Curr Microbiol (2009) 59:147153 DOI 10.

1007/s00284-009-9410-2

Activity of the Antimicrobial Peptide and Thanatin Analog S-thanatin on Clinical Isolates of Klebsiella pneumoniae Resistant to Conventional Antibiotics with Different Structures
Guo-Qiu Wu Jia-Xuan Ding Lin-Xian Li Hai-liang Wang Rui Zhao Zi-Long Shen

Received: 25 November 2008 / Accepted: 6 April 2009 / Published online: 21 May 2009 Springer Science+Business Media, LLC 2009

Abstract The treatment of infections caused by bacteria resistant to the vast majority of antibiotics is a challenge worldwide. To evaluate the effect of S-thanatin (an analog of thanatin, a cationic antimicrobial peptide isolated from the hemipteran insect Podisus maculiventris) against microbial resistant to antibiotics, we studied its bactericidal kinetics, synergistic effect, resistance, and activity on clinical isolates of Klebsiella pneumoniae resistant to conventional antibiotics with different structures. The bactericidal rate of S-thanatin was more than 99% against K. pneumoniae ATCC 700603 when bacterial cultures were monitored for 60 min. The peptide was synergistic with b-lactam cefepime in most of the clinical MDR isolates tested (7/8). An average value of FIC was 0.3708. No synergy was found between the peptide and amoxicillin, gentamycin, tetracycline, or ciprooxacin in all bacteria tested. A total of 48 isolates of K. pneumoniae with different resistance spectrum tested was susceptible to S-thanatin. The MICs were 6.2525 lg/ml. No signicant difference in the MICs of S-thanatin between the sensitive isolates and the resistant isolates to single antibiotic was observed (P [ 0.05). The resistance of K. pneumoniae
G.-Q. Wu (&) H. Wang Center of Clinical Laboratory Medicine of Zhongda Hospital, Southeast University, Nanjing 210009, Peoples Republic of China e-mail: nationball@163.com J.-X. Ding R. Zhao Z.-L. Shen Biotechnology Center, Department of Life Science and Biotechnology, China Pharmaceutical University, Nanjing 210009, Peoples Republic of China L.-X. Li School of Pharmacy, China Pharmaceutical University, Nanjing 210009, Peoples Republic of China

ATCC 700603 to S-thanatin was slightly higher, when cultured at sub-inhibitory concentration for 5 days. S-thanatin may be an attractive candidate for developing into an antimicrobial agent.

Introduction The development of pathogens which have acquired resistance to the vast majority of antibiotics is one of the main problems in clinical settings [14]. An increasing body of evidence shows that prevalence of multi-drug resistant (MDR) Klebsiella pneumoniae in Intensive Care Unit (ICU), such as cephalosporins, carbapenems, uoroquinolones, lincosamides, and aminoglycosides [27]. Despite the many advances in antibiotic researches, bacteria have evolved through adaptation and natural selection of defensive mechanisms that render antimicrobials impotent. Available therapeutic options for antibiotic-resistant organisms are severely limited. Therefore, there is a vital need to develop new effective therapeutics. Antimicrobial peptides (AMPs) have some features that make them good candidates as anti-MDR microbial agents. Recently, thanatin (GSKKPVPIIYCNRRTGKCQRM), an antimicrobial peptide with an anti-parallel b-sheet constrained by disulde bonds, was isolated from the hemipteran insect Podisus maculiventris, showed a broad antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria, and fungi [8]. In the previous research, we synthesized several peptides with amino acid deletion or substitution within the disulde loop of thanatin, and found that S-thanatin (which synthesized by substituting the amino acid of threonine with serine, GSKKPVPIIYCNRRSGKCQRM) exhibited a

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higher antimicrobial activity and less hemolysis toxicity. The process of production by fermentation was also established [9]. Thanatin or its analogs have a strong net cationic (positive) charge, and the three-dimensional folding results in an amphipathic b-sheet structure when interacting with lipidic (membrane) environment [8, 10]. These features are critical for bacterial killing [11, 12]. Recently, our research showed that S-thanatin could apparently reduce lethality of model animals with peritonitis induced by cecal ligation and puncture, and a strong synergy between tigecycline and S-thanatin was observed against Escherichia coli and Enterococcus faecalis [Cirioni O, Wu GQ, et al. Unpublished]. This study was designed to observe the kinetics of bacterial killing of S-thanatin and synergistic effect between the peptide and conventional antibiotics, and evaluate the activity of the peptide on clinical MDR isolates of Klebsiella pneumoniae resistant to conventional antibiotics with different structures.

Double Crane Pharm Corp.), tetracycline (BBI Inc.), ceftazidime (Jiuxin Pharm Inc.), ciprooxacin(Xinchang Pharm Corp.), cefoxitin, and cefepime (Qilu Pharm Corp.) powders were diluted in accordance with the manufacturers recommendations. Solutions were made fresh on the day of use. All other chemicals used were of reagent grade. Microbicidal Assay Exponentially growing bacteria (K. pneumoniae ATCC 700603) were resuspended in fresh MuellerHinton (MH) broth at approximately 107 cells/ml to ensure accurate determination of the 99.9% killing endpoint and exposed to S-thanatin, amoxicillin, cefoxitin, cefepime, cotrimoxazole, gentamycin, and ciprooxacin (the concentrations of all compounds were at 10 lg/ml except for S-thanatin at 6.25 lg/ml) for 0, 15, 30, 60, and 120 min at 37C. After these times, samples were serially diluted in 10 mM of sodium HEPES buffer (pH 7.2) to minimize the carryover effect and plated onto MH agar plates to obtain viable colonies. Synergy Effect Combinations of S-thanatin with antibiotics of different nature were tested for synergistic effect by a checkerboard titration method. Seven clinical MDR isolates (Table 1) were randomly chosen to perform this study. The ranges of drug dilutions used were 0.12564 lg/ml for S-thanatin and 0.250256 lg/ml for conventional antibiotic including amoxicillin, gentamycin, tetracycline, ciprooxacin, and cefepime, which were the representative of penicillins, aminoglycosides, tetracyclines, quinolones, and cephalosporins. The fractional inhibitory concentration (FIC) index for combinations of two antimicrobials was calculated according to the equation FIC index = FIC A ? FIC B = A/MIC A ? B/MIC B, where A and B are the MICs of drug A and drug B in the combination, MIC A and MIC B are the MICs of drug A and drug B alone, and FIC A and FIC B are the FICs of drug A and drug B. The FIC indexes were interpreted as follows: \0.5, synergy; 0.54.0, indifferent; and [4.0, antagonism. Experiments were performed in triplicates. In Vitro Susceptibility Testing Susceptibility testing was performed by the broth microdilution method according to the procedures outlined by the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards [14]. Briey, S-thanatin was prepared as 103 concentrates in PBS. Inocula were prepared by resuspending colonies from a MullerHinton (MH) agar medium and adjusting the

Materials and Methods Preparation of S-thanatin S-thanatin was synthesized by the solid-phase methodology with 9-uorenyl-methoxy-carbonyl as protecting group, and puried by reverse-phase high-performance liquid chromatography (RP-HPLC) using an appropriate 060% acetonitrile gradient in 0.05% triuoroacetic acid. Molecular mass was determined by electrospray mass spectrometry [13]. The peptide in reduced form was taken up in oxidation buffer (1 mg/1 ml) [100 mM ammonium acetate (pH 8.5)], allowed to refold for 3 days at room temperature under stirring and puried by RP-HPLC [13]. Microorganisms and Chemicals For antimicrobial assays in vitro, the commercially available quality control strain of Klebsiella pneumoniae ATCC 700603 was used from Center of Clinical Laboratory, Health Ministry of PRC. The clinical MDR isolates of Klebsiella pneumoniae which was collected from January to July, 2008, was obtained from the Center of Medical Laboratory of Zhongda hospital, Southeast University, China. AST-GN14 cards and GN/CE strips with the VI rieux, Inc., France) were used to TEK2 systems (bioMe conrm organism identities and susceptibilities to antimicrobial agents, and all strains were stored frozen in 10% glycerol at -80C. Amoxicillin (NCPC), Gentamycin (Daxin Pharm Corp.), Trimethoprim/sulfamethoxazole (cotrimoxazole)(Kunshan

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G.-Q. Wu et al.: Activity of the Antimicrobial Peptide and Thanatin Analog S-thanatin Table 1 FIC indexesa for interaction of conventional antibiotics with S-thanatin against clinical MDR isolates of K. pneumoniaeb Organism K34728 K58903 K92003 K97565 K76938 K96679 K50936 K44627
a b c

149

AMO 0.625 0.125 0.688 0.053 1.125 0.125 0.875 0.125 0.813 0.188 0.625 0.125 1.00 0.25 1.125 0.125

GEN 0.563 0.063 0.875 0.125 0.500 0125 0.813 0.188 0.688 0.063 0.875 0.125 1.00 0.25 0.625 0.125

TET 1.00 0.25 1.00 0.25 0.438 0.063 0.625 0.125 0.813 0.188 0.625 0.125 0.875 0.125 1.00 0.25
c

CIP 1.75 0.50 1.625 0.625 0.813 0.187 1.00 0.25 1.75 0.50 0.688 0.063 0.688 0.063 1.375 0.125

FEP 0.343 0.032c 0.438 0.063c 0.438 0.063c 0.281 0.032c 0.313 0.063c 0.75 0.25 0.281 0.032c 0.438 0.063c

FIC indexes were interpreted as follows: \0.5, synergy; 0.504.0, indifferent; [4.0, antagonism The ranges of concentrations tested were 0.12564 mg/l for S-thanatin and 0.250256 mg/l for the other antimicrobial compounds The cases where synergism has been recorded

AMO Amoxicillin, GEN Gentamycin, TET Tetracycline, CIP Ciprooxacin, FEP Cefepime

suspension to match that of a 0.5 McFarland standard with MH broth (MHB). The suspension was diluted into fresh MHB to give 105107 CFU/ml for bacteria. After dispensing 89-ll aliquots of the microbial suspension into each well of a 96-well polypropylene microtiter plate, 11ll of test peptide was added. The MIC was dened as the lowest concentration of drug which prevented visible turbidity after 1620 h. Resistance Study Klebsiella pneumoniae ATCC 700603 harvested from the well with an S-thanatin concentration equal to 0.59 the MIC, was diluted to 1 9 10559 105 CFU/ml in fresh MHB and dispensed into microtiter plates as 100-ll aliquots. S-thanatin was added as described above, and MICs were determined daily for 5 days. Statistical Analysis

of the peptide was 36% at 15 min. The numbers of viable CFU treated with S-thanatin, amoxicillin, gentamycin, and ciprooxacin decreased more than 50% within 30 min. The bactericidal rate of S-thanatin and ciprooxacin was more than 99% when bacterial cultures were monitored for 60 min. Up to 120 min, the cell growth of 20% for amoxicillin, 25% for cefoxitin, and 12.5% for cotrimoxazole was still observed, separately. Interaction of S-thanatin with Antibiotics of Different Structures The FIC indexes for combinations between S-thanatin and amoxicillin, gentamycin, tetracycline, ciprooxacin, and cefepime were summarized in Table 1. A synergistic effect between S-thanatin and cefepime, except for K96679, was observed in all clinical MDR isolates tested. An average
120

Viability % of control

Statistical comparisons between groups were made by analysis of variance (signicance level was xed at 0.05). The difference between data of groups was considered signicant at the level of \0.05.

100 80 60 40 20 0 0 20 40 60

Ts(6.25 g/ml) AMO(10 g/ml) CXT(10 g/ml) FEP(10g/ml) TSU(10 g/ml) GEN(10 g/ml) CIP (10 g/ml)

Results Bactericidal Activity The kinetics of bacterial killing of S-thanatin was evaluated against K. pneumoniae ATCC 700603 as compared to those of conventional antimicrobial agents such as amoxicillin, cefoxitin, cetepime, cotrimoxazole, gentamycin, and ciprooxacin. Cell viability within the rst 2 h was measured. As shown in Fig. 1, the rate of bacterial killing

80

100

120

Minutes
Fig 1 The kinetic assay of bacterial killing of S-thanatin and conventional antimicrobial agents against K. pneumoniae ATCC 700603. TS (6.25 lg/ml), S-thanatin; AMO (10 lg/ml), amoxicillin; CXT (10 lg/ml), cefoxitin; FEP (10 lg/ml), cefepime; TSU (10 lg/ml), Trimethoprim/sulfamethoxazole (cotrimoxazole); GEN (10 lg/ml), gentamycin; CIP (10 lg/ml), ciprooxacin

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value of FIC was 0.3708. No synergy was found between the peptide and amoxicillin, gentamycin, tetracycline, or ciprooxacin in all bacteria tested. The FIC indexes ranged from 0.5 to 2.25(\4.0) except K92003 for gentamycin (0.3750.625) and tetracycline (0.3750.5) (Table 1). The MICs of the antibiotics decreased obviously while the peptide was added (data not shown), indicating that Sthanatin could interact with these antimicrobial agents against K. pneumoniae. In Vitro Anti-MDR Bacterial Activity A total of 48 clinical isolates of K. pneumoniae resistant to conventional antibiotics were tested, including amoxicillin (AMO), amoxicillin/clavulanic acid (AMC), cefepime (FEP), cefoxitin (CXT), trimethoprim/sulfamethoxazole (TSU), piperacilline (PIC), piperacilline/tazobactam (TZP), ticarcilline (TIC), ticarcilline ? clavulanic acid (TCC), cefalotin (CFT), cefotaxime (CTX), ceftazidime (CAZ), cefuroxime (CXM), meropenem (MERO), imipenem (IMI), tobramycine (TOB), amikacine (AKN), gentamycin (GEN), netimicine (NET), and ciprooxacin (CIP). The susceptibility of these isolates to antibiotics and the distributions of MICs of S-thanatin for these organisms are summarized in Table 2. The results showed that the peptide exhibited potency against K. pneumoniae resistant to antimicrobial agents with different structures. All microbes tested were inhibited by the peptide at a concentration of 25 lg/ml or less and the average was 21.22 lg/ml. There was no signicant difference in the MICs of Sthanatin between the sensitive isolates and the resistant isolates to single antibiotic. The P values were 0.5110 (AMC), 0.5499 (TZP), 0.2460 (TCC), 0.6122 (CFT), 0.6338 (CXT), 0.6160 (CTX), 0.4635 (CAZ), 0.7981 (CXM), 0.8800 (FEP), 0.7313 (TSU), 0.1583 (TOB), 0.5667 (AKN), 0.2461 (GEN), 0.7454 (NET), and 0.9754 (CIP), respectively. Evaluation of Resistance When cultures of K. pneumoniae were serially transferred daily, only a little change was observed in the MIC of Sthanatin. When cultured at sub-inhibitory concentration, the nal MIC of S-thanatin for K. pneumoniae was slightly higher when compared with the MIC determined after the standard incubation for 1820 h (data not shown). The MIC of the peptide increased only by two times.

Discussion Although the bactericidal mechanism of S-thanatin still remains to be fully understood, the microbicidal action of cation antimicrobial peptide is generally initiated by

disrupting the integrity of cell membranes through interaction with the phospholipid component [12, 15]. In the case of Gram-negative bacteria such as K. pneumoniae, S-thanatin may interact with lipopolysaccharide (LPS) which is an integral structural component of the outer membrane of Gram-negative bacteria [11]. In the previous study, we had already conrmed that the peptide possessed a LPS-binding and neutralizing activity, and caused the reduction of the plasma endotoxin levels in experimental mouse models of septic shock caused by clinical MDR isolate of Escherichia coli (Wu GQ, Li LX, et al. Unpublished). As suggested by Shai, the cationic peptides interact initially with divalent cation binding sites on surface LPS, displace these divalent cations because they have 103104-fold higher afnity for these sites, and being bulkier than the divalent cations they displace, cause distortion or disruption of the outer membrane structure [16]. Our study showed that antibacterial activity of Sthanatin could competitively be inhibited by monovalent or divalent cation. The peptide lost its activity at 500 mM Na?/K? and partly retained them at 20 mM Ca2?/Mg2? on E. coli ATCC 25922 and B. subtilis ATCC 21332 [13]. However, as suggested by Ulvatne and Vorland [17], these changes of the outer membrane is unlikely to be the main factor leading to cell death. The access of the peptide to the periplasm, from which they reach the inner membrane makes the loss of inner membrane integrity and eventually leads to bacterial death [17]. In bactericide assay, the bactericidal rate of S-thanatin was more than 99% within 60 min, similar with that of ciprooxacin. This was in agreement with the observation that bacteria respiration started to decrease after 1 h and became undetectable at 6 h when S-thanatin was added [10]. These results suggested that action model of S-thanatin on bacteria might be different from those of other cationic antimicrobial peptides possessed an amphipathic a-helice structure such as Protegrin-1 which killed rapidly with a 46 log reduction in survival within 5 min at 4 times the MIC [18]. Most of cationic antimicrobial peptides possessed an amphipathic a-helice structure usually kill bacterial rapidly. The peptides mode of action appears to be by direct lysis of the pathogenic cell membrane such as the barrel-stave model and toroidal-pore model. bsheet structure peptides such as S-thanatin, however, cause distortion or disruption of the outer and reach the inner membrane. It makes the loss of inner membrane integrity and eventually leads to bacterial death. In our experiment, S-thanatin also exhibited a high activity on clinical isolates of K. pneumoniae resistant to conventional antibiotics with different resistance spectrum. The MICs were from 6.25 lg/ml to 25 lg/ml (Table 2), and no signicant difference was found in whatever strains were either sensitive or resistant to a single class of

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Table 2 Drug-resistant spectrums of clinical isolates of K. pneumoniae and the MICs of S-thanatin CAZ FEP S S R S S S R R S R R S R R S R S R R R R S R R R R R R S R R R R R R S R R S R R S S R R I R I I S S R R R S R I R S R R R R S R S R R R S R S S R R S R R R R R R R R R R R R S R R R R S S S S S S S S S S S S S S S S S R R R S 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 R S R S R S S S R S R S S R R R S R R R S R R R R R R S R S R R S I R R R S S R R R R S S S R R R R S R R R S R S R R S R R R S R R S S R S S R R R S S S S R R R S R S S S R S R R S R R S S R R S S R R R S S S S S S S R R S S S S S S S R S R R R S R S R R R R S R R R S R R R R R R R R S R R S S R R R R S S S S S S S S S R S S R S R R S S S S R R R R S R R S S R S R R S R R R S S R S S S R S S S S S R S S R S S R R S S S S R R R S R S R S S R S I R R S R S S S S S S S S S S R R R S S R R S R S R 25 25 25 25 25 12.5 12.5 25 25 12.5 25 25 12.5 25 6.25 25 6.25 25 25 25 25 25 25 25 25 12.5 25 12.5 12.5 25 25 12.5 25 S S S S S S S S S S 25 S S S S R S S S S S 25 CXM MERO IMI TSU TOB AKN GEN NET CIP MICs of S-thanatin(lg/ml)

Bacteria No. Source I S R S R S S S S R S S R I S I S R I R S S R I I S S I I R S R R S S I S R S R I S R R R R R R R R S R R R R R R R S R R R R S R R R S R S R R R R R S R R R S R R R R R R S R R S R R R S R R R R R R R R I S R S S S S R R R R R R R R R R R R R R R R R R R R R I S R R R R R R S R S S S S R R R R R S I R I S S R R R S R S S S R S I S R R R R R I S R S R R S R S R R R S R R S R R R R R I S R S S S S I S R S R S R R S R R R S R I S R S S S S R S R R R R S R S R S S S S R S R R R S R I S R S S S S R R R R R S S

AMO AMC PIC TZP TIC TCC CFT CXT CTX

K30578

Sputum

K41692

K13167

K27317

K12432

K29646

K58903

K042022

K24067

K34728

K42543

K292702

K96679

K14233 K82136

R R

K42232

K254702

F45669

K55336

G.-Q. Wu et al.: Activity of the Antimicrobial Peptide and Thanatin Analog S-thanatin

K76938

K44627

K62697

K50936

K40044

K22405

K21042

K09167

K30873

K94694

K92003 K66621

R R

K50942

K89044

K80387

123

K34532

Urine

151

152

Table 2 continued CAZ FEP CXM MERO IMI TSU TOB AKN GEN NET CIP MICs of S-thanatin(lg/ml) S S R R S S S R R R R S S S S S R R R R R R R S S S R S S S S R R R R S S S S R S S S S I S R R R R R S S R R S I S R S R 25 25 25 12.5 6.25 12.5 25 25 25 25 12.5 12.5 S R S R 25

123
I I S R S S I R R I S S R R R R R R R R R R R S S S R S R R R R R R S R S S R S S R S S S S S S S S S S R R R R R R R R R R S S R R R R R R R R R R R S S R R S R R R R R R I R S S R R S R R R S R R R R S S R R S R S S S S S S S S S S R I S S R R R S R S R S R S I S R S R S S S S S R S I S R S R R R R R R S S S R S R R R R I R R R S S R R S R R R S I R R R S S R

Bacteria No. Source

AMO AMC PIC TZP TIC TCC CFT CXT CTX

K26385

K07842

K06201

K38622 K21254

R R

K33004

Blood

K47684

K97565

K18633

CSF

K43530

Secretion R

K80412

K71994

Pus

K81674

G.-Q. Wu et al.: Activity of the Antimicrobial Peptide and Thanatin Analog S-thanatin

S Sensitive, R Resistant, I Intermediate, AMO Amoxicillin, AMC Amoxicillin/clavulanic acid, FEP Cefepime, GEN Gentamycin, CIP Ciprooxacin, CXT Cefoxitin, TSU Trimethoprim/ sulfamethoxazole(cotrimoxazole), PIC Piperacilline, TZP Piperacilline/tazobactam, TIC Ticarcilline, TCC Ticarcilline/clavulanic acid, CFT Cefalotin, CTX Cefotaxime, CAZ Ceftazidime, CXM Cefuroxime, MERO Meropenem, IMI Imipenem, TOB Tobramycine, AKN Amikacine, NET Netimicine

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antibiotics (all P [ 0.05). The peptide activity was unaffected by the common resistance mechanisms. It may be relative with bactericidal mechanism of the peptide different from conventional antibacterial agents. Based on the mechanism suggested above, antimicrobial cationic peptides can change outer membrane structure and cause loss of inner membrane integrity, and promote the uptake of other agents, e.g., antibiotics and lysozyme, thus show synergy with conventional antibiotics, especially against antibiotic resistant mutants [19]. As shown in Table 1, S-thanatin was synergistic with b-lactam cefepime in most of clinical MDR isolates tested (7/8). In addition, a strong synergy between S-thanatin and tigecycline was also observed against E. coli and E. faecalisin in our another experiment. The remaining question is to explain why synergistic effect of S-thanatin was only observed in some species with some classes of antimicrobial agents instead of all species with all class of antimicrobial agents. The incubation period before subculture was extended from 18 to 20 h for 5 days to enhance the detection of lowfrequency mutations that might engender resistance. Resistance to conventional antibiotics usually developed quite easily, however, only a small increase in the MIC of S-thanatin was observed after being cultured at sub-inhibitory concentration for 5 days. The result implies that the rapid generation of high-level resistance to S-thanatin may hardly be possible. In conclusion, S-thanatin displays a superior performance in anti-MDR isolates of K. pneumoniae, especially when combined with conventional antibiotics such as cefepime, and furthermore it is not easy to induce the resistance, suggesting that it may be a candidate for a new antimicrobial agent.
Acknowledgment The work was partly supported by the Fund of Science Research (No:XY2008341 and 9290002295) from Southeast University, PRC.

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