2.6. Statistical analysis Statistical comparisons between groups were made by analysis of variance (significance level was fixed at 0.05). The difference between data of groups was considered significant at the level of <0.05. 3. Results 3.1. The results of drug susceptibility A total of 97 clinical isolates were tested, of which 66 strains were Gram-negative bacteria. Drug susceptibility was detected to ampicillin (AMP), ampicillin-sulbactam (SAM), piperacillintazobactam (TZP), cefazolin (CFZ), cefotetan (CTT), ceftazidime (CAZ), ceftriaxome (CRO), cefepime (FEP), aztreonam (ATM), ertapenem (ETP), imipenem (IPM), amikacin (AMI), gentamicin (GEN), tobramycin (TOB), ciprofloxacin (CIP), levofloxacin (LEV), furadantin (NIT) and cotrimoxazole/or (SMZ), and 31 strains of Gram-positive bacteria susceptibility to conventional antibiotics were also tested, including penicillin (PEN), oxacillin (OXS), cefotetan (CTT), ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MXF), erythromycin (ERY), clindamycin (CLI), quinupristindalfopristin (SYN), linezoid (LZ), vancomycin (VAN), tetracycline (TET), tigecycline (TC), nitrofurantoin (NIT), rifampin (RIF) and/or cotrimoxazole (SMZ). The susceptibility and resistance of these isolates to antibiotics for these organisms were summarized in Tables 1 and 2 . The results showed 60–100% of tested Gramnegative bacteria strains were resistant to CAZ, FEP, ATM, CRO, SAM, CFZ, and AMP, while 25–60% of them were resistant to AMI, TZP, LEV, CIP, TOB, NIT, CTT, GEN and SMZ. One Gram-negative bacteria strain was resistant to EPT and IPM. Thirty-five to ninety-nine percent of Gram-positive bacteria strains were resistant to TET, MXF, CTT, CIP, LEV, CLI, CRY, OXS and PEN. One Gram-positive bacteria strain was resistant to SMZ, RIF, NIT, TC, VAN, LZ and SYN. The carbapenem antibiotics were exhibited the best antibacteria function against clinical isolate bacterial. Nevertheless, also with exception like E. cloacae CI 101013204, it was resistant to almost all antibiotics including carbapenem antibiotics ETP and IPM (Tables 1 and 2). 3.2. The MICs of S-thanatin The results showed that the MICs of S-thanatin against Gramnegative MDR isolates were more effective than Gram-positive MDR isolates (4–16 g/ml), especially for E. coli, K. pneumoniae, E. cloacae, E. aerogenes, K. ornithinolytica and K. oxytoca. Among these strains, there were all isolates (35/35) resistant to AMP, and 91.4% (32/35) to SAM (6 E. coli, 6 K. pneumoniae, 6 E. cloacae, 7 E. aerogenes, 3 K. ornithinolytica and 4 K. oxytoca), 20.0% (7/35) to TZP (3 E. cloacae, and 4 K. oxytoca), 88.6% (31/35) to CFZ (6 E. coli, 4 K. pneumoniae, 6 E. cloacae, 7 E. aerogenes, 4 K. ornithinolytica and 4 K. oxytoca), 45.7% (16/35) to CTT (1 E. coli, 1 K. pneumoniae, 6 E. cloacae, 7 E. aerogenes, and 1 K. ornithinolytica, 54.3% (19/35) CAZ (6 E. coli, 3 K. pneumoniae, 3 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 57.1% (20/35) CRO
Table 1 Drug-resistant spectrums of clinical isolates of Gram-negative bacteria and the MICs of S-thanatin. Strains and clinical isolate no.a
AMP
SAM
TZP
CFZ
CTT
CAZ
CRO
FEP
ATM
ETP
IPM
AMI
GEN
TOB
CIP
LEV
NIT
SMZb
MICs of Ts (g/ml)c
Sputum Sputum Sputum Sputum Urine Urine Sputum
\ R R R R R R R
\ R R R R S R R
\ S S S S S S S
\ R R R R S R R
\ R S S S S S S
\ R R R R S R R
\ R R R R S R R
\ R R R R S R R
\ R R R R S R R
\ S S S S S S S
\ S S S S S S S
\ R R R S S R R
\ R R R R R R R
\ R R R S I R R
\ R R R R S R R
\ R R R R S R R
\ S S S S S I S
\ R R R R R R R
8 4 8 8 8 8 8 8
Sputum Secretion Secretion Sputum Sputum Sputum Sputum
\ R R R R R R R
\ R R R S R S R
\ I S S S S S S
\ R R R S S S R
\ R S S S S S S
\ R R R S S S I
\ I R R S S S S
\ S R R S S S S
\ R R R S S S I
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S R R S S S S
\ S S S S S S I
\ S S S S R S S
\ S S S S R S S
I I I I R I S
\ R R R S S S R
8 8 8 8 8 8 4 8
Sputum Sputum Sputum Sputum Secretion Sputum Sputum Secretion
\ R R R R R R R R
R S R R R R R R
\ R S I R R R R R
\ R R R R R R R R
\ R R R R R R R R
\ R S R R R R R R
\ R R R R R R R R
\ R S R R R R R R
\ R I R R R R R I
\ \ \ \ \ \ \ \ \
\ S S S S R R R R
\ R S S R R S S S
\ R S I R R R S R
\ R S S R R R S R
\ R S R R R R S R
\ R S I R I I S R
\ R R R R R R R R
\ R S R R R R R R
>128 >128 >128 >128 >128 >128 >128 >128 >128
Sputum Sputum Sputum Blood Blood
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ S S I S S
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ S S S S S
\ \ \ \ \ \
\ S S S S S
>128 >128 >128 >128 >128 >128
Pus Sputum Sputum Sputum Secretion Sputum Sputum
\ R R R R R R R
\ R R R R R R R
\ R S S S S S S
\ R R R R R R R
\ R R R R R R R
\ R S S R I S S
\ R R R R R R R
\ R S S S I S S
\ R S S I R I S
\ \ \ \ \ \ \ \
\ R S S S S I R
\ R S R S I S S
\ R S R S I S S
\ R S R S S S S
\ R S S S S S S
\ R S S S S S S
\ R R R R R S S
\ R R R R R R S
>128 >128 >128 >128 >128 >128 >128 >128
G. Wu et al. / Peptides 32 (2011) 1139–1145
Escherichia coli ATCC 25922 CI 100930206 CI 100928201 CI 100929202 CI 101008201 CI 101014204 CI 100930204 CI 100927201 Klebsiella pneumoniae ATCC 700603 CI 100927209 CI 100928208 CI 100928209 CI 100929203 CI 101013201 CI 101016213 CI 101013203 Acinetobacter baumannii ATCC 19606 CI 100927207 CI 100928204 CI 101014203 CI 101016205 CI 101016208 CI 101016209 CI 101016217 CI 100930202 Stenotrophomonas maltophilia ATCC 13637 CI 100929201 CI 100930105 CI 101016216 CI 101022210 CI 101023202 Pseudomonas aeruginosa ATCC 15442 CI 100927204 CI 100927210 CI 100928207 CI 101007204 CI 101013205 CI 101016211 CI 100930207
Source
1141
1142
Table 1 (Continued) Strains and clinical isolate no.a
a
AMP
SAM
TZP
CFZ
CTT
CAZ
CRO
FEP
ATM
ETP
IPM
AMI
GEN
TOB
CIP
LEV
NIT
SMZb
MICs of Ts (g/ml)c
Sputum Sputum Sputum Sputum Sputum Secretion
\ R R R R R R
\ R R R R R R
\ S I R R R S
\ R R R R R R
\ R R R R R R
\ S S R R R S
\ R R R I R S
\ S R R S R S
\ S I R R R S
\ S S R S S S
\ S S R S S S
\ S R R S S S
\ S R R S I S
\ S R R I R S
\ S R R S I S
\ S R R S I S
\ S I R I R S
\ S R S S R S
8 16 8 8 8 16 8
Secretion Sputum Blood Urine Sputum
\ R R R R R
\ R R R R R
\ S S S R S
\ R R R R R
\ S S R R R
\ S S I R S
\ S R R R R
\ S I R R R
\ S R S S S
\ S R S I R
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
>128 >128 >128 >128 >128 >128
Secretion Secretion Secretion Sputum Sputum Sputum Blood
\ R R R R R R R
\ R R R R R R R
\ S S I I S I S
\ R R R R R R R
\ R R R R R R R
\ S S R I S R R
\ S S R S S R R
\ S S R S S R R
\ S S R S S R R
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S S R S S R R
\ S S R S S R R
\ I R I I R I I
\ S S S S S S S
4 4 4 4 4 4 4 4
\ R R R R S S
\ R R R S S S
\ S S R S S S
\ R R R R S S
\ S S S S S S
\ R R R R S S
\ R R R R S S
\ R R R R S S
\ R R R R S S
\ S S S S S S
\ S S S S S S
\ S S S S S S
\ R R R R S S
\ I I R I S S
\ R R R R S I
\ R R R R S S
\ R R R R R R
\ R R R R S R
>128 >128 >128 >128 >128 >128 >128
Secretion Sputum Urine Sputum
\ R R R R
\ R S R R
\ S S S S
\ R R R R
\ R S S S
\ S I S S
\ S R R S
\ S S S S
\ S I S I
\ S S S S
\ S S S S
\ S S S S
\ S I I S
\ S R R R
\ S S S S
\ S S S S
\ I S S R
\ R R R R
8 8 8 8 8
Sputum Sputum Sputum Sputum
\ R R R R
\ R R R R
\ R R R R
\ R R R R
\ S S S S
\ R R R R
\ R R R I
\ R R R R
\ R R R R
\ S S S S
\ S S S S
\ S S S S
\ I R R R
\ R R R R
\ R R R R
\ R R R R
\ I I I S
\ S S S S
8 8 8 8 8
Sputum Secretion Secretion Sputum Sputum
ATCC, American Type Culture Collection; CI, clinical isolate. S, sensitive; R, resistant; I, intermediate; \, no detected; AMP, ampicillin; SAM, ampicillin-sulbactam; TZP, piperacillin-tazobactam; CFZ, cefazolin; CTT, cefotetan; CAZ, ceftazidime; CRO, ceftriaxome; FEP, cefepime; ATM, aztreonam; ETP, ertapenem; IPM, imipenem; AMI, amikacin; GEN, gentamicin; TOB, tobramycin; CIP, ciprofloxacin; LEV, levofloxacin; NIT, furadantin; SMZ, cotrimoxazole. c Ts, S-thanatin. b
G. Wu et al. / Peptides 32 (2011) 1139–1145
Enterobacter cloacae ATCC 13047 CI 100927203 CI 100928203 CI 101013204 CI 101014205 CI 101016212 CI 100930201 Serratia marcescens ATCC 14041 CI 100928202 CI 101007205 CI 101008205 CI 101014202 CI 101028210 Enterobacter aerogenes ATCC 49701 CI 100928206 CI 101023205 CI 101026211 CI 110111202 CI 110111211 CI 110112203 CI 110112204 Proteus mirabilis ATCC 12453 CI 101016202 CI 101016213 CI 101025205 CI 110118212 CI 110118213 CI 110118216 Klebsiella ornithinolytica ATCC 31898 CI 101016204 CI 101027203 CI 110118210 CI 110119214 Klebsiella oxytoca ATCC 43086 CI 101008202 CI 101014202 CI 110115201 CI 11017202
Source
Table 2 Drug-resistant spectrums of clinical isolates of Gram-positive bacteria and the MICs of S-thanatin. Strains and clinical isolate no.a
PEN
OXS
CTT
CIP
LEV
MXF
ERY
CLI
SYN
LZ
VAN
TET
TC
NIT
RIF
SMZb
MICs of Ts (g/ml)c
Secretion Secretion Secretion Sputum Sputum Sputum Secretion
\ R R R R R R R
\ R R S S S R R
\ R S S S S R R
\ R S S S S R R
\ S S S S S I R
\ S S S S S S R
\ S R S S R R R
\ S R S S R R R
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S R S R S R R
\ R S S S S S S
\ S S S S S S S
\ S S S S S S R
\ R S S S S R S
>128 >128 >128 >128 >128 >128 >128 >128
Secretion Secretion Secretion Secretion Blood Blood Secretion
\ R R R R R R R
\ R R R R R R R
\ R I R S R I S
\ R R R S R R I
\ I R R S R I I
\ R I I S R R S
\ R R R R S R R
\ R R R R S R S
\ S S S S S R S
\ S S S S S S S
\ S S S S S S S
\ S R S S S R R
\ S S S S S S S
\ S S S S S S S
\ R S S S S S S
\ R S S R S S S
>128 >128 >128 >128 >128 >128 >128 >128
Sputum Sputum Urine Sputum Blood Blood Sputum Blood Blood
\ R R R R R R R R R
\ R R R R R R R R R
\ I R R S R S S S S
\ R R R S R R R S S
\ R R I S R R R S S
\ S I R S I R I S S
\ R R R R R R R R R
\ R R S R R R R S R
\ S S S S S S S S S
\ S S S S S S S S S
\ S S S S S S S S S
\ S R S R S R R S S
\ S S S S S S S S S
\ S S S S S S S S S
\ S S S S S S S S S
\ S S S S S S S S S
>128 >128 >128 >128 >128 >128 >128 >128 >128 >128
Secretion Secretion Secretion Urine Blood
\ S R R R R
\ S R R R R
\ S R R R R
\ I R R R R
\ S R R R R
\ S R R R R
\ R R R R R
\ R R R R R
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S R R R R
\ \ \ \ \ \
\ \ \ \ \ \
64 64 64 128 64 64
Blood Blood
R R
R R
S S
R R
R R
S I
S S
S S
S S
S S
S S
S S
S S
S S
S S
S S
>128 >128
G. Wu et al. / Peptides 32 (2011) 1139–1145
Staphylococcus aureus ATCC 29213 CI 100927214 CI 100928211 CI 101013207 CI 101013210 CI 101013211 CI 101013212 CI 100930212 Staphylococcus epidermidis ATCC 12228 CI 100925212 CI 100927215 CI 100928214 CI 101022209 CI 101025207 CI 101025208 CI 101026217 Staphylococcus hominis ATCC 29970 CI 100927213 CI 101016215 CI 101026212 CI 100930211 CI 100930215 CI 100930213 CI 100930214 CI 101013209 CI 101025209 Enterococcus faecium ATCC 29212 CI 101027206 CI 101026212 CI 101028209 CI 110112029 CI 110117204 Streptococcus agalactiae CI 101007206 CI 101014201
Source
a
ATCC, American Type Culture Collection; CI, clinical isolate. S, sensitive; R, resistant; I, intermediate; \, no detected; PEN, penicillin; OXS, oxacillin; CTT, cefotetan; CIP, ciprofloxacin; LEV, levofloxacin; MXF, moxifloxacin; ERY, erythromycin; CLI, clindamycin; SYN, quinupristindalfopristin; LZ, linezoid; VAN, vancomycin; TET, tetracycline; TC, tigecycline; NIT, nitrofurantoin; RIF, rifampin; SMZ, cotrimoxazole. c Ts, S-thanatin. b
1143
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G. Wu et al. / Peptides 32 (2011) 1139–1145
(6 E. coli, 2 K. pneumoniae, 4 E. cloacae, 3 E. aerogenes, 2 K. ornithinolytica and 3 K. oxytoca), 51.4% (18/35) to FEP (6 E. coli, 2 K. pneumoniae, 3 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 54.3% (19/35) to ATM (6 E. coli, 3 K. pneumoniae, 3 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 2.9% (1/35) to ETP and IPM (1 E. cloacae), 20% (7/35) to AMI (5 E. coli and 2 E. cloacae), 40% (14/35) to GEN (7 E. coli, 2 K. pneumoniae, 2 E. cloacae, and 3 K. oxytoca), 42.9% (15/35) to TOB (5 E. coli, 3 E. cloacae, 3 K. ornithinolytica and 4 K. oxytoca), 45.7% (16/35) to CIP (6 E. coli, 1 K. pneumoniae, 2 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 45.7% (16/35) to LFV (6 E. coli, 1 K. pneumoniae, 2 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 17.1% (6/35) to NIT (1 K. pneumoniae, 2 E. cloacae, 2 E. aerogenes, and 1 K. ornithinolytica), 48.6% (17/35) to SMZ (7 E. coli, 4 K. pneumoniae, 2 E. cloacae, and 4 K. ornithinolytica), respectively. The MIC of Sthanatin against different Enterobacter and Klebsiella bacteria was in the range of 4–16 g/ml in Table 1, while almost all the MICs to Gram-positive bacterial were >128 g/ml except Enterococcus faecium in Table 2. The MICs of S-thanatin were no significantly different in the same species regardless of antibiotic-sensitive or -resistant isolates to single or multiple antibiotic (P > 0.05). Likewise, no notable difference could be observed between E. coli, K. pneumoniae, E. cloacae, E. aerogenes, K. ornithinolytica and K. oxytoca, which were sensitive to S-thanatin (P > 0.05). 4. Discussion Pathogenic bacteria from clinical settings are becoming increasingly resistant to conventional antibiotics. Particularly, the increase in resistance of Gram-negative bacteria is more significantly than that in Gram-positive bacteria. There are fewer new and developmental antibiotics active against Gram-negative bacteria and drug development programs seem insufficient to provide therapeutic cover. Recently, clinical isolates producing new subgroup of metallo-beta-lactamase (New Delhi metallo--lactamase 1, NDM1) had already been identified [10,32]. It was urgent to search new generation antibiotics for drug-resistant bacteria instead of conventional antibiotics. As the cationic peptide, S-thanatin was demonstrated that a promising high activity on several clinical MDR isolates such as E. coli and K. pneumoniae in previous study [28,31]. This study showed higher antimicrobial activity of the peptide not only on E. coli and K. pneumoniae, but also on E. cloacae, E. aerogenes, K. ornithinolytica and K. oxytoca. The range of MICs was from 4 to 16 g/ml. The antimicrobial action of S-thanatin is more efficient to Gram-negative bacteria (apart from A. baumannii, S. maltophilia, P. aeruginosa, S. marcescens, and P. mirabilis) than Gram-positive bacteria (apart from Enterococcus faecium). Although the bactericidal mechanism of antimicrobial peptides remains to be fully understood, the microbicidal action of cationic peptides is generally initiated by disrupting the integrity of cell membranes through interaction with the phospholipids component by several models such as “Barrel-stave model”, “Carpet model”, and “Toroidal-pore model” [6,9]. We have already studied the antimicrobial mechanism of S-thanatin. The conclusion about membrane aggregation and perturbation induced and selective toxicity of antimicrobial peptide S-thanatin on bacteria had been reported [29,30]. The different activities may be due to the various mechanisms of S-thanatin. In our previous study, we found the MIC of S-thanatin against L-form strain reached 4fold increase compared with wild type for Gram-negative bacteria. In contrast, the susceptibility to Gram-positive bacteria protoplast decreased. It seemed that S-thanatin interacted with the outer membrane of Gram-negative bacteria, but not cytoplasmic membranes. Cell-wall of Gram-positive bacteria as a physical barrier was responsible for poor susceptibility of S-thanatin [30].
We speculated that S-thanatin must be attracted to LPS and phospholipid of Gram-negative bacteria by the electrostatic and hydrophobic interaction [26]. Then it inserts into the cytoplasmic membrane and membrane starts to destabilize and distort. At last, the perturbation of membrane damages the energization and respiration of cytoplasmic membrane leading to the bacteria becoming tattered and cytoplasm material leaking out. Compared with other peptides, S-thanatin adopted a sheet conformation with novel antibacterial action exhibiting LPS-binding and low hemolytic activities [29]. One obvious mechanism is S-thanatin must be attracted to bacterial surfaces. The Gram-negative bacterial membranes abundantly contain negatively charged lipids in particular of LPS, which can significantly enhance the membrane binding of S-thanatin. The activity of thanatin depends on the size of LPS [18], and the antimicrobial activity of cationic AMPs is compromised when the LPS polysaccharide moieties decreased [12,13]. However, S-thanatin exhibited a poor activity on some Gram-negative strains such as A. baumannii, S. maltophilia, P. aeruginosa, S. marcescens, and P. mirabilis in this study. The reason of differences on susceptibility among Gram-negative strains remained to be further researched. The mechanism of bacteria resistant to conventional antibiotics is diverse, such as active efflux pumps [16,22] and production of -lactamase [20], and etc. However, many agents against drugresistant bacteria may be only useful to one of resistant-drug mechanisms such as a clavulanic acid, which can inhibit the activity of -lactamase produced by bacteria. In present study, our results showed that S-thanatin did not exhibit any preference of killing bacteria in the same species (Table 1). No significantly difference was found between sensitive and resistant isolates to conventional antibiotics with different structures (all P > 0.05). The antibacteria activity of S-thanatin was independent on MDR spectrum of bacteria. It implied that S-thanatin may be useful to be developed as an antimicrobial agent for therapy of infection with MDR bacteria, together with its overall advantageous features such as salt tolerance, low cytotoxicity to eucaryotic cell [27]. Interestingly, we found a clinical isolate of Gram-negative bacteria (Enterobacter cloacae CI 101013204) resistant to almost all antibiotics including ertapenem and imipenem, but it was still susceptible to S-thanatin (the MIC was 8 g/ml). Furthermore, a high activity of S-thanatin on extended-spectrum -lactamases (ESBL)producing strains was also observed in this study. The MICs of S-thanatin against (ESBL)-producing strains, particularly E. coli and K. pneumoniae were the same as those of the international standard strains (4–8 g/ml). It was well known that the majority of NDM1 positive isolates producing carbapenemase was Gram-negative Enterobacteriaceae such as E. coli, K. pneumoniae, and E. cloacae [10,32]. It was reasonable to believe that S-thanatin was potentially useful to anti-NDM-1 positive isolates.
Acknowledgments This work was supported by the Natural Science Foundation of Jiangsu Province, China (Grant No. BK2009274) and Science Foundation of Southeast University (Grant No. 3290000102). We are indebted to Mr. Li for his help in language proof.
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Peptides journal homepage: www.elsevier.com/locate/peptides
The activity of antimicrobial peptide S-thanatin is independent on multidrug-resistant spectrum of bacteria Guoqiu Wu a,∗,1 , Xiaofang Li b,1 , Xiaobo Fan b , Hongbin Wu b , Shenglan Wang b , Zilong Shen b , Tao Xi b,∗∗ a b
Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing 210009, China Biotechnology Center, Department of Life Science and Biotechnology, China Pharmaceutical University, Tongjiaxian 24#, Nanjing 210009, China
a r t i c l e
i n f o
Article history: Received 3 March 2011 Received in revised form 17 March 2011 Accepted 17 March 2011 Available online 29 March 2011 Keywords: Antimicrobial peptide Thanatin Antimicrobial activity Clinical isolate
a b s t r a c t In this study, the activity of S-thanatin (an analog of antimicrobial peptide derived from thanatin) against different bacterial pathogens frequently which can cause therapeutic problems was tested. The result showed minimal inhibitory concentrations (MICs) of S-thanatin against all isolates of the Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Klebsiella ornithinolytica and Klebsiella oxytoca were in the range of 4–16 g/ml, no matter which antibiotic the bacterial was resistant or susceptible, while almost all MICs to Gram-positive bacterial were >128 g/ml except Enterococcus faecium. S-thanatin was more effective toward Gram-negative strains, especially for Enterobacter and Klebsiella. The MICs of S-thanatin were no significantly different in the same species regardless of antibiotic sensitive or -resistant isolates to single or multiple antibiotic (P > 0.05). Likewise, no notable difference could be observed between E. coli, K. pneumoniae, E. cloacae, E. aerogenes, K. ornithinolytica which were sensitive to S-thanatin (P > 0.05). It was implied that the antimicrobial activity of S-thanatin was independent on multi-drug resistance spectrum of bacteria. © 2011 Elsevier Inc. All rights reserved.
1. Introduction One of the most remarkable features of new antimicrobial drugs is their ability to kill microbes efficiently which can cause serious therapeutic problems in clinical therapy, in particular multidrug-resistant (MDR) bacteria has become a severe global health problem for the constant increase of the pathogens resistant to conventional antibiotics [17,19]. In spite of that the whole situation presence fomented severe therapeutic problems, very few antibacterial therapeutic compounds of novel mechanism have been approved over the last 40 years [14]. Thus, there is still an urgent need to develop new antibacterial drugs with potential to kill those resistant pathogens. Fortunately, the naturally occurring peptides termed antimicrobial peptides seem to solve this problem. Antimicrobial peptides (AMPs) have attracted a great deal of attention, which seem to be promising drug candidates for drug-resistant bacteria. Some AMPs are isolated from a wide variety of organisms including plants, prokaryotes, invertebrates
∗ Corresponding author. Tel.: +86 02583272355x808. ∗∗ Corresponding author. Tel.: +86 02583271389. E-mail addresses: guoqiu [email protected] (G. Wu), xitao [email protected] (T. Xi). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.03.019
and mammals [3], and they are important components of natural defenses for most living organisms to resist invading pathogens. They are relatively small, cationic and amphipathic with variable length, sequence and structure [5,7,23,24]. Some of them have already been evaluated in clinical trials [7]. Many AMPs likely contribute to format pores in the plasma membrane then lead to extensive membrane rupture eventually result in energy depletion and microbial lysis [1]. Osmotin, a tobacco pathogenesis-related protein of family 5 (PR-5), has antifungal activity in vitro and in vivo [33], and the action mechanism of this osmotin is heightened by its structural and functional similarities with AMPs. They are considered to be components of the related innate immune response of plants and animals [15,25]. Although many AMPs have the ability of damaging the bacterial membrane, some other bacteriostatic and bactericidal modes of action have been described, which AMPs can affect bacterial growth by binding DNA, inhibiting DNA replication, blocking gene expression or protein synthesis, as well as interfering with other enzymatic activity [1]. Thanatin (GSKKPVPIIYCNRRTGKCQRM), a cationic AMP with antiparallel -sheet structure from 8th amino acid to the Cterminus constrained by disulphide bonds, was isolated from the hemipteran inset Podisus maculiventris, it shows broad antimicrobial activity against bacteria and fungi [8,11]. In this study, S-thanatin, an analog of thanatin, showed higher activity against the Gram-negative bacteria, lower toxicity and better tolerance with cations and PH conditions [27]. The study was designed to
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investigate the activity of S-thanatin on clinical isolates resistant to conventional antibiotics with different structures. 2. Materials and methods 2.1. Bacterial strains and growth conditions A total of 97 clinical isolated bacterial strains were used in this study. The multi-drug resistant strains were isolated from human clinical specimens: (1) Gram-negative bacteria: Escherichia coli (7 strains), Klebsiella pneumoniae (7 strains), Acinetobacter baumannii (8 strains), Stenotrophomonas maltophilia (5 strains), Pseudomonas aeruginosa (7 strains), Enterobacter cloacae (6 strains), Serratia marcescens (5 strains), Enterobacter aerogenes (7 strains), Proteus mirabilis (6 strains), Klebsiella ornithinolytica (4 strains), Klebsiella oxytoca (4 strains); (2) Gram-positive bacteria: Staphylococcus aureus (7 strains), Staphylococcus epidermidis (7 strains), Staphylococcus haemolyticus (9 strains), Enterococcus faecium (5 strain), Streptococcus agalactiae (3 strains). These isolates were obtained from the Center of Medical Laboratory of Zhongda Hospital, Southeast University, China. 15 reference strains from American Type Culture Collection, respectively: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC 19606, Stenotrophomonas maltophilia ATCC 13637, Pseudomonas aeruginosa ATCC 15442, Enterobacter cloacae ATCC 13047, Serratia marcescens ATCC 14041, Enterobacter aerogenes ATCC 49701, Proteus mirabilis ATCC 12453, Klebsiella ornithinolytica ATCC 31898, Klebsiella oxytoca ATCC 43086, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, Staphylococcus hominis ATCC 29970, Enterococcus faecium ATCC 29212. All strains were stored frozen in 10% glycerol at −80 ◦ C. 2.2. Preparation of S-thanatin S-thanatin was synthesized by the solid-phase methodology with 9-fluorenyl-methoxy-carbonyl as protecting group, and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) using an appropriate 0–60% acetonitrile gradient in 0.05% trifluoroacetic acid. Molecular mass was determined by electrospray mass spectrometry using an API instrument (Perkin Elmer SCIEX) as a quality control of synthesis [27]. The peptide in reduced form was taken up in oxidation buffer (1 mg/ml) [100 mM ammonium acetate (pH 8.5)], allowed to refold for 3 days at room temperature under stirring and purified by RP-HPLC [27]. After freeze-drying, S-thanatin was dissolved in phosphatebuffered saline (PBS, PH 7.4) to give a stock solution of 1280 g/ml in −20 ◦ C. 2.3. Other materials MH broth culture was from Oxoid Crop England, polypropylene 96-wells microtiter plates were from CORNIN Crop. All other chemicals used were of reagent grade. 2.4. Drug susceptibility about bacterial strains AST-GN13 cards for Gram-negative bacteria, AST-GP67 cards for Gram-positive bacteria and GN/CE strips with the VITEK2 system (bioMérieux, Marcy l’Etoile, France) were used to confirm isolated bacteria identities and susceptibilities to antimicrobial agents. 2.5. Antimicrobial assays of S-thanatin To test antimicrobial activity of S-thanatin, a microdilution assay was performed in polypropylene 96-wells microtiter plates (PSMicroplates were sterile) [2,4,21]. A 10-l aliquot of the purified
peptide were added to 100 l of a logarithmicphase culture of the tested bacteria which was diluted to 105 –106 colony forming units (cfu) per ml. Control wells were inoculated with 10 l of phosphate-buffered saline. Final concentration of S-thanatin in the test well was 0.5 g/ml, 1 g/ml, 2 g/ml, 4 g/ml, 8 g/ml, 16 g/ml, 32 g/ml, 64 g/ml, and 128 g/ml, respectively. Microbial growth was measured as an increase of optical density at 630 nm (OD630) by a microplate reader (MRX, Dynex) after incubation at 35 ◦ C for 16–20 h. MICs are expressed as where S-thanatin being the lowest concentration causing 100% of growth inhibition [4]. Each concentration was tested in triplicates. 2.6. Statistical analysis Statistical comparisons between groups were made by analysis of variance (significance level was fixed at 0.05). The difference between data of groups was considered significant at the level of <0.05. 3. Results 3.1. The results of drug susceptibility A total of 97 clinical isolates were tested, of which 66 strains were Gram-negative bacteria. Drug susceptibility was detected to ampicillin (AMP), ampicillin-sulbactam (SAM), piperacillintazobactam (TZP), cefazolin (CFZ), cefotetan (CTT), ceftazidime (CAZ), ceftriaxome (CRO), cefepime (FEP), aztreonam (ATM), ertapenem (ETP), imipenem (IPM), amikacin (AMI), gentamicin (GEN), tobramycin (TOB), ciprofloxacin (CIP), levofloxacin (LEV), furadantin (NIT) and cotrimoxazole/or (SMZ), and 31 strains of Gram-positive bacteria susceptibility to conventional antibiotics were also tested, including penicillin (PEN), oxacillin (OXS), cefotetan (CTT), ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MXF), erythromycin (ERY), clindamycin (CLI), quinupristindalfopristin (SYN), linezoid (LZ), vancomycin (VAN), tetracycline (TET), tigecycline (TC), nitrofurantoin (NIT), rifampin (RIF) and/or cotrimoxazole (SMZ). The susceptibility and resistance of these isolates to antibiotics for these organisms were summarized in Tables 1 and 2 . The results showed 60–100% of tested Gramnegative bacteria strains were resistant to CAZ, FEP, ATM, CRO, SAM, CFZ, and AMP, while 25–60% of them were resistant to AMI, TZP, LEV, CIP, TOB, NIT, CTT, GEN and SMZ. One Gram-negative bacteria strain was resistant to EPT and IPM. Thirty-five to ninety-nine percent of Gram-positive bacteria strains were resistant to TET, MXF, CTT, CIP, LEV, CLI, CRY, OXS and PEN. One Gram-positive bacteria strain was resistant to SMZ, RIF, NIT, TC, VAN, LZ and SYN. The carbapenem antibiotics were exhibited the best antibacteria function against clinical isolate bacterial. Nevertheless, also with exception like E. cloacae CI 101013204, it was resistant to almost all antibiotics including carbapenem antibiotics ETP and IPM (Tables 1 and 2). 3.2. The MICs of S-thanatin The results showed that the MICs of S-thanatin against Gramnegative MDR isolates were more effective than Gram-positive MDR isolates (4–16 g/ml), especially for E. coli, K. pneumoniae, E. cloacae, E. aerogenes, K. ornithinolytica and K. oxytoca. Among these strains, there were all isolates (35/35) resistant to AMP, and 91.4% (32/35) to SAM (6 E. coli, 6 K. pneumoniae, 6 E. cloacae, 7 E. aerogenes, 3 K. ornithinolytica and 4 K. oxytoca), 20.0% (7/35) to TZP (3 E. cloacae, and 4 K. oxytoca), 88.6% (31/35) to CFZ (6 E. coli, 4 K. pneumoniae, 6 E. cloacae, 7 E. aerogenes, 4 K. ornithinolytica and 4 K. oxytoca), 45.7% (16/35) to CTT (1 E. coli, 1 K. pneumoniae, 6 E. cloacae, 7 E. aerogenes, and 1 K. ornithinolytica, 54.3% (19/35) CAZ (6 E. coli, 3 K. pneumoniae, 3 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 57.1% (20/35) CRO
Table 1 Drug-resistant spectrums of clinical isolates of Gram-negative bacteria and the MICs of S-thanatin. Strains and clinical isolate no.a
AMP
SAM
TZP
CFZ
CTT
CAZ
CRO
FEP
ATM
ETP
IPM
AMI
GEN
TOB
CIP
LEV
NIT
SMZb
MICs of Ts (g/ml)c
Sputum Sputum Sputum Sputum Urine Urine Sputum
\ R R R R R R R
\ R R R R S R R
\ S S S S S S S
\ R R R R S R R
\ R S S S S S S
\ R R R R S R R
\ R R R R S R R
\ R R R R S R R
\ R R R R S R R
\ S S S S S S S
\ S S S S S S S
\ R R R S S R R
\ R R R R R R R
\ R R R S I R R
\ R R R R S R R
\ R R R R S R R
\ S S S S S I S
\ R R R R R R R
8 4 8 8 8 8 8 8
Sputum Secretion Secretion Sputum Sputum Sputum Sputum
\ R R R R R R R
\ R R R S R S R
\ I S S S S S S
\ R R R S S S R
\ R S S S S S S
\ R R R S S S I
\ I R R S S S S
\ S R R S S S S
\ R R R S S S I
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S R R S S S S
\ S S S S S S I
\ S S S S R S S
\ S S S S R S S
I I I I R I S
\ R R R S S S R
8 8 8 8 8 8 4 8
Sputum Sputum Sputum Sputum Secretion Sputum Sputum Secretion
\ R R R R R R R R
R S R R R R R R
\ R S I R R R R R
\ R R R R R R R R
\ R R R R R R R R
\ R S R R R R R R
\ R R R R R R R R
\ R S R R R R R R
\ R I R R R R R I
\ \ \ \ \ \ \ \ \
\ S S S S R R R R
\ R S S R R S S S
\ R S I R R R S R
\ R S S R R R S R
\ R S R R R R S R
\ R S I R I I S R
\ R R R R R R R R
\ R S R R R R R R
>128 >128 >128 >128 >128 >128 >128 >128 >128
Sputum Sputum Sputum Blood Blood
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ S S I S S
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ \ \ \ \ \
\ S S S S S
\ \ \ \ \ \
\ S S S S S
>128 >128 >128 >128 >128 >128
Pus Sputum Sputum Sputum Secretion Sputum Sputum
\ R R R R R R R
\ R R R R R R R
\ R S S S S S S
\ R R R R R R R
\ R R R R R R R
\ R S S R I S S
\ R R R R R R R
\ R S S S I S S
\ R S S I R I S
\ \ \ \ \ \ \ \
\ R S S S S I R
\ R S R S I S S
\ R S R S I S S
\ R S R S S S S
\ R S S S S S S
\ R S S S S S S
\ R R R R R S S
\ R R R R R R S
>128 >128 >128 >128 >128 >128 >128 >128
G. Wu et al. / Peptides 32 (2011) 1139–1145
Escherichia coli ATCC 25922 CI 100930206 CI 100928201 CI 100929202 CI 101008201 CI 101014204 CI 100930204 CI 100927201 Klebsiella pneumoniae ATCC 700603 CI 100927209 CI 100928208 CI 100928209 CI 100929203 CI 101013201 CI 101016213 CI 101013203 Acinetobacter baumannii ATCC 19606 CI 100927207 CI 100928204 CI 101014203 CI 101016205 CI 101016208 CI 101016209 CI 101016217 CI 100930202 Stenotrophomonas maltophilia ATCC 13637 CI 100929201 CI 100930105 CI 101016216 CI 101022210 CI 101023202 Pseudomonas aeruginosa ATCC 15442 CI 100927204 CI 100927210 CI 100928207 CI 101007204 CI 101013205 CI 101016211 CI 100930207
Source
1141
1142
Table 1 (Continued) Strains and clinical isolate no.a
a
AMP
SAM
TZP
CFZ
CTT
CAZ
CRO
FEP
ATM
ETP
IPM
AMI
GEN
TOB
CIP
LEV
NIT
SMZb
MICs of Ts (g/ml)c
Sputum Sputum Sputum Sputum Sputum Secretion
\ R R R R R R
\ R R R R R R
\ S I R R R S
\ R R R R R R
\ R R R R R R
\ S S R R R S
\ R R R I R S
\ S R R S R S
\ S I R R R S
\ S S R S S S
\ S S R S S S
\ S R R S S S
\ S R R S I S
\ S R R I R S
\ S R R S I S
\ S R R S I S
\ S I R I R S
\ S R S S R S
8 16 8 8 8 16 8
Secretion Sputum Blood Urine Sputum
\ R R R R R
\ R R R R R
\ S S S R S
\ R R R R R
\ S S R R R
\ S S I R S
\ S R R R R
\ S I R R R
\ S R S S S
\ S R S I R
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
>128 >128 >128 >128 >128 >128
Secretion Secretion Secretion Sputum Sputum Sputum Blood
\ R R R R R R R
\ R R R R R R R
\ S S I I S I S
\ R R R R R R R
\ R R R R R R R
\ S S R I S R R
\ S S R S S R R
\ S S R S S R R
\ S S R S S R R
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S S R S S R R
\ S S R S S R R
\ I R I I R I I
\ S S S S S S S
4 4 4 4 4 4 4 4
\ R R R R S S
\ R R R S S S
\ S S R S S S
\ R R R R S S
\ S S S S S S
\ R R R R S S
\ R R R R S S
\ R R R R S S
\ R R R R S S
\ S S S S S S
\ S S S S S S
\ S S S S S S
\ R R R R S S
\ I I R I S S
\ R R R R S I
\ R R R R S S
\ R R R R R R
\ R R R R S R
>128 >128 >128 >128 >128 >128 >128
Secretion Sputum Urine Sputum
\ R R R R
\ R S R R
\ S S S S
\ R R R R
\ R S S S
\ S I S S
\ S R R S
\ S S S S
\ S I S I
\ S S S S
\ S S S S
\ S S S S
\ S I I S
\ S R R R
\ S S S S
\ S S S S
\ I S S R
\ R R R R
8 8 8 8 8
Sputum Sputum Sputum Sputum
\ R R R R
\ R R R R
\ R R R R
\ R R R R
\ S S S S
\ R R R R
\ R R R I
\ R R R R
\ R R R R
\ S S S S
\ S S S S
\ S S S S
\ I R R R
\ R R R R
\ R R R R
\ R R R R
\ I I I S
\ S S S S
8 8 8 8 8
Sputum Secretion Secretion Sputum Sputum
ATCC, American Type Culture Collection; CI, clinical isolate. S, sensitive; R, resistant; I, intermediate; \, no detected; AMP, ampicillin; SAM, ampicillin-sulbactam; TZP, piperacillin-tazobactam; CFZ, cefazolin; CTT, cefotetan; CAZ, ceftazidime; CRO, ceftriaxome; FEP, cefepime; ATM, aztreonam; ETP, ertapenem; IPM, imipenem; AMI, amikacin; GEN, gentamicin; TOB, tobramycin; CIP, ciprofloxacin; LEV, levofloxacin; NIT, furadantin; SMZ, cotrimoxazole. c Ts, S-thanatin. b
G. Wu et al. / Peptides 32 (2011) 1139–1145
Enterobacter cloacae ATCC 13047 CI 100927203 CI 100928203 CI 101013204 CI 101014205 CI 101016212 CI 100930201 Serratia marcescens ATCC 14041 CI 100928202 CI 101007205 CI 101008205 CI 101014202 CI 101028210 Enterobacter aerogenes ATCC 49701 CI 100928206 CI 101023205 CI 101026211 CI 110111202 CI 110111211 CI 110112203 CI 110112204 Proteus mirabilis ATCC 12453 CI 101016202 CI 101016213 CI 101025205 CI 110118212 CI 110118213 CI 110118216 Klebsiella ornithinolytica ATCC 31898 CI 101016204 CI 101027203 CI 110118210 CI 110119214 Klebsiella oxytoca ATCC 43086 CI 101008202 CI 101014202 CI 110115201 CI 11017202
Source
Table 2 Drug-resistant spectrums of clinical isolates of Gram-positive bacteria and the MICs of S-thanatin. Strains and clinical isolate no.a
PEN
OXS
CTT
CIP
LEV
MXF
ERY
CLI
SYN
LZ
VAN
TET
TC
NIT
RIF
SMZb
MICs of Ts (g/ml)c
Secretion Secretion Secretion Sputum Sputum Sputum Secretion
\ R R R R R R R
\ R R S S S R R
\ R S S S S R R
\ R S S S S R R
\ S S S S S I R
\ S S S S S S R
\ S R S S R R R
\ S R S S R R R
\ S S S S S S S
\ S S S S S S S
\ S S S S S S S
\ S R S R S R R
\ R S S S S S S
\ S S S S S S S
\ S S S S S S R
\ R S S S S R S
>128 >128 >128 >128 >128 >128 >128 >128
Secretion Secretion Secretion Secretion Blood Blood Secretion
\ R R R R R R R
\ R R R R R R R
\ R I R S R I S
\ R R R S R R I
\ I R R S R I I
\ R I I S R R S
\ R R R R S R R
\ R R R R S R S
\ S S S S S R S
\ S S S S S S S
\ S S S S S S S
\ S R S S S R R
\ S S S S S S S
\ S S S S S S S
\ R S S S S S S
\ R S S R S S S
>128 >128 >128 >128 >128 >128 >128 >128
Sputum Sputum Urine Sputum Blood Blood Sputum Blood Blood
\ R R R R R R R R R
\ R R R R R R R R R
\ I R R S R S S S S
\ R R R S R R R S S
\ R R I S R R R S S
\ S I R S I R I S S
\ R R R R R R R R R
\ R R S R R R R S R
\ S S S S S S S S S
\ S S S S S S S S S
\ S S S S S S S S S
\ S R S R S R R S S
\ S S S S S S S S S
\ S S S S S S S S S
\ S S S S S S S S S
\ S S S S S S S S S
>128 >128 >128 >128 >128 >128 >128 >128 >128 >128
Secretion Secretion Secretion Urine Blood
\ S R R R R
\ S R R R R
\ S R R R R
\ I R R R R
\ S R R R R
\ S R R R R
\ R R R R R
\ R R R R R
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S S S S S
\ S R R R R
\ \ \ \ \ \
\ \ \ \ \ \
64 64 64 128 64 64
Blood Blood
R R
R R
S S
R R
R R
S I
S S
S S
S S
S S
S S
S S
S S
S S
S S
S S
>128 >128
G. Wu et al. / Peptides 32 (2011) 1139–1145
Staphylococcus aureus ATCC 29213 CI 100927214 CI 100928211 CI 101013207 CI 101013210 CI 101013211 CI 101013212 CI 100930212 Staphylococcus epidermidis ATCC 12228 CI 100925212 CI 100927215 CI 100928214 CI 101022209 CI 101025207 CI 101025208 CI 101026217 Staphylococcus hominis ATCC 29970 CI 100927213 CI 101016215 CI 101026212 CI 100930211 CI 100930215 CI 100930213 CI 100930214 CI 101013209 CI 101025209 Enterococcus faecium ATCC 29212 CI 101027206 CI 101026212 CI 101028209 CI 110112029 CI 110117204 Streptococcus agalactiae CI 101007206 CI 101014201
Source
a
ATCC, American Type Culture Collection; CI, clinical isolate. S, sensitive; R, resistant; I, intermediate; \, no detected; PEN, penicillin; OXS, oxacillin; CTT, cefotetan; CIP, ciprofloxacin; LEV, levofloxacin; MXF, moxifloxacin; ERY, erythromycin; CLI, clindamycin; SYN, quinupristindalfopristin; LZ, linezoid; VAN, vancomycin; TET, tetracycline; TC, tigecycline; NIT, nitrofurantoin; RIF, rifampin; SMZ, cotrimoxazole. c Ts, S-thanatin. b
1143
1144
G. Wu et al. / Peptides 32 (2011) 1139–1145
(6 E. coli, 2 K. pneumoniae, 4 E. cloacae, 3 E. aerogenes, 2 K. ornithinolytica and 3 K. oxytoca), 51.4% (18/35) to FEP (6 E. coli, 2 K. pneumoniae, 3 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 54.3% (19/35) to ATM (6 E. coli, 3 K. pneumoniae, 3 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 2.9% (1/35) to ETP and IPM (1 E. cloacae), 20% (7/35) to AMI (5 E. coli and 2 E. cloacae), 40% (14/35) to GEN (7 E. coli, 2 K. pneumoniae, 2 E. cloacae, and 3 K. oxytoca), 42.9% (15/35) to TOB (5 E. coli, 3 E. cloacae, 3 K. ornithinolytica and 4 K. oxytoca), 45.7% (16/35) to CIP (6 E. coli, 1 K. pneumoniae, 2 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 45.7% (16/35) to LFV (6 E. coli, 1 K. pneumoniae, 2 E. cloacae, 3 E. aerogenes, and 4 K. oxytoca), 17.1% (6/35) to NIT (1 K. pneumoniae, 2 E. cloacae, 2 E. aerogenes, and 1 K. ornithinolytica), 48.6% (17/35) to SMZ (7 E. coli, 4 K. pneumoniae, 2 E. cloacae, and 4 K. ornithinolytica), respectively. The MIC of Sthanatin against different Enterobacter and Klebsiella bacteria was in the range of 4–16 g/ml in Table 1, while almost all the MICs to Gram-positive bacterial were >128 g/ml except Enterococcus faecium in Table 2. The MICs of S-thanatin were no significantly different in the same species regardless of antibiotic-sensitive or -resistant isolates to single or multiple antibiotic (P > 0.05). Likewise, no notable difference could be observed between E. coli, K. pneumoniae, E. cloacae, E. aerogenes, K. ornithinolytica and K. oxytoca, which were sensitive to S-thanatin (P > 0.05). 4. Discussion Pathogenic bacteria from clinical settings are becoming increasingly resistant to conventional antibiotics. Particularly, the increase in resistance of Gram-negative bacteria is more significantly than that in Gram-positive bacteria. There are fewer new and developmental antibiotics active against Gram-negative bacteria and drug development programs seem insufficient to provide therapeutic cover. Recently, clinical isolates producing new subgroup of metallo-beta-lactamase (New Delhi metallo--lactamase 1, NDM1) had already been identified [10,32]. It was urgent to search new generation antibiotics for drug-resistant bacteria instead of conventional antibiotics. As the cationic peptide, S-thanatin was demonstrated that a promising high activity on several clinical MDR isolates such as E. coli and K. pneumoniae in previous study [28,31]. This study showed higher antimicrobial activity of the peptide not only on E. coli and K. pneumoniae, but also on E. cloacae, E. aerogenes, K. ornithinolytica and K. oxytoca. The range of MICs was from 4 to 16 g/ml. The antimicrobial action of S-thanatin is more efficient to Gram-negative bacteria (apart from A. baumannii, S. maltophilia, P. aeruginosa, S. marcescens, and P. mirabilis) than Gram-positive bacteria (apart from Enterococcus faecium). Although the bactericidal mechanism of antimicrobial peptides remains to be fully understood, the microbicidal action of cationic peptides is generally initiated by disrupting the integrity of cell membranes through interaction with the phospholipids component by several models such as “Barrel-stave model”, “Carpet model”, and “Toroidal-pore model” [6,9]. We have already studied the antimicrobial mechanism of S-thanatin. The conclusion about membrane aggregation and perturbation induced and selective toxicity of antimicrobial peptide S-thanatin on bacteria had been reported [29,30]. The different activities may be due to the various mechanisms of S-thanatin. In our previous study, we found the MIC of S-thanatin against L-form strain reached 4fold increase compared with wild type for Gram-negative bacteria. In contrast, the susceptibility to Gram-positive bacteria protoplast decreased. It seemed that S-thanatin interacted with the outer membrane of Gram-negative bacteria, but not cytoplasmic membranes. Cell-wall of Gram-positive bacteria as a physical barrier was responsible for poor susceptibility of S-thanatin [30].
We speculated that S-thanatin must be attracted to LPS and phospholipid of Gram-negative bacteria by the electrostatic and hydrophobic interaction [26]. Then it inserts into the cytoplasmic membrane and membrane starts to destabilize and distort. At last, the perturbation of membrane damages the energization and respiration of cytoplasmic membrane leading to the bacteria becoming tattered and cytoplasm material leaking out. Compared with other peptides, S-thanatin adopted a sheet conformation with novel antibacterial action exhibiting LPS-binding and low hemolytic activities [29]. One obvious mechanism is S-thanatin must be attracted to bacterial surfaces. The Gram-negative bacterial membranes abundantly contain negatively charged lipids in particular of LPS, which can significantly enhance the membrane binding of S-thanatin. The activity of thanatin depends on the size of LPS [18], and the antimicrobial activity of cationic AMPs is compromised when the LPS polysaccharide moieties decreased [12,13]. However, S-thanatin exhibited a poor activity on some Gram-negative strains such as A. baumannii, S. maltophilia, P. aeruginosa, S. marcescens, and P. mirabilis in this study. The reason of differences on susceptibility among Gram-negative strains remained to be further researched. The mechanism of bacteria resistant to conventional antibiotics is diverse, such as active efflux pumps [16,22] and production of -lactamase [20], and etc. However, many agents against drugresistant bacteria may be only useful to one of resistant-drug mechanisms such as a clavulanic acid, which can inhibit the activity of -lactamase produced by bacteria. In present study, our results showed that S-thanatin did not exhibit any preference of killing bacteria in the same species (Table 1). No significantly difference was found between sensitive and resistant isolates to conventional antibiotics with different structures (all P > 0.05). The antibacteria activity of S-thanatin was independent on MDR spectrum of bacteria. It implied that S-thanatin may be useful to be developed as an antimicrobial agent for therapy of infection with MDR bacteria, together with its overall advantageous features such as salt tolerance, low cytotoxicity to eucaryotic cell [27]. Interestingly, we found a clinical isolate of Gram-negative bacteria (Enterobacter cloacae CI 101013204) resistant to almost all antibiotics including ertapenem and imipenem, but it was still susceptible to S-thanatin (the MIC was 8 g/ml). Furthermore, a high activity of S-thanatin on extended-spectrum -lactamases (ESBL)producing strains was also observed in this study. The MICs of S-thanatin against (ESBL)-producing strains, particularly E. coli and K. pneumoniae were the same as those of the international standard strains (4–8 g/ml). It was well known that the majority of NDM1 positive isolates producing carbapenemase was Gram-negative Enterobacteriaceae such as E. coli, K. pneumoniae, and E. cloacae [10,32]. It was reasonable to believe that S-thanatin was potentially useful to anti-NDM-1 positive isolates.
Acknowledgments This work was supported by the Natural Science Foundation of Jiangsu Province, China (Grant No. BK2009274) and Science Foundation of Southeast University (Grant No. 3290000102). We are indebted to Mr. Li for his help in language proof.
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