Pone.0029439 1.6

Novel Bacterial Metabolite Merochlorin A Demonstratesin vitro Activity against Multi-Drug Resistant Methicillin-Resistant Staphylococcus aureus George Sakoulas1*, Sang-Jip Nam2, Sandra Loesgen2, William Fenical2,3, Paul R. Jensen2, Victor Nizet1,3, 1 Department of Pediatrics, School of Medicine, University of California San Diego, La Jolla, California, United States of America, 2 Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California, United States of America, 3 School of Pharmacy and Pharmaceutical Sciences, School of Medicine, University of California San Diego, La Jolla, California, United States of America Background: We evaluated the in vitro activity of a merochlorin A, a novel compound with a unique carbon skeleton,against a spectrum of clinically relevant bacterial pathogens and against previously characterized clinical and laboratoryStaphylococcus aureus isolates with resistance to numerous antibiotics.
Methods: Merochlorin A was isolated and purified from a marine-derived actinomycete strain CNH189. Susceptibility testingfor merochlorin A was performed against previously characterized human pathogens using broth microdilution and agardilution methods. Cytotoxicity was assayed in tissue culture assays at 24 and 72 hours against human HeLa and mousesarcoma L929 cell lines.
Results: The structure of as new antibiotic, merochlorin A, was assigned by comprehensive spectroscopic analysis.
Merochlorin A demonstrated in vitro activity against Gram-positive bacteria, including Clostridium dificile, but not againstGram negative bacteria. In S. aureus, susceptibility was not affected by ribosomal mutations conferring linezolid resistance,mutations in dlt or mprF conferring resistance to daptomycin, accessory gene regulator knockout mutations, or thedevelopment of the vancomycin-intermediate resistant phenotype. Merochlorin A demonstrated rapid bactericidal activityagainst MRSA. Activity was lost in the presence of 20% serum.
Conclusions: The unique meroterpenoid, merochlorin A demonstrated excellent in vitro activity against S. aureus and C.
dificile and did not show cross-resistance to contemporary antibiotics against Gram positive organisms. The activity was,however, markedly reduced in 20% human serum. Future directions for this compound may include evaluation for topicaluse, coating biomedical devices, or the pursuit of chemically modified derivatives of this compound that retain activity inthe presence of serum.
Citation: Sakoulas G, Nam S-J, Loesgen S, Fenical W, Jensen PR, et al. (2012) Novel Bacterial Metabolite Merochlorin A Demonstrates in vitro Activity against Multi-Drug Resistant Methicillin-Resistant Staphylococcus aureus. PLoS ONE 7(1): e29439. doi:10.1371/journal.pone.0029439 Editor: Michael Chaussee, University of South Dakota, United States of America Received August 15, 2011; Accepted November 28, 2011; Published January 18, 2012 Copyright: ß 2012 Sakoulas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by National Institutes of Health Grant RO1-GM08555. The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
estimates of about 20% for bacteremia [3]. Currently, the burden ofMRSA infections in the United States is enormous, with almost The constant evolution of bacterial pathogens with resistance to 100,000 annual cases of invasive infections translating to almost clinically available antimicrobial agents drives a continuous demand 20,000 deaths, representing a leading cause of infection-related for the development of novel antimicrobial agents. In the United mortality by a single pathogen [4]. In addition, S. aureus has States, Staphylococcus aureus is the leading cause of hospital-associated historically demonstrated a propensity to develop resistance to and community-associated bacterial infections involving the antibiotics quite rapidly after they are introduced clinically, bloodstream, skin and soft tissue and other sites, with a the majority frequently within 1–2 years [5]. In this setting, the development of these pathogens being methicillin-resistant S. aureus (MRSA) of novel antimicrobial agents against MRSA continues to be in great [1,2]. MRSA is endemic in most hospitals, and invasive MRSA demand. We have examined the in vitro properties of a novel infections have been associated with higher mortality rates than meroterpenoid, merochlorin A (Figure 1), against MSSA and comparable infections caused by methicillin-susceptible S. aureus MRSA with a wide range of resistance determinants against most (MSSA), even when adjusting for host factors, with mortality currently available anti-staphylococcal antibiotics.
January 2012 | Volume 7 | Issue 1 | e29439 characterized in cited references in these tables. Susceptibilitytesting was performed in duplicate using Mueller-Hinton broth(MHB) and Mueller-Hinton agar (MHA) according to CLSImethods. Susceptibility of merochlorin A and vancomycin againstMRSA ATCC33591 was also determined in the presence of 20%activated-pooled human serum (obtained from a pool from healthydonor serum) and 80% MHB. Using 96-well tissue culture treatedplates (MICROTESTTM 96, Becton-Dickinson, Franklin Lakes,NJ), MICs in the presence and absence of serum were determinedthrough the broth microdilution method. It was noted that in thepresence of serum, the endpoints were difficult to read visually orvia a spectrophotometer. Thus, the color-change indicator,resazurin (Sigma-Adrich, St. Louis, MO), was used as an endpointsignal for cell growth as previously shown [16,17]. Resazurinsodium salt powder was solubilized in sterile water to a finalconcentration of 270 mg/40 mL, filtered using a Costar #8160Spin-XH Centrifuge Tube Filter by centrifugation at 10,000 rpmfor 10 min, and added to each well of the test plate in a finalconcentration of 10%. After incubation for 2 hours at 37uC, theplates were visually evaluated for color change from the blueindicator to the pink resorufin, a sign of bacterial growth.
Figure 1. Chemical structure of merochlorin A.
Overnight cultures of test strains in MHB were diluted 1:1000 in fresh broth (inoculum approximately 56105 cfu/ml) containing merochlorin A at 46 MIC (8–16 mg/L) and incubated withshaking at 200 RPM at 37uC. Samples at time 0, 4, and 24 hours were serially diluted 100–106, and 10 microliters were plated on The actinobacterium (strain CNH-189) was isolated from a near- TSA plates. Colonies were counted after 20 hours at 37uC to shore marine sediment collected off Oceanside, California. It was identified as a new Streptomyces sp. based on 16S rRNA genesequence analysis (accession number HQ214120). The strain was cultured in sixty 2.8 L Fernbach flasks each containing 1 L of A1 production medium (10 g starch, 4 g yeast extract, 2 g peptone, 1 g ATCC33591 was incubated for 1 hour in MHB containing 3, 40 mg Fe2(SO4)3 4H2O, 100 mg KBr) and shaken at merochlorin A 2 mg/L (16 MIC) at 37uC with shaking at 200 230 rpm at 27uC. After seven days of cultivation, sterilized XAD-16 RPM in a 1.5 mL Eppendorf tube. The bacteria were pelleted by resin (20 g/L) was added to adsorb the organic products, and the microcentrifugation, washed twice in PBS, and resuspended in an culture and resin were shaken at 215 rpm for 2 hours. The resin was equal volume of fresh antibiotic-free MHB. Samples were filtered through cheesecloth, washed with deionized water, and obtained at various time points, serially-diluted, and 10 microliters eluted with acetone. The acetone was removed under reduced were plated on TSA plates. Colonies were counted at 20 hours to pressure, and the resulting aqueous layer was extracted with EtOAc calculate cfu/ml over time. The PAE was calculated according to (36500 mL). The EtOAc-soluble fraction was dried in vacuo to yield the Craig and Gudmundsson formula: PAE = T – C. In this 4.5 g of crude extract. Note that no specific permits were required formula, T refers to the time it takes the treated culture to recover for the collection of ocean floor sediment. The location of sediment by one-log10 CFU greater then immediately observed after drug collection was not privately-owned or protected territories and there removal (time 0), and C refers to the corresponding recovery time was no impingement on endangered or protected species.
observed for the untreated control [18].
Purification and Structure Assignment of Merochlorin A The crude extract was purified by open column chromatogra- An inoculum of approximately 56107 cfu/mL of MRSA phy on silica gel (25 g), eluted with a step gradient ofdichloromethane and methanol. The dichloromethane/methanol ATCC33591 was prepared in fresh MHB by transferring several 100:1 fraction contained a mixture of metabolites, which were colonies of overnight growth on MHA plates using sterile purified by reversed-phase HPLC (Phenomenex Luna C-18 (2), applicators. Ten microliters of serial 10-fold dilutions (100–107) were plated in triplicate on MHA plates containing 0, 0.25, 0.5, 1, 2, 4, 8, and 16 mg/L of merochlorin A. Colonies were counted at merochlorin A. The structure of merochlorin A was assigned by 20 hours, and surviving bacteria were enumerated for each comprehensive spectroscopic analysis involving interpretation of HRMS data (assignment of molecular formula), infrared and UVspectroscopy (functional groups and chromophore analyses), and by comprehensive 1D and 2D NMR studies at 500 MHz.
Cytotoxcity was assessed by incubation of HeLa and L929 (American Type Culture Collection (ATCC), Manassas, VA) Bacterial strains and in vitro susceptibility testing mammalian cell lines (26104 cells per well) in sterile 96 well tissue Bacterial strains used in this study are described in Tables 1 and culture-treated plates in the presence of decreasing concentrations 2 [6–15]. Many of these strains have been previously well- of merochlorin A and incubation at 37uC with 5% CO2 for 24 h.
January 2012 | Volume 7 | Issue 1 | e29439 Cytotoxicity was assayed by MTS (3-(4,5-dimethylthiazol-2-yl)-5- Table 1. In vitro susceptibility against representative strains (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium) using of clinically relevant bacterial pathogens for merochlorin A.
the CellTiter 96H Aqueous non-radioactive cell proliferation assayaccording to manufacturer’s instructions (Promega Madison, WI).
This assay measures the reducing potential of viable cells using a colorimetric reaction, which is quantitated spectrophotometrically at 490 nm. Cytotoxicity was defined as the concentration which showed a .50% reduction in absorbance at 490 nm compared tothe merochlorin A-free negative control. Etoposide at 10 mg/L was used as the positive cytotoxicity control.
Isolation and structure assignment of merochlorin A The crude extract (4.5 g) was fractionated by open column chromatography on silica gel (25 g), eluted with a step gradient of dichloromethane and methanol. The dichloromethane/methanol 100:1 fraction contained a mixture of metabolites, which were purified by reversed-phase HPLC (Phenomenex Luna C-18 (2), an isocratic solvent system from 85% CH3CN to afford merochlorin A (1, 12.0 mg), as a pale yellow oil. The molecular formula for merochlorin A was deduced as C on analysis of HRESIMS data (a pseudomolecular ion peak at m/z429.1821 [M+H]+) and on interpretation of 13C NMR data.
Merochlorin A showed strong UV absorptions at 240, 296, and 330 nm, indicating a conjugated aromatic or phenolic functional group. The IR spectrum showed broad absorptions for multiplehydroxyl groups (3380 cm21) and characteristic carbonyl groups (1704 cm21). The 1H NMR spectrum of merochlorin A displayeda pair of meta-coupled aromatic protons [H-4 (dH 6.16), H-6 (dH6.38)], one olefin proton H-18 (dH 4.92), five methyl singlets H-21 Table 2. In vitro susceptibility of merochlorin A against multi-drugresistant S. aureus strains, including those resistant tocontemporary antibiotics.
Progenitor of 853b, VAN MIC 1 mg/L (this study) In vitro selected VISA, VAN MIC 8 mg/L (this study) Bloodstream Endocarditis, mecA+, VAN MIC 2 mg/L [9] Selected in vivo VISA, mecA+, VAN MIC 4 mg/L [9] Endocarditis, SA701 Progenitor, DAP MIC 0.25 mg/L [10,11] SA502A, tetM, agr group II prototype [12] VISA selected in vivo from RN9120, VAN MIC 4 mg/L [12] Note: Isolates are grouped together above in cases where they have been isolated from the same patient and/or are the results of manipulations in the laboratory of anoriginal strain and have been confirmed identical to each other by pulsed-field gel electrophoresis (PFGE).
doi:10.1371/journal.pone.0029439.t002 January 2012 | Volume 7 | Issue 1 | e29439 [(dH 1.53), H-20 (dH 1.45), H-24 (dH 1.65), H-22 (dH 1.56), H-25 Susceptibility testing using various MRSA and MSSA with (dH 0.81)], and an exchangeable proton 3-OH (dH 11.9). The 13C resistance to contemporary antibiotics revealed no significant NMR and HSQC spectroscopic data revealed two carbonyl, ten change in MIC to merochlorin A with the acquisition of resistance quaternary, four methine, four methyene, and five methyl carbons.
to vancomycin (both intermediate-level VISA and high–level Interpretation of data from 2D NMR experiments allowed three vanA-mediated VRSA), linezolid, or daptomycin (Table 2). The fragments, a 1,2,3,5 tetrasubstituted benzene moiety, the C-10 side MIC of all S. aureus strains tested against merochlorin A was 2– chain of the molecule, and a propan-2-ylidenecyclopentane 4 mg/L. Population analysis of ATCC 29213 (MSSA) and ATCC moiety, to be assembled. The first fragment was established from 33591 (MRSA) showed a slightly heterogeneous type of suscep- analysis of a 1H-1H coupling constant and 2D NMR spectral data.
tibility for the MSSA and a homogeneous susceptibility for MRSA The meta-coupling of two aromatic protons [H-4 (dH 6.16, d, J = 2.0 Hz), H-6 (dH 6.38, d, J = 2.0 Hz)] indicated a 1,2,3,5 Examination of an accessory gene regulator (agr) knockout of tetrasubstituted benzene moiety. A long range HMBC correlation MSSA RN9120, derived from strain RN6390b (SA502A) (Table 2) of the aromatic proton H-4 to carbons C-3 (dC 165.4) and C-5 (dC which has previously demonstrated a proclivity towards develop- 166.5) and their carbon chemical shifts suggested the substitution ment of vancomycin intermediate resistant phenotype also did not of the two hydroxyl groups at C-3 and C-5, respectively. The show a change in merochlorin A MIC. Agar dilution susceptibility presence of two hydroxyl groups was confirmed by the testing showed one dilution lower MIC than broth-based methods methylation of 5-OH followed by the acetylation of 3-OH. A chelated hydroxyl proton (dH 11.59, 3-OH) strongly suggested the Cytotoxicity, defined as a 50% reduction in absorbance at presence of a carbonyl group at C-1, which revealed the ketone 490 nm measuring the reduced MTS reagent, was noted at group based on the carbon chemical shift of C-1 (dC 193.2). The 64 mg/L and 2 mg/L in HeLa cells at 24 and 72 hours, second fragment, the C-10 side chain of the molecule, was respectively, and at 32 mg/L and 4 mg/L in L929 cells at 24 established from COSY crosspeaks and HMBC correlations (Figure 1b). The COSY crosspeaks from the protons H-16 However, merochlorion A susceptibility testing in MHB through H-18 [H-16 (dH 1.40, 1.14)- H-17 (dH 2.03, 1.75)- H-18 containing 10% and 20% human serum resulted in complete (dH 4.92)], and the long-range HMBC correlations from an inactivation of in vitro activity (MIC.64 mg/L) for all strains olefinic proton H-18 to carbons C-19 (dC 131.6), C-20 (dC 18.1), tested. The post antibiotic effect against MRSA ATCC33591 was C-21 (dC 26.1) permitted the assignment of the side chain of the determined to be 8 and 9 hours at 2 mg/L (16MIC) in 2 separate fragment (C-16 to C-21). The last fragment, a propan-2- ylidenecyclopentane moiety, was assigned by analysis of COSY Killing assays were performed in Mueller-Hinton broth against and HMBC spectroscopic data. The COSY crosspeaks between linezolid-resistant MRSA and 2 VISA (one mecA+ and one H-9 and H-15, and long-range HMBC correlations from H-15 to mecA2). For the VISA strains, fully vancomycin-susceptible C-8, C-9, C-13, C-14, and C-23: from two dimethyl singlet H-22 progenitor parent strains were compared in parallel. For all strains to carbons C-14 and C-23: from H-24 to carbons C-14 and C-23 tested, merochlorin A exhibited potent bactericidal activity, allowed the construction of the propan-2-ylidenecyclopentane achieving the limit of detection at 24 hours (approximately 104 cfu/mL reduction). For both pairs tested, the VISA isolate The three fragments were connected by analysis of HMBC showed increased killing at 4 hours compared to the vancomycin spectroscopic data. The observation of the three bond HMBC susceptible parent strain (data not shown).
correlations from H-6 to C-2, C-4, and C-8 along with two bondHMBC correlations from H-6 to C-5 and C-7 permitted the connection of C-7 to C-8. The connection of C-16 to C-10, of C-10 to C-11, and of C-10 to C-9 was established by the long-range We report a novel natural product, merochlorin A, with in vitro HMBC correlations from H-16 to C-9, C-10, C-11, from H-15 to activity against multi-drug resistant Gram-positive organisms, C-9 and C-10, and from a singlet methyl H-25 to C-9, C-10, C-11, including C. dificile and MRSA. Merochlorin A (Figure 1) possesses and C-16. The connection of C-12 with C-8 was determined by a new carbon skeleton unrelated to any antibacterial agents long-range HMBC correlations from H-13 to C-8 and C-12 and reported to date. This is particularly noteworthy given the paucity from H-9 to C-8 and C-12. Establishing the connectivity of the of novel antibiotic classes currently in development for clinical use.
remaining part of the molecule was difficult due to the absence of Resistance to commonly-used MRSA agents, including vancomy- any protons correlating with carbons C-1, C-11, and C-12. Thus, cin (both intermediate and high-level vanA-mediated), linezolid, we considered carefully the chemical shifts of C-1 (dC 193.2), C-12 and daptomycin did not affect in vitro susceptibility. We evaluated (dC 200.1) and C-11 (dC 91.1): these data led us to place the resistant isolates to contemporary anti-staphylococcal antibiotics remaining chlorine atom at C-11 and to connect C-1 to C-11 and because future agents must be able to counter resistance to these C-11 to C-12, thus completing the structure assignment of drugs, which is to be expected in the years ahead with increased utilization. While resistance to linezolid and daptomycin is rare In vitro susceptibility testing using both broth and agar-based among clinical isolates, we anticipate higher resistance rates in the CLSI methods against single strain representatives of clinically- future. Furthermore, reduced susceptibility across classes that has relevant Gram-positive and Gram-negative bacteria revealed been determined between the lipopeptide daptomycin and activity of compound merochlorin A against MSSA, MRSA, intermediate-level glycopeptides resistance in S. aureus, as well as and VRE but no activity against Gram-negatives (Table 1). For reduced susceptibility to telavancin mediated by vanA, further bacteria for which activity was present, agar dilution consistently heighten concern about the future of antimicrobial resistance in S.
gave a single dilution lower MIC than broth microdilution aureus. This lack of cross-resistance of merochlorin A to (Table 1). Of particular interest was the high potency against contemporary antibiotics with specific mechanisms of action is Clostridium dificile representative strains, including the contempo- consistent with our preliminary mechanism of action studies rary virulent BI strain (also known as the NAP1 or ribotype 027 suggesting global inhibition of DNA, RNA, protein, and cell wall January 2012 | Volume 7 | Issue 1 | e29439 Figure 2. Merochlorin A population analysis of ATCC 29213 (MSSA) and ATCC 33591 (MRSA). Significantly higher viable bacteria at 0.5and 1 mg/L for MSSA compared to MRSA (p,0.01).
doi:10.1371/journal.pone.0029439.g002 Merochlorin A showed bactericidal activity and had an 8– and subsequently infected with resistant Gram-positive pathogens 9 hour post antibiotic effect. Cytotoxicity was favorable in the 24 hour assay but was increased to concentrations close to its In summary, we present merochlorinA, a novel meroterpenoid antimicrobial range in the 72 hour assay. Population analyses of a compound which may provide a foundation for developing viable representative MRSA and MSSA showed a more heterogeneous antimicrobial compounds against MRSA and Clostridium dificile susceptibility profile for MSSA and a homogeneous profile for that are unaffected by resistance to currently utilized antimicrobial In light of inhibition by serum, several avenues exist for merochlorin A. First would be chemical modification of the compound in order to eliminate serum inactivation whilepreserving in vitro activity against MRSA. Our laboratories have We are grateful to Diane M. Cintron of R.M. Alden Research Laboratories(Culver City, CA) for performing susceptibility testing against Clostridium started to develop derivatives based on merochlorin A structure dificile and to Karen Shaw of Trius Therapeutics (San Diego, CA) for that are not inhibited by serum and achieve compounds better suited for systemic use. Other strategies may include development No specific permits were required for the collection of ocean floor and formulation as an enterally administered agent against C.
sediment. The location of sediment collection was not privately-owned or dificile, topical S. aureus decolonization, or the topical therapy of protected territories and there was no impingement on endangered or wound infections, particularly in the setting of rising resistance to mupirocin in S. aureus [19]. Note that prolonged treatment ofinfected devitalized wounds with systemic vancomycin was a feature of some cases in which patients became colonized with Conceived and designed the experiments: GS S-JN SL WF PJ VN MH.
vancomycin-resistant S. aureus [14,15]. Considerations can also be Performed the experiments: GS S-JN SL. Analyzed the data: GS S-JN SL made for using this compound to coat biomedical devices such as WF PJ VN MH. Contributed reagents/materials/analysis tools: GS S-JN central venous catheters that are at risk for becoming colonized SL WF PJ VN MH. Wrote the paper: GS S-JN SL WF PJ VN MH.
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