ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 2010, p. 683–688 Vol. 54, No. 20066-4804/10/$12.00 doi:10.1128/AAC.00702-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Anti-Clostridium difficile Potential of Tetramic Acid Derivatives fromPseudomonas aeruginosa Quorum-Sensing Autoinducers�†

Chihiro Ueda,1 Kazuhiro Tateda,1* Manabu Horikawa,2 Soichiro Kimura,1 Yoshikazu Ishii,1

Kaoru Nomura,2 Kanako Yamada,3 Takashi Suematsu,4 Yasuhisa Inoue,4 Masaji Ishiguro,2Shinichi Miyairi,5 and Keizo Yamaguchi1

Department of Microbiology and Infectious Diseases, Toho University School of Medicine, 5-21-16 Ohmori-nishi, Ohta-ku,Tokyo 143-8540, Japan1; Suntory Institute for Bioorganic Research, Osaka 618-8503, Japan2; Graduate School of

Science and Technology, Nagasaki University, Nagasaki, Japan3; Department of Anatomy and Physiology,Faculty of Wellness, Kwassui Women’s College, Nagasaki, Japan4; and Nihon University College of

Pharmacy, Laboratory of Bio-organic Chemistry, Chiba, Japan5

Received 26 May 2009/Returned for modification 15 June 2009/Accepted 4 November 2009

We have examined the potential bactericidal activities of several tetramic acids derived from Pseudo-monas autoinducers against Clostridium difficile, a cause of antibiotic-associated pseudomembranouscolitis. Clinical isolates of C. difficile (n � 4) were incubated in broth with a chemically synthesizedPseudomonas autoinducer and its tetramic acid derivatives. The structure-activity relationship and themechanisms of action were examined by a time-killing assay and by determination of the morphological/staining characteristics. The use of some tetramic acids derived from N-3-oxododecanoyl L-homoserinelactone resulted in more than 3-log reductions in the viability of C. difficile within 30 min at 30 �M. Theouter membrane was suggested to be one of the targets for the bactericidal activity of tetramic acid,because disturbance of the bacterial outer surface was demonstrated by alteration of the Gram-stainingcharacteristic and electron microscopy. The data for the tetramic acid derivatives demonstrate that theketo-enol structure and the length of the acyl side chain of tetramic acid may be essential for theantibacterial activity of this molecule. These results suggest the potential for tetramic acid derivatives tobe novel agents with activity against C. difficile.

Clostridium difficile is an anaerobic, Gram-positive, spore-forming rod and is well recognized as an etiologic agent for C.difficile-associated diarrhea (CDAD) (13, 15, 18). This organ-ism is known to produce several toxins, such as toxins A and B,which are associated with the pathogenesis of this disease.CDAD is one of the most common nosocomial infections andis a frequent cause of morbidity and mortality among elderlyhospitalized patients (15, 18). In recent years, dramatic in-creases in the incidence of CDAD have been documented inmany hospitals internationally (16, 23, 27). In addition, approx-imately 3 to 8% of CDAD patients have been reported todevelop fulminant diseases, defined as diseases whose coursesare complicated by perforation, severe ileus with toxicmegacolon, and hypotension (12, 17). The rates of mortalityfrom fulminant CDAD are reported to be 30 to 80% (12).Although vancomycin and metronidazole are the first-linetreatment options for CDAD, significant numbers of casesof treatment failure after treatment with these agents havebeen detected in the United States, Europe, and other coun-tries (12). There is a need to develop more effective choicesof treatments against CDAD.

Quorum-sensing systems that allow communication inbacterial societies through the production of autoinducermolecules have been the subject of much recent researchactivity (5, 19). Among human pathogens, the quorum-sens-ing system in Pseudomonas aeruginosa is one of the mostinvestigated (2, 25). The production of virulence factors bythis organism, such as exotoxins, pigments, and exopolysac-charide, is finely regulated by the coordinated production ofautoinducer molecules, such as N-3-oxododecanoyl L-homo-serine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homo-serine lactone (C4-HSL) (5, 22). Furthermore, recentprogress in this field demonstrated that autoinducer mole-cules may play a role not only in intraspecies communicationbut also in interspecies and interkingdom communication,suggesting the multifunctional potential of autoinducers.Importantly, Kaufmann and colleagues have reported thatthe tetramic acid (TA) degradation products of 3-oxo-C12-HSL possess antibacterial activity against Gram-positive or-ganisms, such as Staphylococcus aureus (14). These datasuggest the antibacterial potential of bacterial autoinducermolecules and their TA degradation products, although un-derstanding of their antibacterial spectra, in addition totheir mechanisms of actions, is limited. Furthermore, thestructure-activity relationship of TAs remains fully un-known.

In the present study, we examined the antibacterial activitiesof P. aeruginosa autoinducers and several synthetic TAs againstC. difficile. The structure-activity relationship was explored tosearch for TA derivatives with stronger activity.

* Corresponding author. Mailing address: Department of Microbi-ology and Infectious Diseases, Toho University School of Medicine,5-21-16 Ohmorinishi, Ohtaku, Tokyo 143-8540, Japan. Phone: 81-3-3762-4151, ext. 2396. Fax: 81-3-5493-5415. E-mail: kazu@med.toho-u.ac.jp.

† Supplemental material for this article may be found at http://aac.asm.org/.

� Published ahead of print on 16 November 2009.

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(The results of parts of this study were presented at the 48thInterscience Conference on Antimicrobial Agents and Chemo-therapy, October 2008, Washington, DC [abstr. B-3803].)

MATERIALS AND METHODS

Bacterial strains and culture method used. C. difficile strains (n � 4) isolatedfrom patients with pseudomembranous colitis in the hospital of the Toho Uni-versity School of Medicine were used. The bacteria were grown under anaerobicconditions (Anaero Pack-Anaero; Mitsubishi Gas Chemical, Co., Inc., Japan) inbrain heart infusion broth (BHI; Difco, Becton-Dickinson) supplemented with1% (wt/vol) yeast extract (Difco, Becton-Dickinson) and 0.1% (wt/vol) L-cysteine(Sigma) at 37°C. For agar plate culture, Mueller-Hinton agar supplemented with5% defibrinated sheep blood was used.

P. aeruginosa autoinducers and TAs used. P. aeruginosa autoinducers (3-oxo-C12-HSL, C4-HSL) and other acyl-HSL analogs as precursors of TAs (TA1 toTA7) were chemically synthesized, as described previously (11, 20). Conversionof the acyl-HSL analogs into TAs (TA1 to TA7) was basically done by theprocedure previously described for the preparation of TA1 (14) (Fig. 1). Thestructures of the Z-isofom and the E-isoform of the tetramic acid derivatives areshown in Fig. 1A and B, respectively (21).

General procedure for synthesis of TAs. The condensation of 1 equivalent of3-oxocarboxylic acid (1.0 mmol) and 1.2 equivalents of amino acid ester hydro-chloride or hydrobromide (1.2 mmol) in the presence of 1-hydroxy-7-azabenzo-triazole (1.2 mmol), N-methylmorpholine (1.5 mmol), and 1-(3-dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (EDCI � HCl) in acetonitrile at roomtemperature for 12 h furnished N-3-oxo-acyl-amino acid ester at a moderateyield. The acylamide obtained was converted into the corresponding TA bytreatment with 1 equivalent of sodium tert-butoxide in methanol (0.2 mol/liter) at55°C for 2 h. Purification of the TAs was performed by reverse-phase high-pressure liquid chromatography with a Daisopak SP-120-5-ODS-AP column (20mm by 250 mm; Daiso Co., Ltd.) and a linear gradient of 36% to 72% (vol/vol)CH3CN containing 0.1% (vol/vol) trifluoroacetic acid for 30 min, followed by anisocratic of 72% (vol/vol) CH3CN containing 0.1% (vol/vol) trifluoroacetic acidfor 15 min at a flow rate of 10 ml/min. Monitoring for the TAs was at 254 nm withan SPD-M10A photodiode array detector (Shimadzu, Kyoto, Japan), and the

TAs were collected in fractions of 10 ml each. Each fraction was confirmed to bepure by Shimadzu liquid chromatography (LC)–ion-trap–time-of-flight massspectrometry (MS) with a Cadenza CD-C18 column (2 mm by 100 mm; ImtaktCorp.) and a linear gradient of 30% to 80% (vol/vol) CH3CN containing 0.1%(vol/vol) formic acid over 20 min. The pure fractions were collected and lyoph-ilized. 1H nuclear magnetic resonance (NMR) spectra were recorded at 400 and500 MHz (Jeol EX-400 and Burker DMX-500 NMR spectrometers). The chem-ical shifts were measured relative to the signals for residual CHCl3 (7.26 ppm).The data from 1H-NMR and LC-MS characterizing each compound are de-scribed in the supplemental material.

Alterations of Gram-staining characteristics and morphology of bacteria.After incubation at 37°C for 24 h, C. difficile was harvested by centrifugation at5,000 rpm for 5 min. The bacteria were incubated with TAs or P. aeruginosaautoinducers for the indicated times. The bacteria were stained by Gram’smethod and were observed by light microscopy.

Effects of TAs on viability of bacteria. C. difficile was cultured in the presenceof TAs or Pseudomonas autoinducers at 37°C for the indicated times. Afterincubation, the numbers of viable bacteria were evaluated by cultivation of thesamples after they were serially diluted 10-fold. In some experiments, bacteriol-ysis by TAs was monitored by observing the optical density of the bacterialsuspensions at 600 nm.

Changes in cell surface structures and accumulation of TA1 by electronmicroscopy. For electron microscopy, bacterial cells were immediately immersedin a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phos-phate buffer (pH 7.4) at 4°C for 1 h. After the cells were rinsed in phosphate-buffered saline (PBS), they were postfixed for 1 h in 1% OsO4 in PBS, followedby dehydration in ethanol, and they were then embedded in Epon 812. For theimmunocytochemical observation, bacterial cells were immersed in a mixture of0.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH7.4) for 1 h at 4°C. After the cells were washed in PBS for 1 h with no osmiumtreatment, all cells were dehydrated in graded ethanol and embedded in Unicrylresin. Ultrathin sections were incubated with 1.5% bovine serum albumin in 0.01M PBS for 20 min, followed by incubation with rabbit anti-dansyl antibody(Molecular Probes) diluted 1:100 with 1% BSA and PBS for 2 h at roomtemperature. Negative control sections were incubated with nonimmune rabbitserum at the same concentration. After incubation, the sections were rinsed with

FIG. 1. Chemical structures of P. aeruginosa autoinducers (3-oxo-C12-HSL, C4-HSL) and synthetic TAs (TA1 to TA7, dansyl-TA).

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PBS and incubated with a goat anti-rabbit IgG conjugate with 10-nm colloidalgold particles (BioCell Research Laboratories, Cardiff, United Kingdom) diluted1:20 with PBS at room temperature for 30 min.

Preparation of multilamellar vesicles and solid-state NMR spectroscopy. Inthe model membrane, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)and 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) were utilized aslipids at a molar ratio of 4:1. The membrane system was made of lipids andcosolubilized with 3-oxo-C12-HSL or TA1 at an additive/lipid (A/L) molar ratioof 1:20 in chloroform-methanol (2:1). After evaporation of the solvent undervacuum for one night, the lipid film was hydrated with buffer (20 mM Tris-HCl,100 mM NaCl [pH 7.6]) and vortex mixed. The suspension was freeze-thawed for10 cycles and centrifuged. The supernatant was removed to adjust the watercontent to �80% (wt/wt), and the suspension was transferred to NMR tubescapped to prevent dehydration.

Solid-state NMR spectra were acquired on a CMX Infinity 300 spectrometer(Chemagnetics, Varian, Palo Alto, CA) operating at a proton resonance fre-quency of 300 MHz. 31P spectra were acquired by using a 5-�s single-excitationpulse with 30-kHz continuous-wave (CW) 1H decoupling during acquisition. Thedwell time was 50 ms, and 256 to 1,024 transient readings were accumulated foreach free induction decay (FID) with a 3-s relaxation delay. The 31P chemicalshifts were referenced externally to that of 85% H3PO4 (0 ppm).

RESULTS

Effects of TA on viability of C. difficile. We evaluated theactivities of TA1 and the Pseudomonas autoinducers againstfour strains of C. difficile after 24 h of incubation. As shown inFig. 2, no reduction in bacterial numbers was observed for anystrains examined in the presence of 3-oxo-C12-HSL (50 �M)and C4-HSL (50 �M). In contrast, TA1 reduced the numbersof all bacterial isolates examined in a concentration-dependentmanner.

Effects of TA on Gram-staining characteristics and cell sur-face structures of C. difficile. We compared the Gram-stainingcharacteristics of the C. difficile strains treated with TA1 (50�M) or not treated. Drastic alterations in the Gram-stainingcharacteristics and morphological changes to the cells were

observed (see Fig. S1 in the supplemental material). Even abrief treatment with TA1 (30 min) clearly induced a change inthe Gram-staining characteristics.

Therefore, we next examined the cell surface structures of C.difficile cells treated with TA1 or 3-oxo-C12-HSL by electronmicroscopy. There was no change in the surface structures ofthe cells treated with 3-oxo-C12-HSL compared with those ofthe control bacterial cells. In contrast, TA1 treatment inducedalterations in the bacterial cell surface structures, such as ir-regular margins on the cell surface and a peptidoglycan layerwith an increased width (see Fig. S2 in the supplemental ma-terial). The deposition and accumulation of dansyl-TA weredemonstrated on some areas of the cell surfaces, and scattereddansyl-TA was detected in the cytoplasmic spaces of the bac-teria (Fig. 3a and b). Collectively, these data suggest that TA1accumulates in the cells, probably in the cell surface layer,which may be associated with changes to the Gram-stainingcharacteristics and a loss of viability.

Structure and activity relationship of TAs. To obtain anunderstanding of the structure-activity relationship and tosearch for TAs with stronger activity, we have synthesizedseven TAs with different structures and compared their activ-ities on the viability of C. difficile (Fig. 1). As shown in Fig. 4,the present data suggest that there are structurally criticalpoints for the antibacterial activities of the TAs. We clearlyobserved that TA2 and TA7 had less activity than TA1, sug-gesting that the keto-enol structure and the length of the acylside chain play critical roles. The TAs were generally observedto have antibacterial effects at concentrations of 3 to 50 �Mand in a concentration-dependent manner. Interestingly, TA6was observed to have the strongest effects, in which a morethan 105-fold reduction in bacterial numbers was demonstratedwith TA6 at 10 �M.

FIG. 2. Effect of TA1 on growth of C. difficile. The bacteria (C. difficile 1 to 4) were grown in broth for 24 h with 50 �M autoinducers3-oxo-C12-HSL (C12), C4-HSL (C4), or various concentrations of TA1. The bacterial numbers were then examined (n � 3). Cont., control.

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Induction of bacteriolysis by TAs. We further examined theeffects of the TAs on the growth and bacteriolysis of C. difficile.As shown in Fig. 5, a rapid increase in the optical density at 600nm was observed for the C. difficile control, and the opticaldensity reached a maximum at 6 h after incubation. The TAsexamined suppressed increases in the optical density by 24 hafter incubation. In particular, TA6 demonstrated the stron-gest activity, in which an abrupt reduction in the optical densitywas observed within a couple of hours of incubation. Theseresults suggest that the TAs may induce bacteriolysis in C.difficile, probably through disorganization of the cell surfacestructures.

Analysis of multilamellar vesicles by solid-state NMR spec-troscopy. To examine the effect of tetramic acid on the lipidmembrane, the 31P NMR spectra of the lipid membranes in theabsence and the presence of 3-oxo-C12-HSL or TA1 were mea-sured under static conditions. For the control experiment inthe absence of 3-oxo-C12-HSL or TA1, an axially symmetric,motionally averaged powder pattern was observed (see Fig. S3in the supplemental material). This pattern indicates that the

lipids from the multilamellar vesicles and lipid bilayers aremainly oriented in the magnet, with their normal pattern beingperpendicular to the magnetic field (3). In the presence of3-oxo-C12-HSL, a peak similar to that in the control samplewas observed. The external appearance was also milky whitefor the control sample. In the presence of TA1, however, anisotropic peak was observed. Furthermore, the external ap-pearance was a little see-through white rather than milkywhite. This isotropic component may be ascribable to the fastisotopic tumbling of the phospholipids, which is likely to becaused by the formation of micelles, small unilamellar vesicles,or small discoidal bilayers (4).

DISCUSSION

Kaufmann and collaborators have reported that TA1, a deg-radation product of the P. aeruginosa autoinducer 3-oxo-C12-HSL, has activity against Gram-positive organisms, such asBacillus cereus and Staphylococcus aureus (14). The concentra-tions of TA1 effective against C. difficile (10 to 50 �M) are

FIG. 3. Localization of TA in C. difficile. Dansyl-TA-treated bacteria were reacted with anti-dansyl-TA antibody and gold-labeled anti-rabbitantibody and were then examined by electron microscopy. Arrows, the accumulation of TA1 on the bacterial surface. Magnifications, �25,000 (a)and �80,000 (b).

FIG. 4. Comparison of activities of TAs against C. difficile. Thebacteria were grown for 24 h in BHI broth in the presence of variousconcentrations of TAs. The bacterial numbers were then examined bycultivation of the samples after serial 10-fold dilutions. The detectionlimit is 2 log CFU/ml. Dashed line, bacterial numbers without TAs; �,TA1; �, TA2; ‚, TA3; E, TA4; f, TA5; ●, TA6; �, TA7.

FIG. 5. Induction of bacteriolysis by TAs. The bacteria were incu-bated with several TAs at different concentrations for 26 h. At eachtime point, the optical density (OD) at 600 nm was determined.Dashed line, inoculum dose of C. difficile; ●, control; f, TA1 (50 �M);‚, TA3 (50 �M); �, TA4 (30 �M); E, TA5 (50 �M); �, TA6 (30 �M).

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similar to those described in the previous report (10 to 30 �Mfor B. cereus and S. aureus [14]). These concentrations may bebiologically relevant, because high concentrations of 3-oxo-C12-HSL (�600 �M) have previously been detected in a P.aeruginosa biofilm (1). It is plausible that P. aeruginosa mayutilize TA as an interference strategy to preclude encroach-ment by competing bacteria in a microbial community, al-though it is not known whether this organism produces anautoinducer under anaerobic conditions. In relation to thispoint of view, C. difficile is an inhabitant of the human intes-tinal tract and may have a chance to colocalize and interactwith a variety of intestinal organisms. Although it is likely thatnormal commensal bacteria suppress the overgrowth of C.difficile and protect the intestinal tract from pseudomembra-nous colitis, the activities of endogenous TAs and TA-produc-ing bacteria in the gut and their ability to prevent CDAD arenot known.

Even a brief TA1 treatment induced changes in the Gram-staining characteristics, suggesting an alteration of the cellsurface peptidoglycan structures. Consistent with these find-ings, electron microscopy revealed that bacteria exposed toTA1 bacteria had an ill-defined cell surface margin and anincrease in the width of the peptidoglycan layer. Furthermore,a fluorescent derivative of TA1 and gold-labeled anti-TA1antibody were found to be accumulated and deposited in C.difficile. The 31P NMR spectrum of a model membrane in thepresence of TAs indicated the existence of an isotropic com-ponent, which may be caused by the formation of micelles.These data indicate that TAs directly interact with lipids, asdetermined by use of a model membrane; i.e., the enolateanion of TAs may interact with the ammonium cation of phos-phatidylcholine, DMPC. This finding is consistent with the factthat anti-C. difficile activity is eliminated in the presence ofexcessive Fe3� (data not shown). On the other hand, an inter-action between 3-oxo-C12-HSL and the ammonium cation ofphospholipids would not occur because of the lower acidity ofthe enolate. The interaction of TAs and lipids may destabilizethe cytoplasmic membrane and seriously affect the control ofpeptidoglycan metabolism. Collectively, these data suggest thatcertain types of TAs may be bactericidal for C. difficile, prob-ably because of the damage to the cell surface structures of C.difficile, such as the peptidoglycan layer and cytoplasmic mem-brane, that they cause.

Several natural products containing a tetramic acid motif,such as reutericycline, streptolygidin, and tenuazonic acid,have been demonstrated to possess mycotoxic, antibacterial,antiviral, and antioxidant activities (9, 24, 26). Reutericycline isa well-known tetramic acid isolated from the sourdough isolateLactobacillus reuteri LTH2584 (7, 10). This compound hasbeen demonstrated to be bactericidal against Gram-positivebacteria by acting as a proton ionophore, thereby dissipatingthe transmembrane change in pH and leading to cell lysis (8).The efficacy of this mechanism is a function of the high degreeof hydrophobicity of reutericycline, which therefore favors par-titioning into the cytoplasmic membrane (6, 8). Consistent withthese data, the structure-activity relationship found in thepresent study clearly indicates the importance of the hydro-phobic acyl side chain in TAs. In addition, the presence of aketo-enol structure at the C-3 position was shown to be essen-tial for the bactericidal activities of the TAs against C. difficile.

These data further suggest that tetramic acid may destabilizethe membrane, which results in antibacterial activity.

It may be important to consider the potential applicationand limitation of TAs in the clinical setting. Elimination of theanti-C. difficile activity by metal cations may prevent the clinicalapplication of this compound. On the other hand, it is possiblethat TAs may act in synergy with other classes of antibiotics. Tothis end, we observed an exaggeration of the anti-C. difficileactivity of aminoglycosides in the presence of TAs at sub-MICs(data not shown). Further investigations of the effects of TAsin an in vivo model of C. difficile infection may be necessary tocorrectly define the potential of TAs to be therapeutic agentsagainst this disease.

ACKNOWLEDGMENTS

We thank Barbara H. Iglewski for the kind gift of P. aeruginosaPAO1. We express our deep appreciation to Tse-Hsien Koh for hiscritical comments and careful review of the manuscript.

This study was supported by a grant for strategic research for non-governmental schools of the Heisei 20th from the Ministry of Educa-tion, Culture, Sports, Science and Technology of Japan. No specificfunding was received for this study.

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